SOCS1 kinase inhibitory region peptide mimics regulate interferon gamma and TLR7-induced inflammatory signatures in murine macrophages.

SOCS1 kinase inhibitory region peptide mimics regulate interferon gamma and TLR7-induced inflammatory signatures in murine macrophages. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SOCS1 kinase inhibitory region peptide mimics regulate interferon gamma and TLR7-induced inflammatory signatures in murine macrophages. Jatin Sharma, Valeria Vicuna, Lauren Stafford, Thais Ortiz Rodriguez, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3925558/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although it is known that SOCS1 can modulate JAK/STAT signaling through both its kinase inhibitory region (KIR) and SOCS box domain, and limit toll-like receptor (TLR) induced inflammation via the SOCS1 box domain, the relative contribution of the KIR domain to TLR regulation is not well understood. In this study, we utilized peptide mimics of SOCS1 KIR to study the effect of the KIR domain in modulating TLR7 and interferon γ (IFNγ) signaling in murine primary macrophages and cell lines. We found that SOCS1 KIR mimetics were able to inhibit, by up to 50%, the inflammatory signatures associated with TLR7 stimulation, IFNγ stimulation, and the enhanced IFNγ-induced gene signature, mediated by TLR7 and IFNγ co-treatment. While inhibition of IFNg mediated activation correlated with reduced Y701 phosphorylation on STAT1 and Y705 phosphorylation on STAT3, the inhibition of TLR7-induced inflammation and the TLR7-enhanced IFNγ-induced gene signature coincided with a reduction in both Y701 and S727 phosphorylation on the STAT1 transactivation domain. Altogether, we report for the first time a novel role of the SOCS1 KIR domain in regulating TLR7-mediated, and TLR7-enhanced IFNγ-mediated, inflammation. Biological sciences/Cell biology/Cell growth Biological sciences/Cell biology/Cell signalling Biological sciences/Cell biology Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages Biological sciences/Immunology Biological sciences/Immunology/Chemokines Biological sciences/Immunology/Cytokines Biological sciences/Immunology/Inflammation Biological sciences/Immunology/Innate immune cells Biological sciences/Immunology/Signal transduction therapeutic cytokine SOCS mimetic translation jak/stat signal transduction chemokine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Macrophages exhibit plasticity in the elicitation of effector functions, which allows them to play critical roles in diverse functions including the clearance of apoptotic tissue debris, immune cell trafficking, destruction of pathogenic microbes, and tissue repair( 1 ). To manage revolving tissue specific demands, a combination of external stimuli and intrinsic regulatory mechanisms serve to drive the magnitude, duration, and type of effector functions within macrophages. The importance of Toll-like receptors (TLRs) in the regulation of macrophage effector functions was first demonstrated when it was shown that mice deficient in MyD88 (myeloid differentiation factor 88) or TLR4 were resistant to fatal doses of lipopolysaccharide (LPS)( 2 – 4 ). TLRs are a family of membrane bound or cytosolic receptors that recognize conserved pathogen associated molecular patterns (PAMPS). TLR stimulation is critical in many processes including the transcription of NF-κB responsive genes Il6, Tnfa, and Il1 ,( 5 , 6 ) and the production of microbe lytic compounds( 3 , 7 ). TLR7, a cytosolic TLR that recognizes ssRNA, is highly expressed in macrophages and plays a role in antiviral immunity( 8 ). Consistent with its stated roles, deregulated or excessive TLR7 signal dysregulation has been implicated in driving autoimmune pathogenesis( 9 – 11 ). Indeed, alterations in TLR7 expression have been associated with systemic lupus erythematosus (SLE) pathogenesis in both humans and preclinical disease models ( 12 – 16 ). Additionally, tumor necrosis factor α (TNFα), a downstream product of TLR7 signaling, has been proposed as an important therapeutic target due to its association with autoimmune diseases( 17 – 19 ). Nitric oxide and nitric oxide synthase, additional products of TLR stimulation, have also been associated with Type 1 diabetes (T1D) and SLE pathogenesis.( 20 – 24 ). In addition to TLR signaling, signaling through the Janus kinase/signal transducers and activation of transcription (Jak/STAT) pathway can elicit effector functions in macrophages. For example, interferon gamma (IFNg) promotes the phosphorylation and activation of Jak2, which then facilitates the phosphorylation, dimerization, and nuclear translocation of STAT1. Nuclear translocated STAT1 promotes the transcription of interferon stimulated genes( 25 ). Crosstalk between the JAK/STAT and the TLR signaling pathways has been well documented( 26 ). TLR ligands, such as LPS, have been shown to signal through STAT1; specifically, through the STAT1 transactivation domain (TAD). Activation of the STAT1-TAD depends on serine 727 (S727) residue phosphorylation ( 27 ). Alanine substitution S727 at in STAT1 significantly reduced TLR mediated TNFa production in macrophages( 28 – 30 ). While phosphorylation of the Y701 site on STAT1 is requisite for IFNg mediated effects, TAD activation is essential for full STAT1 transcriptional activity as it both assists in the recruitment of transcriptional cofactors and enhances STAT1 transcriptional output( 31 ). IFNg, apart from signaling through the Y701 residue on STAT1, can also mediate the activation of STAT1 TAD, albeit through a pathway distinct from the TLRs( 32 ). Notably, TLR7 signaling has been shown to synergize with IFNγ in a STAT1-S727 dependent manner to potentiate autoreactive B cell development in a murine model of SLE ( 33 ). Previous research has shown that suppressor of cytokine signaling 1 (SOCS-1) is a critical regulator of both TLR and JAK/STAT signaling (reviewed in ( 34 )). SOCS1 acts as a classical feedback inhibitor, by attenuating the signal that mediated its transcription. Three domains of SOCS1 largely contribute to the regulation of JAK/STAT and TLR signaling – The kinase inhibitory region (KIR), the SH2 domain, and the SOCS box domain. The SH2 domain significantly contributes to the binding of SOCS1 to its target molecule and is required for binding to phosphorylated JAK1 ( 35 , 36 ). While the SOCS box targets accessory proteins in the JAK/STAT and TLR pathways for proteasomal degradation, the KIR directly inhibits STAT protein activation ( 35 , 37 , 38 ). TLR ligands induce the expression of SOCS1 as part of a negative feedback loop, which then critically regulates the magnitude and duration of myeloid cell (e.g. macrophages) effector functions ( 39 ) ( 40 ). Although the contribution of the SOCS box in SOCS1-mediated TLR inhibition has been well characterized, the significance of the KIR is less understood ( 41 – 44 ). To study the contribution of the kinase inhibitory region of SOCS1 to the regulation of macrophage effector functions in response to TLR7 and IFNγ signaling, we utilized peptide mimics of the kinase inhibitory region (SOCS1-KIR). It has previously been shown that peptide mimics of SOCS-KIR inhibit autoimmune disease progression in experimental disease models, with a dimeric variant possessing enhanced efficacy( 34 , 45 – 47 ) We observed reduced STAT (STAT1 and STAT3) activation, and generation of STAT-associated downstream effector functions, in murine primary peritoneal macrophages and RAW264.7 macrophages treated with SOCS1-KIR mimetics. Together, these results implicate a novel mechanism by which the kinase inhibitory region of SOCS1 regulates both TLR7 and combinatorial TLR/IFNg signaling in macrophages. Additionally, our findings suggest that the use of peptide mimetics of SOCS1-KIR may represent a novel therapeutic strategy for the regulation of macrophage responsiveness to external stimulation. MATERIALS AND METHODS Peritoneal macrophage isolation 2 mL 3% Brewer thioglycolate medium was injected into the peritoneal cavity of 6–8 weeks old female BALB/c mice, obtained from the University of Florida animal care breeding services in strict accordance with approved protocols by the Institutional Animal Care and use Committee-accredited Association of Assessment and Accreditation for Laboratory Animal Care. After 4 days, mice were humanely euthanized through compressed carbon dioxide gas (CO 2 ) gas exposure with the use of a flow meter to achieve a 30–70% chamber displacement rate per minute. Death was confirmed by cervical dislocation. 5mL of PBS was injected into the peritoneal cavity and withdrawn to collect peritoneal exudate cells (PECs). PECs were enriched for peritoneal macrophages by seeding 4x10 5 cells per well in a 12-well tissue culture-treated plates (Alkali Scientific) in complete DMEM (10% FBS) with 1x antibiotic-antimycotic (Sigma) and 5% CO 2 overnight and washing out suspended cells. IACUC statement All procedures performed on animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida and were conducted in strict accordance of the approved guidelines. This study did not adhere to the essential 10 ARRIVE guidelines as mice were purchased strictly for the purpose of peritoneal macrophage generation and isolation. LDH release assay 20,000 RAW264.7 or HeLa cells were seeded into a 96-well plate and treated under varying conditions. 50µL of cell supernatants were collected, and Lactate dehydrogenase activity was measured using CyQuant LDH Cytotoxicity assay kit (Invitrogen cat# C20301) as per the manufacturer’s recommendation. Sample Absorbance values, read at 490nm, were normalized to spontaneous release control (water) and maximum release control (manufacturer supplied lysis buffer) to yield % cytotoxicity values. Values were reported as % Cytotoxicity. Peptide Synthesis The SOCS1 KIR mimetic peptide also called SOCS1 KIR (53DTHFRTFRSHSDYRRI), SOCS1-KIR dimeric variant also called SOCS1 KIR dimer (DTHFRTFRSHSDYRRIGGGGGDTHFRTFRSHSDYRRI), pJAK2 (1001LPQDKEYYKVKEP), or Scrambled KIR or control peptide (KHRTDSRHSDRIYTFRF) was generated in-house using Applied Biosystems 431a automated peptide synthesizer (Applied Biosystems, Carlsbad, CA) by conventional fluorenylmethylcarbonyl chemical methods as described( 48 , 49 ), or purchased from GenScript (Piscataway, NJ) at 95% purity. A palmitoyl-lysine (a lipophilic group) was added to the N-terminus of the peptides during the final step to assist in cell penetration. Peptides were characterized by high-performance liquid chromatography (HPLC) and mass spectrometry. Peptides were dissolved dropwise in DMSO, then suspended to final administration volume in sterile PBS (Sigma Aldrich St. Louis, MO), or dissolved in Nanopure water prior to use. Immunoblotting 4x10 5 PECs, RAW264.7, U3A, or HeLa cells were seeded overnight on 12-well plates in complete DMEM with 10% FBS and 1X antibiotic-antimycotic (ABAM). Next day, cells were washed and treated with human IL-6 (10 or 100 ng/mL), mouse IFNγ (10 ng/mL (R&D Systems cat# 485-MI-100), human IFNγ (R&D systems cat# 285-IF), or Resiquimod (11 µM) (Sigma cat# SML0196-10MG) alongside a 2-hour pre-treatment with 33 µM of SOCS1 mimetics, control peptides, Stattic (Sigma cat# S7947), or AG490 (Sigma cat# T3434-5MG) for 30 minutes. The cells were lysed in RIPA lysis buffer system (Santa Cruz biotechnology cat# sc-24948); thereafter, equal amounts of the lysates were run on 4–12% polyacrylamide gels followed by transfer to a nitrocellulose membrane. Membranes were probed with the following primary antibodies: anti-phospho S727 Stat1, anti-phospho Y701-Stat1 (Clone 58D6), anti-Stat1 (Clone D1K9Y), anti-phospho Y705-Stat3 (Clone D3A7), or anti-Stat3 (Clone 79D7) antibodies. Goat anti-rabbit HRP (Biorad cat# 1706515) was used for detection. The blots were developed with clarity western ECL substrate (Biorad cat# 1705061) and read on iBright imaging system (ThermoFisher Scientific, MA). Image analysis for relative quantification of protein bands was performed in iBright Analysis Software (ThermoFisher Scientific, MA). All antibodies were purchased from cell signaling technology unless stated otherwise. Intracellular staining HeLa cells were seeded overnight in complete DMEM on a 12-well plate (4x10 5 Cells/well). On the next day, the cell monolayer was washed with 1X DPBS, supplied with fresh media, and co-treated with human IFNγ (100 ng/mL) in the presence or absence of 33 µM of SOCS1 mimetic peptides, control peptide, or AG490. Cells were incubated for 30min, washed, and fixed in 200 µL warm fixation buffer (BioLegend cat #420801). Fixed cells were washed and permeabilized with TruePhos perm buffer (Biolegend cat# 425401) overnight at -20°C. The cells were incubated with PE anti-STAT1 Phospho (Y701) Antibody (1/20; Biolegend cat# 666404) for 30 minutes in the dark. 20,000 events were acquired by BD Accuri C6. All flowcytometry analysis was performed in FlowJo v10 (Tree Star, San Carlos, CA). Cytokine secretion analysis A total of 5x10 5 RAW264.7 or PECs were seeded overnight on a 12-well plate, followed by washing and incubation with 11 or 33 µM of SOCS1 mimetic peptides, control peptide, or AG490 for 24 hours. After incubation, the macrophages were treated with Resiquimod (11 µM) or LPS (2 nM) for 24 hours. Supernatants were collected on Day 3 and assayed for mouse TNFα by ELISA (BD biosciences cat# 555268) or a multiplexed cytokine array (Biolegend LegendPlex Mouse macrophage panel cat# 740845) as per the manufacturer’s recommendation. The LegendPlex data was analyzed using the QOGNIT webtool ( https://legendplex.qognit.com/ ). RNA isolation and RT-qPCR Total RNA was extracted from 4x10 5 cells using the MicroPlus kit (Qiagen cat# 74034). First strand cDNA synthesis was done using the iScript gDNA clear cDNA synthesis kit (Biorad cat# 1725035). SSO SYBR Green (Biorad cat# 1725272) and gene-specific primers ( Table 1 ) were utilized to amplify relative amounts of cDNA on a CFX connect Real Time system (Biorad). The fold change expression was calculated using the value 2−∆∆CT method, with Bio-Rad CFX manager software and RPLP0 as the reference gene. Luciferase reporter assay A total of 5x10 5 HeLa (STAT1-Luc) or U3A (STAT3-Luc) reporter cells were cultured for 24 or 4 hours on a 12-well plate under varying treatment conditions (like cytokine secretion assay) and were lysed in passive lysis buffer (Promega) for 15min at room temperature. The luciferase activity of the lysate was measured as per the kit (Promega Cat# E1910) manufacturer’s recommendation. Statistical analysis GraphPad Prism v9 was used to calculate statistical significance using one-way ANOVA or two-way ANOVA coupled with Dunnett’s multiple comparison tests with an α of 0.05. p values are indicated within each figure. Bars indicate mean + standard deviation (SD). RESULTS SOCS1 KIR mimetics reduce TLR7-induced inflammatory mediators To gain greater insight on the regulation of TLR7 signaling by the kinase inhibitory region of SOCS1, the RAW264.7 murine macrophage cell line was stimulated with TLR7 agonist Resiquimod (R848) for 24 hours in the presence of SOCS1 KIR peptide mimetics (SOCS1 KIR and SOCS1 KIR dimer 10 µM), or AG490, a known JAK1/2 inhibitor. Additionally, a peptide corresponding to region 1001–1019 of murine JAK2 (pJAK2(1001–1019)) was used as a peptide control. Multiplex cytokine analysis showed that while both SOCS1 KIR (p = 0.0053) and AG490 (p = 0.0006) significantly reduced IL-6 secretion, SOCS1 KIR dimer failed to do so (Fig. 1 A). The SOCS1 KIR dimer significantly reduced TNFa production (p = 0.0013), by 70% on average while TNFa secretion was not significantly impacted by either AG490 or SOCS1 KIR (Fig. 1 B). Neither SOCS1 mimetic modulated IL-10 secretion, while AG490 reduced IL-10 to baseline levels (p = 0.0002) (Fig. 1 C). pJAK2 (1001–1019), previously shown to antagonize endogenous SOCS1 function ( 50 , 51 ), significantly enhanced IL-6 production almost two-fold beyond R848 stimulation but had no additional effect on TNFa or IL-10 production (Fig. 1 A-C). Together, these results suggested regulation of TLR7-mediated effector functions in macrophages by SOCS1 KIR and SOCS1 KIR dimer, which prompted additional experiments. Considering differences between the effects of the monomeric and dimeric variants of SOCS1 KIR mimetics on TNFα production, it was likely that dosing of these mimetics had not been optimized. As such, we next ran a dose response experiment with the mimetics, and measured TNFa secretion by ELISA (Fig. 1 D). Although SOCS1 KIR failed to inhibit TNFa production initially (Fig. 1 B), increasing concentrations of SOCS1 KIR were effective in reducing TNFa production. Consistent with the result in Fig. 1 B, the SOCS1 KIR dimer had a lower effective half max compared to SOCS1 KIR; however, SOCS1 KIR, SOCS1 KIR dimer, and AG490 all inhibited TNFa in a dose dependent manner reaching maximum effective dose at 33µM (Fig. 1 D). At the newly defined maximum dose (33µM), SOCS1 KIR [RAW264.7, p = 0.0029 and PECs, p = 0.0932)] and SOCS1 KIR dimer [RAW264.7, p < 0.0001 and PECs, p = 0.0518] reduced TNFα secretion induced by R848 in all macrophages to near unstimulated levels. (Fig. 1 E, 1 G). Considering that previous reports using SOCS1 deficient macrophages demonstrated the importance SOCS1 in the regulation of LPS-induced TNFα ( 52 , 53 ), we assessed the ability of the SOCS1 KIR mimetics to modulate LPS stimulation. We observed that both SOCS1 KIR mimetics were effective in inhibiting LPS-induced TNFα (Fig. 1 F), while the control peptide had no effect on R848 (TLR7) or LPS (TLR4) mediated activation of the RAW264.7 cell line or PECs (Fig. 1 D-G). Significantly, minimal cellular toxicity was observed with the SOCS1 mimetic peptides, which contrasted with cellular cytotoxicity by AG490 and Stattic, measured by lactate dehydrogenase ( Fig. S1 ). Together these results showed inhibition of TLR induced TNFa production in a dose-dependent manner by the SOCS1 KIR mimetic peptide, which was comparable to the effect of AG490, but without toxicity. Although chemokine and nitric oxide synthase production upon TLR stimulation play a critical role in the trafficking of leucocytes to the site of infection and subsequent microorganism clearance, overproduction of these molecules is associated with autoimmunity and cancer( 54 ). As such, we next examined the effect of SOCS1 KIR mimetics on TLR-7 mediated chemokine and Nos2 transcript profiles in RAW264.7 cells and PECs through qPCR [primers in Table 1]. As expected, chemokines ccl2, ccl3, ccl5 , cxcl10 , and nos2 transcripts were minimally expressed in either RAW264.7 or PECs in the absence of TLR-7 agonist R848 stimulation (as indicated by the white blocks), but their levels were markedly elevated upon R848 administration (as denoted by the conversion of transcriptomic levels to black) (Fig. 2 A, B). Consistent with Fig. 1 , R848 stimulation also enhanced tnfa and il6 transcript levels in RAW264.7 cells and murine macrophages (Fig. 2 A, B). The administration of either SOCS1 KIR, or SOCS1 KIR dimer, attenuated the TLR-7 agonist mediated transcript levels of ccl2, ccl3 , cxcl10, nos2 , and tnfa in both macrophage populations. Also consistent with the results shown in Fig. 1 , tnfa inhibition was more potent with the SOCS1 KIR dimer compared to SOCS1 KIR in both macrophage populations. The quantitative graphical representations show that while chemokine and nos2 down-modulation was consistently more pronounced by the SOCS1 KIR dimer within PECs, as compared to SOCS1 KIR, the results were more variable in the RAW264.7 cell line (Fig. 2 A, B, and S2A). Indeed, SOCS1 KIR dimer administration reduced ccl2, cxcl10 , and nos2 transcripts to near baseline in PECs (Fig. 2 B). SOCS1 KIR dimer-mediated reduction of R848-induced ifnb and ccl5 was also more pronounced in the RAW264.7 cell line compared to SOCS1 KIR, while the level of ccl4 reductions were comparable (Fig. 2 A, B, and S2B). It is also notable that SOCS1 KIR regulated R848-induced il6 transcript levels in RAW264.7 macrophages, but not in PECs. In contrast, SOCS1 KIR dimer effectively mitigated il6 transcript levels in both the cell line and primary macrophages. As expected, control peptide had no effect on R848-induced transcript up-regulation. Additionally, it should be noted that TLR7 stimulation with R848 had no effect on TATA binding protein (TBP), which served as a control non-TLR inducible gene. ( Fig. S2 C ). Taken together, this data indicates that while SOCS1 KIR dimer and SOCS1 KIR both attenuated TLR7-induced inflammatory genes, the SOCS1 KIR dimer demonstrated a higher attenuation efficacy, particularly in primary macrophages. SOCS1 KIR mimetics attenuate STAT1 transcriptional activity IFNγ canonically signals through JAK2-mediated phosphorylation of STAT1 and is also a potent activator of macrophage effector functions such as phagocytosis, antigen presentation, and NO synthesis ( 55 – 57 ). SOCS1 KIR has been previously shown to bind JAK2 and attenuate STAT1 activation ( 46 , 58 ) however, the regulation of downstream transcriptional activity by both the monomeric and dimeric variants remains poorly understood. To better understand the effects of the SOCS1 mimetics on STAT1 transcriptional activity, we utilized a HeLa-reporter cell line transfected with a STAT1 responsive luciferase construct. The reporter HeLa cells were treated with IFNγ for 30min and were intracellularly stained for Y701-STAT1 phosphorylation followed by flow cytometry (Fig. 3 A). The effect of the SOCS1 mimetic peptides on the inhibition of Y701 phosphorylation was assessed by western blot analysis (Fig. 3 B). Statistically significant inhibition was observed for SOCS1 KIR dimer (p = 0.0263) and AG490 (p = 0.0111) (Fig. 3 B). To assess the effect of SOCS1 mimetics on STAT1 transcriptional activity, we treated the HeLa reporter cells with IFNγ for 24 hours and co-treated with inhibitors or control peptide. We observed a dose-dependent inhibitory effect in relative luminescence after treatment with the SOCS1 mimetics (Fig. 3 C), with the highest concentration of SOCS1 KIR inhibiting 50% (p < 0.0001) luminescence compared to 70% with SOCS1 KIR dimer (Fig. 3 D). These results provided an impetus to more closely evaluate the effect of SOCS1 mimetics on the regulation of STAT1-induced genes mediated by IFNg stimulation. We treated RAW264.7 cells and PECs with IFNγ in the presence or absence of SOCS1 KIR dimer and SOCS1 KIR and analyzed selected STAT1 targeted genes. In the absence of IFNγ stimulation, our panel of STAT1 targeted transcripts were minimally activated in RAW264.7 and PECs as denoted by the lightly colored heat map panel which became near black in response to IFNγ signaling (Fig. 4 A, B). Although some distinct transcripts were utilized between RAW264.7 macrophages and PECs, based on differential expression levels, the administration of the SOCS1 KIR mimetics reduced the color intensity of the heat map, indicative of reduced transcript levels for multiple targets in both RAW264.7 cells and PECs. The quantitative graphical representations revealed statistically significant reductions in Nos2 and CD274 (PDL1) by SOCS1 KIR peptides within both RAW264.7 and primary macrophages. In contrast to the overall results obtained from TLR stimulation, SOCS1 mimetic peptide efficacy varied between primary and RAW264.7 macrophages after IFNg stimulation. While Irf1 and Stat1 were consistently reduced upon administration of the SOCS1 KIR mimetics, statistical significance was only achieved by SOCS1 KIR dimer mediated reduction of stat1 transcripts in primary macrophages. Within PECs, the SOCS1 KIR dimer significantly attenuated the expression of C xcl9, Nos2 , and Stat1 . Notably SOCS1 KIR effects were more modest within PECs compared to RAW264.7 cell line; however, a similar trend of inhibition could still be seen. Together, these results highlight the inhibitory effect of the SOCS1 KIR mimetics on STAT1-induced transcription in response to interferon gamma stimulation. SOCS1 KIR dimer promotes enhanced inhibition of IFNγ-mediated STAT1 & STAT3 signaling Given our previously published preclinical data showing enhanced reduction of SLE associated pathologies by the SOCS1 KIR dimer compared to SOCS1 KIR,( 50 ) combined with our current data showing enhanced efficacy of the dimer in mitigating R848 and IFNg signaling, we next tested the hypothesis that the attenuation of R848- and IFNγ-induced inflammatory markers in macrophages would be associated with reduced STAT phosphorylation levels. We assessed Y701 and Y705 phosphorylation status on STAT1 and STAT3 respectively in RAW264.7 cells and peritoneal macrophages after co-incubation with IFNγ and the SOCS1 mimics. As can be seen, the SOCS1 KIR dimer, but not SOCS1 KIR, significantly reduced IFNg mediated STAT1 and STAT3 phosphorylation at Y701 and Y705 in the RAW264.7 cell line. (Fig. 5 A, B). IFNg-mediated STAT1 and STAT3 phosphorylation at tyrosine Y701 and Y705 was not reduced through SOCS1 KIR administration, while SOCS1 KIR dimer had a trending, though not statistically significant, effect within PECs (Fig. 5 C, D). The control, AG490, caused significant reductions in IFNg mediated STAT1 and STAT3 phosphorylation at Y701and Y705 in both the RAW264.7 cell line and in PECs (Fig. 5 A-D). Together these results show that although the SOCS1 KIR and SOCS1 KIR dimer consistently decreased IFNγ-mediated STAT1 activation at Y701, and SOCS1 KIR dimer consistently decreased IFNγ-mediated STAT3 activation at Y705 in RAW264.7 macrophages, only SOCS1 KIR dimer inhibited IFNγ-mediated activation of STAT1 and STAT3 to levels comparable to AG490 in primary peritoneal macrophages. SOCS1 KIR dimer regulates IL-6 mediated STAT1 and STAT3 phosphorylation in macrophages . IL-6 activates Jak2, promoting the phosphorylation and activation of transcription factors STAT1 and STAT3 ( 59 ). Since SOCS1 was originally discovered as a regulator of IL-6 signaling ( 60 – 62 ), we next investigated whether the peptide mimics of the KIR region of SOCS1 could inhibit STAT3 activation with respect to IL-6 signaling. To test this, we first treated the U3A reporter cell line, transfected with a STAT3-responsive luciferase construct, with IL-6. SOCS1 KIR (p < 0.0027), SOCS1 KIR dimer (p < 0.0072), and AG490 comparably reduced IL-6 mediated STAT3 transcriptional activity and STAT3 phosphorylation (Fig. 6 A, B). Stattic, a STAT3 irreversible competitive inhibitor used as a positive control, reduced IL-6 mediated transcriptional levels and STAT3 phosphorylation at Y705 to near background. We also tested the STAT1 transcriptional activity using the HeLa STAT1-luciferase reporter cells. While the luminescence increase was only two-fold with IL-6 (Fig. 6 D), compared to more than 15-fold with IFNγ (Fig. 3 D), we observed a dose dependent inhibitory effect with SOCS1 KIR dimer, but not with SOCS1 KIR (Fig. 6 C). The SOCS1 KIR dimer had a strong and significant effect (p < 0.0001) on IL-6 induced STAT1 transcriptional activity at the highest concentration tested that was consistently more pronounced than AG490 (Fig. 6 D). Next, we analyzed the inhibitory activity of the SOCS1 mimetics on macrophages treated with IL-6. While the reporter assays suggested regulation of IL-6 mediated STAT3 but not STAT1 activation by SOCS1 KIR, the SOCS1 mimetic monomer failed to inhibit either IL-6 mediated STAT1 or STAT3 phosphorylation in either RAW264.7 cells or PECs (Fig. 6 E-H). AG490 treatment significantly reduced Y701-STAT1 phosphorylation in PECs but not RAW264.7, while inhibition of Y705-STAT3 failed to reach significance in either cell type. In contrast to SOCS1 KIR, SOCS1 KIR dimer significantly inhibited both IL-6 mediated Y701-pSTAT1 and Y705-pSTAT3 in RAW264.7 cells (Fig. 6 E, F) and PECs (Fig. 6 G, H). These data suggest that SOCS1 KIR dimer, and not SOCS1 KIR, effectively inhibited IL-6 mediated Y701-pSTAT1 and Y705-pSTAT3 phosphorylation. The inhibition by SOCS1 KIR dimer was also consistently more potent than Jak inhibitor AG490, while lacking any cytotoxicity observed with AG490 at equivalent concentration ( Fig. S1 ). SOCS1 KIR inhibits TLR7 and combined IFNγ-induced expression of IFNγ gene signature While phosphorylation of Y701 drives STAT1 nuclear translocation ( 63 ), full transcriptional and biological activity requires phosphorylation of S727. Additionally, it has been shown that the induction of effector functions, such as TNFa secretion and Nos2 expression, in RAW264.7 by TLR signaling is dependent on S727-STAT1 phosphorylation ( 30 , 64 ). We next assessed the ability of the SOCS1 mimetics to regulate this process. As such, we stimulated RAW264.7 and murine primary macrophages with either R848 or IFNγ in the presence or absence of SOCS1 KIR, SOCS1 KIR dimer, a control peptide, or AG490. As can be seen within the representative western blots, and graphically compiled from several experiments, treatment of RAW264.7 and PECs with either R848 or IFNγ significantly enhanced the phosphorylation of STAT1 at S727 (Fig. 7 A-D). While SOCS1 KIR, SOCS1 KIR dimer, and AG490 significantly reduced R848 induced S727 phosphorylation in RAW264.7 cells, only SOCS1 KIR dimer and AG490 consistently reduced R848-induced S727 phosphorylation in PECS (Fig. 7 A-D). Notably IFNγ induced phosphorylation of S727 was unaffected by either SOCS1 mimetic or AG490. As expected, the control peptide had no effect on IFNγ or R848 stimulation in either the RAW264.7 cells or the primary macrophages (Fig. 7 A-D). Given that TLR signaling can modulate cellular responsiveness to IFNg in a S727-STAT1 dependent manner ( 65 ), we next assessed the ability of the SOCS1 mimetic peptides to regulate the combinatorial effects of TLR7 and IFNγ signaling within primary murine macrophages. As expected, IFNγ, but not TLR7 stimulation, promoted the phosphorylation of Y701 on STAT1. Additionally, Y701 phosphorylation by the combination of R848 and IFNγ stimulation was statistically indistinct from IFNγ stimulation alone (Fig. 7 E). In contrast, while both R848 and IFNγ enhanced S727-STAT1 phosphorylation individually, western blot data clearly show enhanced phosphorylation with the combination of R848 and IFNγ treatment (Fig. 7 E). Notably, murine macrophages cultured in the presence of the combinatorial stimulation consistently had lower levels of Y701 and S727 stimulation in the presence of the SOCS1 KIR mimetic peptides, which reached statistical significance with tyrosine 701 phosphorylation when co-cultured with SOCS1 KIR dimer. While the control peptide had no effect on either Y701 or S727 phosphorylation in response to combinatorial stimulation, AG490 only significantly inhibited S727 phosphorylation. Together these data show that the SOCS1 KIR mimetics, in particular SOCS1 KIR dimer, mediated inhibitory effects on the phosphorylation of S727 within macrophages in response to R848, combined R848 and IFNγ stimulation, but not IFNγ alone. We next assessed the ability of the SOCS1 mimetic peptides to modulate transcriptional activity induced by combined R848 and IFNγ stimulation by designing a panel of responsive transcripts ( Fig. S3 ). Baseline transcript levels for S tat1, Nos2, Cd274 (PDL1), Socs1 , and Socs3 were more elevated in primary macrophages than the RAW264.7 macrophage cell line, as can be seen with darker shading of grey within the PECs compared to the RAW264.7 cells. Except for Socs3 transcript levels within primary macrophages, the addition of 30 µM R848 minimally affected other transcripts. The addition of 1ng/ml of IFNg alone induced transcript changes within RAW264.7 cells after 24 hours of culture. However, the combined treatment of 1ng/ml IFNγ and 30 µM R848 mediated statistically significant up-regulation of Stat1, Nos2, Cd274, Socs1 , and Socs3 in both primary macrophages and the RAW264.7 cell line (Fig. 8 A, B). The enhanced transcriptional activity was consistent with the increased S727-STAT1 phosphorylation in response to the combined treatment ( Fig. 7 E), suggesting S727-STAT1 dependent enhancement of IFNg responsiveness by TLR signaling( 30 , 65 ). The administration of SOCS1 KIR dimer, but not the monomer consistently reduced Stat1 transcription in both the RAW264.7 cell line and primary macrophages. Nos2, Cd274, Socs1 , and Socs3 gene transcription was significantly reduced in the macrophage cell line and consistently reduced in PECs (Fig. 8 A, B). As expected, the control peptide had no effect on the transcription activity mediated by the combinatorial stimulation. Notably, the reduction in transcription by SOCS1 KIR dimer was comparable to the reduction mediated by AG490. However, unlike AG490, the transcriptional inhibition mediated by SOCS1 KIR dimer was at a concentration that did not promote cellular toxicity. To dissect the role of the S727 residue on STAT1 in this dual signal-enhanced transcription, we transfected RAW264.7 cells with a S727A-STAT1 plasmid and evaluated transcript levels in response to R848 and IFNγ co-treatment. An approximate 50% reduction in all transcript levels except PDL1 was noted, highlighting at least a partial role of the S727-STAT1 residue in mediating R848 and IFNγ-mediated transcription ( Fig. S4 ). Together, these data show that SOCS1 KIR dimer administration ameliorated transcriptional activity promoted by TLR7/IFNg. DISCUSSION Recent advances in RNA sequencing technologies have confirmed the results of numerous rodent studies, and preliminary studies involving human samples ( 34 , 66 , 67 ), demonstrating the importance of SOCS1 in the regulation of human diseases ( 68 – 70 ). Indeed, early seminal studies, conducted shortly after the discovery of SOCS1, demonstrated the critical role of SOCS1 in regulating the magnitude and duration of macrophage effector functions ( 53 ). It is therefore no surprise that SOCS1 deficiencies, and/or aberrations in the magnitude and duration macrophage effector functions have been associated with autoimmunity, asthma, and cancer ( 34 , 71 ). While it is well established that the SOCS box region of SOCS1 is crucial to the regulation of TLR and cytokine signaling, the role of the kinase inhibitory region (KIR) is less well understood ( 35 , 46 ). In this study we have expanded upon previous work by our group and others, by exploring specific macrophage signaling pathways that are regulated by peptide mimics of the SOCS1 kinase inhibitory region and potential mechanisms of action. We show that peptide mimetics of SOCS1 KIR attenuated TLR7, IFNg, and TLR7-enhanced IFNg induced gene expression in murine macrophages. Mechanistically we show that the SOCS1 KIR dimer variant attenuated IFNg and IL-6 mediated phosphorylation of STAT1 Y701 and STAT3 Y705 more potently than the SOCS1 KIR monomeric variant. Additionally, the SOCS1 KIR dimer reduced TLR7, but not IFNg induced phosphorylation of the S727 transactivating domain of STAT1. Macrophage recognition of viral ssRNA by TLR7 is critical for the induction of an antiviral state within an individual cell, and the killing of the internalized pathogen. TLR7 is widely utilized as an endosomal sensor for ssRNA and 2’,3’-cGMP to activate the innate immune system, which includes the induction of type 1 interferons. The TLR7 agonist imiquimod is currently used to treat certain infections and cancers in immunodeficient individuals ( 72 , 73 ). Additionally, recent works have suggested that stimulation of the TLR7 pathway could enhance immune activation against hepatitis B virus (HBV) infections and to combat SARS CoV-2 ( 74 ). Conversely, pre-clinical work and human studies have indicated that dysregulated TLR7 signaling likely has important relevance in the autoimmune diseases lupus and psoriasis. It has been shown recently that a TLR7 gain-of-function genetic variation enhanced aberrant survival of activated B lymphocytes and caused human lupus ( 15 ). In this study, we have shown that SOCS1 KIR mimetics reduce TLR7-associated inflammatory markers in primary macrophages with a dose-dependent effect on TNFα at both protein and transcript levels. Our results show that AG490 inhibited the production of both IL-6 and IL-10, consistent with another JAK inhibitor Ruxolitinib ( 75 ). Meanwhile, both SOCS1 KIR mimetic peptides inhibited IL-6 production but left IL-10 unaffected. While IL-6 is intimately associated with the progression of inflammatory processes, IL-10 is involved in the restoration of tolerance. Notably, it has been previously shown that the differential regulation of IL-6 and IL-10 production by macrophages can be mediated in a SOCS1 dependent manner. Several PRR pathways signal through MyD88 to induce transcription of cytokines such as IL-6 and IL-10 and while it is known that SOCS1 KIR domain modulates MyD88 signaling through MAL degradation, evidence suggests that LPS-mediated IL-10 production is not completely MyD88 dependent which could provide insight into the differential regulation seen with SOCS1 KIR mimetics ( 76 , 77 ). It is tempting to speculate that therapeutic targeting of the SOCS1 kinase inhibitory region may exert a biological effect that is distinct from commercially available small molecule targets of janus kinases given their ability to maintain IL-10 levels. In this study, we have shown that SOCS1 KIR mimetics reduce TLR7-associated inflammatory markers in primary macrophages with a dose-dependent effect on TNFα at both protein and transcript levels. This has potential biological relevance as macrophages are the major cellular producer of TNFα. Although TLR-mediated production of TNFα is critical in maintaining proper immune functions, uncontrolled TNFα production is associated with autoimmune/auto-inflammatory diseases including rheumatoid arthritis, Crohn’s disease, and psoriasis ( 5 , 6 , 78 ). Increased expression of IFNγ and TNFα in the liver can also mediate chronic hepatitis ( 79 – 82 ). Notably, it has been recently shown that PEDGA gel-encapsulated SOCS1 KIR peptide reduced M1 macrophage associated markers such as TNFα and Nos2 in the RAW264.7 macrophage cell line ( 83 ). Our results are consistent with SOCS1 conditional knockout experiments in macrophages which led to increased sensitivity to LPS and incessant production of TNFα, IL-6, and CCL2 ( 69 ). We have previously shown that SOCS1-KIR mimetics can reduce TNFα production by both CD4 and CD8 T lymphocytes, suggesting that SOCS1 KIR regulation of TNFα may extend to multiple cell types. Although additional, definitive studies are necessary to establish the mechanism(s) by which SOCS1 KIR mimetics inhibit TLR7 mediated TNFα production, numerous reports support the notion that the regulation may be a secondary effect of interferon beta regulation ( 44 , 84 ). TLR7 possesses Toll/interleukin-1 receptor domain, which serves as a docking site for MyD88, which recruits serine/threonine kinases that drive the cascade culminating in the activation of transcription factors such as NF-kB, activating protein-1(AP-1), and interferon regulatory factors ( 85 ). In turn, the transcription of NF-kB responsive genes such as TNFα ensues, as well as interferon regulatory factors IRF7 and IRF3 contributing to the production of IFNβ. IFNβ promotes the up-regulation of IRF7, in a Jak/STAT dependent manner, forming a feed forward loop to produce more IFNβ. In addition to driving IFNβ production, IRF7 forms a complex with MyD88 and other molecules to drive inflammatory mediators such as TNFα ( 85 ). Notably, the SOCS1-KIR mimetics also inhibited IFNβ production. As such, it is quite possible that SOCS1-KIR mimic peptide may inhibit TNFα production by inhibiting IFNβ mediated production of IRF7 by targeting the Jak/STAT pathway. TLR7 stimulation also promotes the generation of a localized inflammatory response which drives the trafficking of immune cells to the area of microbial insult and elicitation of effector functions necessary for clearance. In this study we show that the administration of SOCS1 KIR and SOCS1 KIR dimer attenuated transcript levels of ccl2, ccl3, ccl5 , and cxcl10. It has been previously shown that the administration of the SOCS1 KIR monomer reduced chemokine expression in the retinae in a rat model of uveitis ( 47 ). Intriguingly, it has been observed that the inhibition of miR155, a post transcriptional regulator of SOCS1, in microglia reduces LPS-induced Nos2, TNFα, IL-6, CCL2, CXCL10, and CXCL9 expression ( 86 – 88 ). In our study, we have shown that SOCS1 mimetics reduced these gene products under both TLR7- and IFNγ-stimulation conditions in peritoneal macrophages. The results observed fit well with our current understanding that SOCS1 deficiency can cause anterior uveitis, marked by increased immune cell infiltration where chemokines play a marked role in the pathology. Interestingly, SOCS1 can reduce chemokine expression, under homeostasis and in response to pathogenic threats and therefore, may be an essential factor in maintaining the ocular immune privilege ( 89 ). Interferon-inducible CXCR3-responsive chemokines such as CXCL9 and CXCL10 have been implicated in inflammatory endocrine, neuronal, and ocular disorders ( 90 – 93 ). Moreover, monocyte chemotactic factors such as CCL2-5 are potent recruiters of dendritic cells and macrophages ( 94 ). Therefore, chemokine regulation could be an alternative approach to ameliorating chronic inflammation. We and others have previously demonstrated that peritoneal, and even topical administration, of SOCS1 KIR was efficacious in prolonging the survival of SOCS1 deficient mice, reducing paralysis in a murine model of multiple sclerosis, and mitigating uveitis that was induced in rodent models ( 95 ). In translational studies, we have recently demonstrated that the administration of topical eye drops containing SOCS1 KIR reduced clinical pathology in horses bearing equine recurrent uveitis, one of the leading causes of equine blindness ( 96 ). It is well established that combination of bio-active peptide regions, using peptide linkers, can generate molecules with increased therapeutic efficacy. Peptide linkers often consist of glycines and/or serines as their flexibility and hydrophilic properties are often beneficial and not inhibitory to the main protein domains. We generated a dimeric SOCS1 KIR mimic (KIR dimer), using a glycine repeat linker, and found that KIR dimer possessed increased efficacy in comparison to SOCS1 KIR in a murine model of lupus ( 50 ). In this study we show that while SOCS1 KIR statically inhibited IL-6 and IFNγ, KIR dimer was more efficacious in the inhibition of TNFα and chemokine mRNA production in PECs. Although both SOCS1 KIR and SOCS1 KIR dimer reduced IFNγ mediated Y701 phosphorylation of STAT1 and Y705 of STAT3, SOCS1 KIR dimer was a more effective inhibitor. KIR dimer was also more effective in the inhibition of IL-6 mediated phosphorylation of STAT1 and STAT3 tyrosine phosphorylation. We hypothesize that the dimer has increased efficacy through enhanced STAT1 regulation. Abbreviations CCL: CC chemokine ligand CCR: CC chemokine receptor cGMP: Cyclic guanosine monophosphate CNS: Central nervous system CXCL: C-X-C motif chemokine ligand CXCR: C-X-C motif chemokine receptor HBV: Hepatitis B virus IFN: Interferon IL: Interleukin JAK: Janus kinase KIR: Kinase inhibitory region LPS: Lipopolysaccharide MAL: MyD88-adapter-like MAPK: Mitogen-activated protein kinase PDL1: Programmed death-ligand 1 PEC: Peritoneal exudate cells PRR: Pattern recognition receptor R848: Resiquimod SLE: Systemic lupus erythematosus SOCS1: Suppressor of cytokine signaling-1 ssRNA: Single-stranded ribonucleic acid STAT: Signal transducer and activator of transcription T1D: Type I diabetes TAD: Transactivation domain Tem: Effector memory T cells TLR: Toll-like receptor TNF: Tumor necrosis factor Treg: Regulatory T cells Declarations ACKNOWLEDGEMENTS JL3 was partially supported by a grant from The Grayson Jockey Research Foundation, Private Funding, and the University of Florida. AUTHOR CONTRIBUTIONS J.S., P.E.K., and J.L.3. designed the experiments. J.S. and J.L.3. conducted experiments and performed data analysis. J.S. and J.L.3. wrote the manuscript. L.S., W.C.S., P.E.K., and J.L.3. edited the paper. V.V., and T.O.R. assisted in experiments. All authors approve the manuscript. CONFLICTS OF INTEREST SOCS mimetic technology is protected by patents 15/113,725 and 11,603,387. The research was supported, in part, by a company currently licensing the technology. Funding support : University of Florida, The Grayson Jockey Research Foundation, and private funding. References Hirayama D, Iida T, Nakase H. The phagocytic function of macrophage-enforcing innate immunity and tissue homeostasis. Vol. 19, International Journal of Molecular Sciences. 2018. Medzhitov R, Preston-Hurlburt P, Janeway CA. A human homologue of the Drosophila toll protein signals activation of adaptive immunity. Nature. 1997;388(6640). Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol. 1999;162(7). Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999;11(1). Zhang Y, Broser M, Rom WN. Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-κB. Proc Natl Acad Sci U S A. 1994;91(6). Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Vol. 13, Annual Review of Immunology. 1995. Doyle SE, O’Connell RM, Miranda GA, Vaidya SA, Chow EK, Liu PT, et al. Toll-like Receptors Induce a Phagocytic Gene Program through p38. Journal of Experimental Medicine. 2004;199(1). Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-Specific Recognition of Single-Stranded RNA via Till-like Receptor 7 and 8. Science (1979). 2004;303(5663). Wirth JR, Molano I, Ruiz P, Coutermarsh-Ott S, Cunningham MA. TLR7 Agonism Accelerates Disease and Causes a Fatal Myeloproliferative Disorder in NZM 2410 Lupus Mice. Front Immunol. 2020;10. Jenks SA, Cashman KS, Zumaquero E, Marigorta UM, Patel A V., Wang X, et al. Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus. Immunity. 2018;49(4). Pawar RD, Ramanjaneyulu A, Kulkarni OP, Lech M, Segerer S, Anders HJ. Inhibition of Toll-like receptor-7 (TLR-7) or TLR-7 plus TLR-9 attenuates glomerulonephritis and lung injury in experimental lupus. Journal of the American Society of Nephrology. 2007;18(6). Pisitkun P. Autoreactive B Cell Responses to RNA-Related Antigens Due to TLR7 Gene Duplication. Science (1979) [Internet]. 2006;312(5780):1669–72. Available from: http://www.sciencemag.org/cgi/doi/ 10.1126/science.1124978 Mohan C, Putterman C. Genetics and pathogenesis of systemic lupus erythematosus and lupus nephritis. Vol. 11, Nature Reviews Nephrology. 2015. Lee YH, Choi SJ, Ji JD, Song GG. Association between toll-like receptor polymorphisms and systemic lupus erythematosus: A meta-analysis update. Lupus. 2016;25(6). Brown GJ, Cañete PF, Wang H, Medhavy A, Bones J, Roco JA, et al. TLR7 gain-of-function genetic variation causes human lupus. Nature [Internet]. 2022;605(7909):349–56. Available from: https://doi.org/10.1038/s41586-022-04642-z Christensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RAA, Shlomchik MJ. Toll-like Receptor 7 and TLR9 Dictate Autoantibody Specificity and Have Opposing Inflammatory and Regulatory Roles in a Murine Model of Lupus. Immunity. 2006;25(3). Aringer M, Smolen JS. The role of tumor necrosis factor-alpha in systemic lupus erythematosus. Vol. 10, Arthritis Research and Therapy. 2008. Manara M, Sinigaglia L. Bone and TNF in rheumatoid arthritis: Clinical implications. Vol. 1, RMD Open. 2015. Lee LF, Xu B, Michie SA, Beilhack GF, Warganich T, Turley S, et al. The role of TNF-α in the pathogenesis of type 1 diabetes in the nonobese diabetic mouse: Analysis of dendritic cell maturation. Proc Natl Acad Sci U S A. 2005;102(44). Suarez-Pinzon WL, Mabley JG, Strynadka K, Power RF, Szabó C, Rabinovitch A. An inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite prevents diabetes development in NOD mice. J Autoimmun. 2001;16(4). Pan L, Yang S, Wang J, Xu M, Wang S, Yi H. Inducible nitric oxide synthase and systemic lupus erythematosus: A systematic review and meta-analysis. Vol. 21, BMC Immunology. 2020. Benaglio P, Zhu H, Okino ML, Yan J, Elgamal R, Nariai N, et al. Type 1 diabetes risk genes mediate pancreatic beta cell survival in response to proinflammatory cytokines. Cell Genomics. 2022;2(12). Nagy G, Perl A. The role of nitric oxide in abnormal T cell signal transduction in systemic lupus erythematosus. Vol. 118, Clinical Immunology. 2006. Nagy G, Koncz A, Phillips P, Perl A. Mitochondrial Signal Transduction Abnormalities in Systemic Lupus Erythematosus. Curr Immunol Rev. 2005;1(1). Ahmed CMI, Larkin J, Johnson HM. SOCS1 mimetics and antagonists: A complementary approach to positive and negative regulation of immune function. Vol. 6, Frontiers in Immunology. 2015. Chmielewski S, Olejnik A, Sikorski K, Pelisek J, Błaszczyk K, Aoqui C, et al. STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis. PLoS One. 2014;9(12). Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, et al. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol. 2002;3(1529–2908). Najjar I, Fagard R. STAT1 and pathogens, not a friendly relationship. Vol. 92, Biochimie. 2010. Schindler C, Fu XY, Improta T, Aebersold R, Darnell JE. Proteins of transcription factor ISGF-3: One gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon α. Proc Natl Acad Sci U S A. 1992;89(16). Luu K, Greenhill CJ, Majoros A, Decker T, Jenkins BJ, Mansell A. STAT1 plays a role in TLR signal transduction and inflammatory responses. Immunol Cell Biol. 2014;92(9). Varinou L, Ramsauer K, Karaghiosoff M, Kolbe T, Pfeffer K, Müller M, et al. Phosphorylation of the Stat1 Transactivation Domain Is Required for Full-Fledged IFN-γ-Dependent Innate Immunity. Immunity. 2003;19(6). Kovarik P, Stoiber D, Eyers PA, Menghini R, Neininger A, Gaestel M, et al. Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway. Proc Natl Acad Sci U S A. 1999;96(24). Chodisetti SB, Fike AJ, Domeier PP, Singh H, Choi NM, Corradetti C, et al. Type II but Not Type I IFN Signaling Is Indispensable for TLR7-Promoted Development of Autoreactive B Cells and Systemic Autoimmunity. The Journal of Immunology. 2020;204(4). Sharma J, Larkin J. Therapeutic implication of SOCS1 modulation in the treatment of autoimmunity and cancer. Frontiers in Pharmacology. 2019. Liau NPD, Laktyushin A, Lucet IS, Murphy JM, Yao S, Whitlock E, et al. The molecular basis of JAK/STAT inhibition by SOCS1. Nat Commun [Internet]. 2018;9(1):1558. Available from: http://www.nature.com/articles/s41467-018-04013-1 Doggett K, Keating N, Dehkhoda F, Bidgood GM, Meza Guzman LG, Leong E, et al. The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo. Cytokine. 2023;165. Zhang JG, Farley A, Nicholson SE, Willson TA, Zugaro LM, Simpson RJ, et al. The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proceedings of the National Academy of Sciences [Internet]. 1999;96(5):2071–6. Available from: http://www.pnas.org/cgi/doi/ 10.1073/pnas.96.5.2071 Ritz O, Guiter C, Dorsch K, Dusanter-Fourt I, Wegener S, Jouault H, et al. STAT6 activity is regulated by SOCS-1 and modulates BCL-XL expression in primary mediastinal B-Cell lymphoma. Leukemia [Internet]. 2008;22(11):2106–10. Available from: http://www.nature.com/articles/leu200885 Dalpke AH, Opper S, Zimmermann S, Heeg K. Suppressors of Cytokine Signaling (SOCS)-1 and SOCS-3 Are Induced by CpG-DNA and Modulate Cytokine Responses in APCs. The Journal of Immunology. 2001;166(12). Piñeros Alvarez AR, Glosson-Byers N, Brandt S, Wang S, Wong H, Sturgeon S, et al. SOCS1 is a negative regulator of metabolic reprogramming during sepsis. JCI Insight. 2017;2(13). Yu CF, Peng WM, Schlee M, Barchet W, Eis-Hübinger AM, Kolanus W, et al. SOCS1 and SOCS3 Target IRF7 Degradation To Suppress TLR7-Mediated Type I IFN Production of Human Plasmacytoid Dendritic Cells. The Journal of Immunology. 2018;200(12). Zhou X, Liu Z, Cheng X, Zheng Y, Zeng F, He Y. Socs1 and Socs3 degrades Traf6 via polyubiquitination in LPS-induced acute necrotizing pancreatitis. Cell Death Dis [Internet]. 2015;6(12):e2012. Available from: http://www.nature.com/doifinder/ 10.1038/cddis.2015.342 Strebovsky J, Walker P, Lang R, Dalpke AH. Suppressor of cytokine signaling 1 (SOCS1) limits NFκB signaling by decreasing p65 stability within the cell nucleus. The FASEB Journal [Internet]. 2011;25(3):863–74. Available from: http://www.fasebj.org /doi/10.1096/fj.10-170597 Prêle CM, Woodward EA, Bisley J, Keith-Magee A, Nicholson SE, Hart PH. SOCS1 Regulates the IFN but Not NFκB Pathway in TLR-Stimulated Human Monocytes and Macrophages. The Journal of Immunology. 2008;181(11). Collins EL, Jager LD, Dabelic R, Benitez P, Holdstein K, Lau K, et al. Inhibition of SOCS1 –/– Lethal Autoinflammatory Disease Correlated to Enhanced Peripheral Foxp3 + Regulatory T Cell Homeostasis. The Journal of Immunology. 2011; Jager LD, Dabelic R, Waiboci LW, Lau K, Haider MS, Ahmed CMI, et al. The kinase inhibitory region of SOCS-1 is sufficient to inhibit T-helper 17 and other immune functions in experimental allergic encephalomyelitis. J Neuroimmunol [Internet]. 2011;232(1–2):108–18. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0165572810004686 He C, Yu CR, Mattapallil MJ, Sun L, Larkin J, Egwuagu CE. SOCS1 Mimetic Peptide Suppresses Chronic Intraocular Inflammatory Disease (Uveitis). Mediators Inflamm. 2016; Thiam K, Loing E, Verwaerde C, Auriault C, Gras-Masse H. IFN-γ-derived lipopeptides: Influence of lipid modification on the conformation and the ability to induce MHC class II expression on murine and human cells. J Med Chem. 1999;42(18). Szente BE, Soos JM, Johnson HM. The C-Terminus of IFN-γ Is Sufficient for Intracellular Function. Biochem Biophys Res Commun. 1994;203(3). Sharma J, Collins TD, Roach T, Mishra S, Lam BK, Mohamed ZS, et al. Suppressor of cytokine signaling-1 mimetic peptides attenuate lymphocyte activation in the MRL/lpr mouse autoimmune model. Sci Rep. 2021;11(1). Ahmed CMI, Dabelic R, Martin JP, Jager LD, Haider SM, Johnson HM. Enhancement of Antiviral Immunity by Small Molecule Antagonist of Suppressor of Cytokine Signaling. The Journal of Immunology. 2010;185(2). Nakagawa R, Naka T, Tsutsui H, Fujimoto M, Kimura A, Abe T, et al. SOCS-1 Participates in Negative Regulation of LPS Responses. Immunity [Internet]. 2002;17(5):677–87. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761302004491 Kinjyo I, Hanada T, Inagaki-Ohara K, Mori H, Aki D, Ohishi M, et al. SOCS1/JAB Is a Negative Regulator of LPS-Induced Macrophage Activation. Immunity [Internet]. 2002;17(5):583–91. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761302004466 Ghafouri-Fard S, Shahir M, Taheri M, Salimi A. A review on the role of chemokines in the pathogenesis of systemic lupus erythematosus. Vol. 146, Cytokine. 2021. Herbst S, Schaible UE, Schneider BE. Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis. PLoS One. 2011;6(5). BoseDasgupta S, Pieters J. Inflammatory Stimuli Reprogram Macrophage Phagocytosis to Macropinocytosis for the Rapid Elimination of Pathogens. PLoS Pathog. 2014;10(1). Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Vol. 8, Nature Reviews Immunology. 2008. Plummer CE, Polk T, Sharma J, Bae SS, Barr O, Jones A, et al. Open label safety and efficacy pilot to study mitigation of equine recurrent uveitis through topical suppressor of cytokine signaling-1 mimetic peptide. Sci Rep [Internet]. 2022;12(1):7177. Available from: https://doi.org/10.1038/s41598-022-11338-x Narazaki M, Witthuhn BA, Yoshida K, Silvennoinen O, Yasukawa K, Ihle JN, et al. Activation of JAK2 kinase mediated by the interleukin 6 signal transducer gp130. Proc Natl Acad Sci U S A. 1994;91(6). Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature [Internet]. 1997;387(6636):924–9. Available from: http://www.nature.com/articles/43219 Starr R, Willson TA, Viney EM, Murray LJL, Rayner JR, Jenkins BJ, et al. A family of cytokine-inducible inhibitors of signalling. Nature [Internet]. 1997;387(6636):917–21. Available from: http://www.nature.com/articles/43206 Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature [Internet]. 1997;387(6636):921–4. Available from: http://www.nature.com/articles/43213 Sadzak I, Schiff M, Gattermeier I, Glinitzer R, Sauer I, Saalmüller A, et al. Recruitment of Stat1 to chromatin is required for interferon-induced serine phosphorylation of Stat1 transactivation domain. Proc Natl Acad Sci U S A. 2008;105(26). Varinou L, Ramsauer K, Karaghiosoff M, Kolbe T, Pfeffer K, Muller M, et al. Phosphorylation of the STAT1 transactivation domain is required for full-fledged IFN-gamma-dependent innate immunity. In: Epitheorese Klinikes Farmakologias kai Farmakokinetikes. 2005. Dalpke AH, Eckerle S, Frey M, Heeg K. Triggering of toll-like receptors modulates IFN signalling: Involvement of serine 727 STAT1 phosphorylation and suppressors of cytokine signaling. Eur J Immunol. 2003;33(7). Yoshimura A, Suzuki M, Sakaguchi R, Hanada T, Yasukawa H. SOCS, inflammation, and autoimmunity. Vol. 3, Frontiers in Immunology. 2012. Naka T, Fujimoto M. SOCS1, a negative regulator of cytokine signals and TLR responses, in human liver diseases. Gastroenterology Research and Practice. 2010. Hadjadj J, Castro CN, Tusseau M, Stolzenberg MC, Mazerolles F, Aladjidi N, et al. Early-onset autoimmunity associated with SOCS1 haploinsufficiency. Nat Commun. 2020;11(1). Sachithanandan N, Graham KL, Galic S, Honeyman JE, Fynch SL, Hewitt KA, et al. Macrophage Deletion of SOCS1 Increases Sensitivity to LPS and Palmitic Acid and Results in Systemic Inflammation and Hepatic Insulin Resistance. Diabetes [Internet]. 2011;60(8):2023–31. Available from: http://diabetes.diabetesjournals.org/cgi/doi/ 10.2337/db11-0259 Guo H, Li R, Wang M, Hou Y, Liu S, Peng T, et al. Multiomics Analysis Identifies SOCS1 as Restraining T Cell Activation and Preventing Graft-Versus-Host Disease. Advanced Science [Internet]. 2022;n/a(n/a):2200978. Available from: https://doi.org/10.1002/advs.202200978 Misharin A V., Morales-Nebreda L, Reyfman PA, Cuda CM, Walter JM, McQuattie-Pimentel AC, et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. Journal of Experimental Medicine. 2017;214(8). Bubna A. Imiquimod-Its role in the treatment of cutaneous malignancies. Vol. 47, Indian Journal of Pharmacology. 2015. Edwards L, Ferenczy A, Eron L, Baker D, Owens ML, Fox TL, et al. Self-administered topical 5% imiquimod cream for external anogenital warts. Arch Dermatol. 1998;134(1). Poulas K, Farsalinos K, Zanidis C. Activation of TLR7 and Innate Immunity as an Efficient Method Against COVID-19 Pandemic: Imiquimod as a Potential Therapy. Vol. 11, Frontiers in Immunology. 2020. Das R, Guan P, Sprague L, Verbist K, Tedrick P, An QA, et al. Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis. Blood. 2016;127(13). Serezani CH, Lewis C, Jancar S, Peters-Golden M. Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression. Journal of Clinical Investigation. 2011;121(2). Mansell A, Smith R, Doyle SL, Gray P, Fenner JE, Crack PJ, et al. Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation. Nat Immunol [Internet]. 2006;7(2):148–55. Available from: http://www.nature.com/articles/ni1299 Holbrook J, Lara-Reyna S, Jarosz-Griffiths H, McDermott MF. Tumour necrosis factor signalling in health and disease. F1000Res. 2019;8. Federici M, Giustizieri ML, Scarponi C, Girolomoni G, Albanesi C. Impaired IFN- -Dependent Inflammatory Responses in Human Keratinocytes Overexpressing the Suppressor of Cytokine Signaling 1. The Journal of Immunology [Internet]. 2002;169(1):434–42. Available from: http://www.jimmunol.org/cgi/doi/ 10.4049/jimmunol.169.1.434 Madonna S, Scarponi C, Doti N, Carbone T, Cavani A, Scognamiglio PL, et al. Therapeutical potential of a peptide mimicking the SOCS1 kinase inhibitory region in skin immune responses. Eur J Immunol. 2013; Okamoto T, Furuya M, Yamakawa T, Yamamura K ichi, Hino O. TNF-α gene expression in the liver of the IFN-γ transgenic mouse with chronic active hepatitis. Biochem Biophys Res Commun. 1996;226(3). Toyonaga T, Hino O, Sugai S, Wakasugi S, Abe K, Shichiri M, et al. Chronic active hepatitis in transgenic mice expressing interferon-γ in the liver. Proc Natl Acad Sci U S A. 1994;91(2). Jha A, Larkin J, Moore E. SOCS1-KIR peptide in PEDGA Hydrogels Reduces Pro‐Inflammatory Macrophage Activation. Macromol Biosci. 2023; Baetz A, Frey M, Heeg K, Dalpke AH. Suppressor of cytokine signaling (SOCS) proteins indirectly regulate Toll-like receptor signaling in innate immune cells. Journal of Biological Chemistry. 2004;279(52). Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Vol. 5, Frontiers in Immunology. 2014. Cardoso AL, Guedes JR, Pereira de Almeida L, Pedroso de Lima MC. miR-155 modulates microglia-mediated immune response by down-regulating SOCS-1 and promoting cytokine and nitric oxide production. Immunology. 2012;135(1). Pathak S, Grillo AR, Scarpa M, Brun P, D’Incà R, Nai L, et al. MiR-155 modulates the inflammatory phenotype of intestinal myofibroblasts by targeting SOCS1 in ulcerative colitis. Exp Mol Med. 2015;47. Ye J, Guo R, Shi Y, Qi F, Guo C, Yang L. MiR-155 Regulated Inflammation Response by the SOCS1-STAT3-PDCD4 Axis in Atherogenesis. Mediators Inflamm. 2016;2016. Yu CR, Hayashi K, Lee YS, Mahdi RM, Shen DF, Chan CC, et al. Suppressor of Cytokine Signaling 1 (SOCS1) Mitigates Anterior Uveitis and Confers Protection Against Ocular HSV-1 Infection. Inflammation. 2015; Fallahi P, Ferrari SM, Ragusa F, Ruffilli I, Elia G, Paparo SR, et al. Th1 chemokines in autoimmune endocrine disorders. Vol. 105, Journal of Clinical Endocrinology and Metabolism. 2020. Howard OMZ, Hui FD, Shao BS, Caspi RR, Chen X, Plotz P, et al. Autoantigens signal through chemokine receptors: Uveitis antigens induce CXCR3- and CXCR5-expressing lymphocytes and immature dendritic cells to migrate. Vol. 105, Blood. 2005. Borjini N, Fernández M, Giardino L, Calzà L. Cytokine and chemokine alterations in tissue, CSF, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (EAE), in a rat model of multiple sclerosis. J Neuroinflammation. 2016;13(1). Christen U, Kimmel R. Chemokines as Drivers of the Autoimmune Destruction in Type 1 Diabetes: Opportunity for Therapeutic Intervention in Consideration of an Optimal Treatment Schedule. Vol. 11, Frontiers in Endocrinology. 2020. Greaves DR, Schall TJ. Chemokines and myeloid cell recruitment. Vol. 2, Microbes and Infection. 2000. He C, Yu CR, Sun L, Mahdi RM, Larkin J, Egwuagu CE. Topical administration of a suppressor of cytokine signaling-1 (SOCS1) mimetic peptide inhibits ocular inflammation and mitigates ocular pathology during mouse uveitis. J Autoimmun [Internet]. 2015;62:31–8. Available from: https://www.sciencedirect.com/science/article/pii/S0896841115000827 Plummer C, Polk T, Sharma J, Bae S, Barr O, Jones A, et al. Mitigation of Equine Recurrent Uveitis Through Topical Suppressor of Cytokine Signaling-1 Mimetic Peptide: Open Label Safety and Efficacy Pilot Study [Internet]. Research Square; 2022. Available from: http://europepmc.org/abstract/PPR/PPR442089 Tables Table 1 is available in the Supplementary Files section. Additional Declarations Competing interest reported. SOCS mimetic technology is protected by patents 15/113,725 and 11,603,387. J. Larkin is co-inventor on the patent on the SOCS mimetic technologies. J. Larkin was supported, in part, by a company currently licensing the technology. J. Sharma, L. Stafford, W.C. Smith, T.O. Rodriguez, V. Vicuna, P.E. Kima declare that they have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Supplementary Files Table1serinekinsaepaper.pdf Table 1: Primers used in study FigureS1.pdf FigureS2.pdf FigureS3.pdf FigureS4.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3925558","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":274975421,"identity":"51356ad7-95d8-4b4f-95ac-5169a6b12794","order_by":0,"name":"Jatin Sharma","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Jatin","middleName":"","lastName":"Sharma","suffix":""},{"id":274975422,"identity":"df98d24e-c004-4a22-83e6-076ff455c288","order_by":1,"name":"Valeria Vicuna","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Vicuna","suffix":""},{"id":274975423,"identity":"d332e49a-b9f2-461e-a527-8136d5179cf6","order_by":2,"name":"Lauren Stafford","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Lauren","middleName":"","lastName":"Stafford","suffix":""},{"id":274975424,"identity":"a2b822e0-61eb-48b8-a13d-231e5529cda4","order_by":3,"name":"Thais Ortiz Rodriguez","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Thais","middleName":"Ortiz","lastName":"Rodriguez","suffix":""},{"id":274975425,"identity":"93afe71f-7957-45f3-8837-17be67d2bc41","order_by":4,"name":"W. Clay Smith","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"W.","middleName":"Clay","lastName":"Smith","suffix":""},{"id":274975426,"identity":"665f52a8-6de6-4470-91fb-4fe273e81e58","order_by":5,"name":"Peter E. Kima","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"E.","lastName":"Kima","suffix":""},{"id":274975427,"identity":"fb9ed7fa-80a2-4e86-acd1-76ab018b31be","order_by":6,"name":"joseph Larkin III","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACxnYgbjBgsONnYHx4gIGBmQgtzRAtyZINzAbEaQGpYWwA4g0HiNXC3Mz87OOMgjvMxjeSGQ4wVFgnNhB2GJvxzA0Gz/jMwFrOpBOjhcGY8YHBYWazG/kHDjC2HSZGC/tnkBbGzTOAtjD+I0oLjzHjBqCWDRIgLQ3EaSlmnGFwOFnizGOGAwnH0o0JajFsb9/M2PPnsB1/ezLjgw811rKEtaCoSCCkHATkiVE0CkbBKBgFIxwAAGtXQMvS6V+mAAAAAElFTkSuQmCC","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"joseph","middleName":"Larkin","lastName":"III","suffix":""}],"badges":[],"createdAt":"2024-02-03 22:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3925558/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3925558/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51778080,"identity":"2ec872cd-33b3-48de-8d6c-24e9a695d0f6","added_by":"auto","created_at":"2024-02-28 21:15:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":298171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR mimetics inhibit TLR7-induced IL-6 and TNF\u003c/strong\u003eα\u003cstrong\u003e, but not IL-10 in primary macrophages and the RAW264.7 macrophage cell line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 cells and primary peritoneal murine macrophages (PECs) were treated with TLR7 agonist Resiquimod (R848) (11 μM) or TLR4 agonist LPS (2 nM) for 24hr in the presence of varied doses of SOCS1 KIR, SOCS1 KIR dimer, AG490, pJAK2, or a control peptide. Bar graphs shown for LegendPlex bead-based multiplex cytokine assay (n=6) for \u003cstrong\u003e(A) \u003c/strong\u003eIL-6, \u003cstrong\u003e(B) \u003c/strong\u003eTNFa, and \u003cstrong\u003e(C) \u003c/strong\u003eIL-10 production in RAW264.7 cells in response to 11 μM R848 in the presence or absence of SOCS1 KIR, SOC1 KIR dimer, AG490, or pJak2. \u003cstrong\u003e(D) \u003c/strong\u003eRAW264.7 cells were treated with R848 (11 μM) and varying doses of SOCS1 KIR mimics or AG490 to generate a dose-curve response for TNFa secretion measured with ELISA, depicted as a scatter plot. \u003cstrong\u003e(E)\u003c/strong\u003eRAW264.7 cells were stimulated with R848 (11 μM) or \u003cstrong\u003e(F)\u003c/strong\u003e LPS (2 nM) and co-treated with 33μM doses of SOCS1 KIR mimics, AG490, or control peptide for 24 hours and TNFα secretion was measured using ELISA. Data depicted on bar graphs (n=3). \u003cstrong\u003e(G) \u003c/strong\u003ePECs were treated with R848 for 24hr with or without the SOCS1 mimetics, JAK1/2 inhibitor AG490, or a control peptide. TNFαsecretion was assessed using ELISA, data reported on bar graph (n=3). One-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/da21b21734a2d7591a14f0a6.png"},{"id":51778082,"identity":"3129443f-cd12-4eaf-b38b-b3ff12d1b825","added_by":"auto","created_at":"2024-02-28 21:15:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":387544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR mimetic peptides inhibit TLR7-induced inflammatory gene signature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eRAW264.7 (n=3) (left) or \u003cstrong\u003e(B)\u003c/strong\u003e PECs (n=3) (right) were treated with the TLR7 agonist Resiquimod (R848) (11 μM) and with SOCS1 mimetics or a control peptide for 24hr. Heatmaps showing gene expression relative to RPLP0 on a grey scale gradient and bar graphs showing average gene expression relative to RPLP0. Two-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/428d649ffa5e8f716f5e0f36.png"},{"id":51778089,"identity":"9a447f60-7345-474b-aac6-ab0239344ab9","added_by":"auto","created_at":"2024-02-28 21:15:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR mimetics attenuate IFNγ-mediated STAT1-associated transcriptional activity in HeLa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa STAT1-luciferase reporter cells were treated with IFNγ for 0.5 or 24 hours with or without SOCS1 mimetics, AG490, or a control peptide. \u003cstrong\u003e(A) \u003c/strong\u003eIntracellular staining for Y701-pSTAT1. A representative histogram and bar graph denoting individual experiments after 0.5hr IFNγ (100ng/ml) stimulation (n=3-6).\u003cstrong\u003e (B) \u003c/strong\u003eRepresentative Western blot for Y701-pSTAT1 and STAT1, and bar graph depicting individual experiments shown for HeLa STAT1-Luc reporter cells after 0.5hr \u0026nbsp;IFNγ (10ng/ml) stimulation (n=3). \u003cstrong\u003e(C) \u003c/strong\u003eGraph showing IFNγ (10ng/ml) treated HeLa STAT1-luciferase reporter cells cultured with graded doses of SOCS1 KIR, SOCS1 KIR dimer, or control peptide for 24 hours. \u003cstrong\u003e(D)\u003c/strong\u003e Relative luminescence at the highest inhibitor concentration after 24 hr was plotted on bar graph (n=3). One-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values are shown on the bar graphs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/8776d6783e272b6a2dc7f343.png"},{"id":51778091,"identity":"592c1e7f-b734-47b8-9347-4b076fe30b28","added_by":"auto","created_at":"2024-02-28 21:15:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":290139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR mimetics attenuate IFNγ-mediated gene expression in macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRAW264.7 cells (n=4) and \u003cstrong\u003e(B)\u003c/strong\u003e PECs (n=3) were treated with 10ng/mL of IFNγ for 24 hours with or without SOCS1 mimetics, AG490, or a control peptide. Average gene expression relative to RPLPL0 for STAT1 target genes depicted as heatmap and bar graphs. One-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/78f2f8cfb1693ff99dce3451.png"},{"id":51778090,"identity":"05204fda-d493-460b-b610-a901a438d65e","added_by":"auto","created_at":"2024-02-28 21:15:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":375832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR dimer inhibits \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eIFNγ -mediated STAT1 \u0026amp; STAT3 tyrosine phosphorylation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7, or PECs were treated for 30min with 10ng/mL IFNγ in the presence of the SOCS1 mimetics, AG490, or a control peptide. \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative Western blot for Y701-pSTAT1 and STAT1, and bar graph depicting individual experiments are shown for RAW264.7 cell line cultured under varied conditions. (n=6). \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative Western blot for Y705-pSTAT3 and STAT3, and bar graph shown for RAW264.7 cell line cultured under described conditions (n=6).\u003cstrong\u003e (C)\u003c/strong\u003e Representative Western blot for Y701-pSTAT1 and STAT1 (loading control) and bar graph shown for PECs (n=3). \u003cstrong\u003e(D) \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eWestern blot for Y705-pSTAT3 and STAT3 and bar graph shown for PECs (n=3).\u003cstrong\u003e \u003c/strong\u003eOne-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/12ba67664a806d59ec0bab77.png"},{"id":51778086,"identity":"43f971ba-682e-425b-9a1d-f97ffa625db8","added_by":"auto","created_at":"2024-02-28 21:15:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":548581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR dimer attenuates IL-6 mediated STAT1 and STAT3 tyrosine activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eU3A STAT3-Luciferase reporter cells, RAW264.7 cells and PECs were treated with IL-6 for varied times alongside SOCS1 mimetics, AG490, or STAT3 irreversible inhibitor Stattic. \u003cstrong\u003e(A) \u003c/strong\u003eBar graph showing luciferase activity after 4 hours of IL-6 treatment in U3A STAT3 reporter cells (n=5). \u003cstrong\u003e(B) \u003c/strong\u003eWestern blot for Y705-pSTAT3 and STAT3 (loading control) after 15 min treatment with IL-6 in U3A STAT3 reporter cells (n=4-6). Bar graph (left) and a representative blot provided (right). \u003cstrong\u003e(C)\u003c/strong\u003e A dosage curve for the luciferase activity measured after 24 hours of IL-6 treatment and \u003cstrong\u003e(D)\u003c/strong\u003e a bar graph at the highest dose used was reported for HeLa STAT1 reporter cells (n=2-4). \u003cstrong\u003e(E) \u003c/strong\u003eY705-pSTAT3 to total STAT3 relative intensity demonstrated as a representative blot and bar graph for RAW264.7 (n=3). \u003cstrong\u003e(F) \u003c/strong\u003eY701-pSTAT1 to total STAT3 relative intensity reported as a representative blot and bar graph for RAW264.7 cells (n=3). \u003cstrong\u003e(G) \u003c/strong\u003eY705-pSTAT3 to total STAT3 relative intensity demonstrated as a representative blot and bar graph for PECs (n=3). \u003cstrong\u003e(H) \u003c/strong\u003eY701-pSTAT1 to total STAT3 relative intensity reported as a representative blot and bar graph for PECs (n=3). One way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/9f9aec4c8a59400b3ba58f57.png"},{"id":51778506,"identity":"62830dc2-45d2-4ee1-8ba2-635ebd39193e","added_by":"auto","created_at":"2024-02-28 21:23:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":565858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR mimetics attenuate TLR7, but not, IFNγ-mediated STAT1 phosphorylation at S727.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 and PECs were treated with TLR7 agonist R848 (A, C), IFNγ(B, D), or both (E) for 30min in the presence of SOCS1 mimetics, AG490, or a control peptide. Representative western Blot for S727-pSTAT1 and STAT1 (loading control) and bar graphs in \u003cstrong\u003e(A, B) \u003c/strong\u003eRAW264.7 cells (n=3) and \u003cstrong\u003e(C, D) \u003c/strong\u003ePECs\u003cstrong\u003e. (E) \u003c/strong\u003eRepresentative western Blot for Y701-STAT1, S727-pSTAT1, and STAT1 (loading control) and bar graphs in PECs (n=3)\u003cstrong\u003e. \u003c/strong\u003eOne-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/6de4c39c5b0fec278652fd2f.png"},{"id":51778505,"identity":"9fa0c462-baa5-4b4b-95a1-67435f7d968d","added_by":"auto","created_at":"2024-02-28 21:23:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":400385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOCS1 KIR mimetics modulate combinatorial TLR7-enhanced IFNγ-induced gene signature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 and PECs were treated with 1ng/mL IFNγ, 30uM R848, or both for 24 hours alongside SOCS1 mimetics, AG490, or a control peptide. Average expression of IFNγ-induced genes relative to RPLP0 (reference gene) depicted as heatmap and bar graphs for \u003cstrong\u003e(A) \u003c/strong\u003eRAW264.7 cells and \u003cstrong\u003e(B)\u003c/strong\u003e PECs. Two-way ANOVA followed by Dunnett’s multiple comparison test was used for statistical testing in GraphPad Prism v9. P-values less than 0.05 are shown on the bar graphs. (Bars indicate mean, error bars S.D.)\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/96ecd37ac94f4422f675b9e3.png"},{"id":84096033,"identity":"c81871b5-42a5-48df-bc76-7e37d2a77478","added_by":"auto","created_at":"2025-06-06 17:31:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4213349,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/7a6851a6-8d9c-42b3-b636-4daa14f7a71a.pdf"},{"id":51778081,"identity":"f7cce925-c7de-4e34-9d11-1f05b94aa6d6","added_by":"auto","created_at":"2024-02-28 21:15:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: Primers used in study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table1serinekinsaepaper.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/83fc82414a13fc8b182f4447.pdf"},{"id":51778092,"identity":"06d1ec0a-6762-4ee1-a953-0fc1a3b56212","added_by":"auto","created_at":"2024-02-28 21:15:07","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":38407,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/c9bbddc511adb7bec3fc8e3f.pdf"},{"id":51778084,"identity":"de098598-1af5-48c6-b1e4-07e7fe356de5","added_by":"auto","created_at":"2024-02-28 21:15:06","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":75472,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/35429c99615b9443697bc545.pdf"},{"id":51778083,"identity":"43f311a1-1594-47ee-ae58-73caa421fc56","added_by":"auto","created_at":"2024-02-28 21:15:06","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17987,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/216a51f2a3dc4869553e4930.pdf"},{"id":51778093,"identity":"dc2e8cf6-3481-4555-8e59-1f85322fb936","added_by":"auto","created_at":"2024-02-28 21:15:07","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":44401,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3925558/v1/3ddea889cf49197c173b25cc.pdf"}],"financialInterests":"Competing interest reported. SOCS mimetic technology is protected by patents 15/113,725 and 11,603,387. J. Larkin is co-inventor on the patent on the SOCS mimetic technologies. J. Larkin was supported, in part, by a company currently licensing the technology.\n\nJ. Sharma, L. Stafford, W.C. Smith, T.O. Rodriguez, V. Vicuna, P.E. Kima declare that they have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper.","formattedTitle":"SOCS1 kinase inhibitory region peptide mimics regulate interferon gamma and TLR7-induced inflammatory signatures in murine macrophages.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMacrophages exhibit plasticity in the elicitation of effector functions, which allows them to play critical roles in diverse functions including the clearance of apoptotic tissue debris, immune cell trafficking, destruction of pathogenic microbes, and tissue repair(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). To manage revolving tissue specific demands, a combination of external stimuli and intrinsic regulatory mechanisms serve to drive the magnitude, duration, and type of effector functions within macrophages. The importance of Toll-like receptors (TLRs) in the regulation of macrophage effector functions was first demonstrated when it was shown that mice deficient in MyD88 (myeloid differentiation factor 88) or TLR4 were resistant to fatal doses of lipopolysaccharide (LPS)(\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). TLRs are a family of membrane bound or cytosolic receptors that recognize conserved pathogen associated molecular patterns (PAMPS). TLR stimulation is critical in many processes including the transcription of NF-κB responsive genes \u003cem\u003eIl6, Tnfa, and Il1\u003c/em\u003e,(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and the production of microbe lytic compounds(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). TLR7, a cytosolic TLR that recognizes ssRNA, is highly expressed in macrophages and plays a role in antiviral immunity(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Consistent with its stated roles, deregulated or excessive TLR7 signal dysregulation has been implicated in driving autoimmune pathogenesis(\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Indeed, alterations in TLR7 expression have been associated with systemic lupus erythematosus (SLE) pathogenesis in both humans and preclinical disease models (\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Additionally, tumor necrosis factor α (TNFα), a downstream product of TLR7 signaling, has been proposed as an important therapeutic target due to its association with autoimmune diseases(\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Nitric oxide and nitric oxide synthase, additional products of TLR stimulation, have also been associated with Type 1 diabetes (T1D) and SLE pathogenesis.(\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to TLR signaling, signaling through the Janus kinase/signal transducers and activation of transcription (Jak/STAT) pathway can elicit effector functions in macrophages. For example, interferon gamma (IFNg) promotes the phosphorylation and activation of Jak2, which then facilitates the phosphorylation, dimerization, and nuclear translocation of STAT1. Nuclear translocated STAT1 promotes the transcription of interferon stimulated genes(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Crosstalk between the JAK/STAT and the TLR signaling pathways has been well documented(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). TLR ligands, such as LPS, have been shown to signal through STAT1; specifically, through the STAT1 transactivation domain (TAD). Activation of the STAT1-TAD depends on serine 727 (S727) residue phosphorylation (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Alanine substitution S727 at in STAT1 significantly reduced TLR mediated TNFa production in macrophages(\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). While phosphorylation of the Y701 site on STAT1 is requisite for IFNg mediated effects, TAD activation is essential for full STAT1 transcriptional activity as it both assists in the recruitment of transcriptional cofactors and enhances STAT1 transcriptional output(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). IFNg, apart from signaling through the Y701 residue on STAT1, can also mediate the activation of STAT1 TAD, albeit through a pathway distinct from the TLRs(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Notably, TLR7 signaling has been shown to synergize with IFNγ in a STAT1-S727 dependent manner to potentiate autoreactive B cell development in a murine model of SLE (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious research has shown that suppressor of cytokine signaling 1 (SOCS-1) is a critical regulator of both TLR and JAK/STAT signaling (reviewed in (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)). SOCS1 acts as a classical feedback inhibitor, by attenuating the signal that mediated its transcription. Three domains of SOCS1 largely contribute to the regulation of JAK/STAT and TLR signaling \u0026ndash; The kinase inhibitory region (KIR), the SH2 domain, and the SOCS box domain. The SH2 domain significantly contributes to the binding of SOCS1 to its target molecule and is required for binding to phosphorylated JAK1 (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). While the SOCS box targets accessory proteins in the JAK/STAT and TLR pathways for proteasomal degradation, the KIR directly inhibits STAT protein activation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). TLR ligands induce the expression of SOCS1 as part of a negative feedback loop, which then critically regulates the magnitude and duration of myeloid cell (e.g. macrophages) effector functions (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Although the contribution of the SOCS box in SOCS1-mediated TLR inhibition has been well characterized, the significance of the KIR is less understood (\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo study the contribution of the kinase inhibitory region of SOCS1 to the regulation of macrophage effector functions in response to TLR7 and IFNγ signaling, we utilized peptide mimics of the kinase inhibitory region (SOCS1-KIR). It has previously been shown that peptide mimics of SOCS-KIR inhibit autoimmune disease progression in experimental disease models, with a dimeric variant possessing enhanced efficacy(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) We observed reduced STAT (STAT1 and STAT3) activation, and generation of STAT-associated downstream effector functions, in murine primary peritoneal macrophages and RAW264.7 macrophages treated with SOCS1-KIR mimetics. Together, these results implicate a novel mechanism by which the kinase inhibitory region of SOCS1 regulates both TLR7 and combinatorial TLR/IFNg signaling in macrophages. Additionally, our findings suggest that the use of peptide mimetics of SOCS1-KIR may represent a novel therapeutic strategy for the regulation of macrophage responsiveness to external stimulation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePeritoneal macrophage isolation\u003c/h2\u003e \u003cp\u003e 2 mL 3% Brewer thioglycolate medium was injected into the peritoneal cavity of 6\u0026ndash;8 weeks old female BALB/c mice, obtained from the University of Florida animal care breeding services in strict accordance with approved protocols by the Institutional Animal Care and use Committee-accredited Association of Assessment and Accreditation for Laboratory Animal Care. After 4 days, mice were humanely euthanized through compressed carbon dioxide gas (CO\u003csub\u003e2\u003c/sub\u003e) gas exposure with the use of a flow meter to achieve a 30\u0026ndash;70% chamber displacement rate per minute. Death was confirmed by cervical dislocation. 5mL of PBS was injected into the peritoneal cavity and withdrawn to collect peritoneal exudate cells (PECs). PECs were enriched for peritoneal macrophages by seeding 4x10\u003csup\u003e5\u003c/sup\u003e cells per well in a 12-well tissue culture-treated plates (Alkali Scientific) in complete DMEM (10% FBS) with 1x antibiotic-antimycotic (Sigma) and 5% CO\u003csub\u003e2\u003c/sub\u003e overnight and washing out suspended cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIACUC statement\u003c/h2\u003e \u003cp\u003e All procedures performed on animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida and were conducted in strict accordance of the approved guidelines. This study did not adhere to the essential 10 ARRIVE guidelines as mice were purchased strictly for the purpose of peritoneal macrophage generation and isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLDH release assay\u003c/h2\u003e \u003cp\u003e20,000 RAW264.7 or HeLa cells were seeded into a 96-well plate and treated under varying conditions. 50\u0026micro;L of cell supernatants were collected, and Lactate dehydrogenase activity was measured using CyQuant LDH Cytotoxicity assay kit (Invitrogen cat# C20301) as per the manufacturer\u0026rsquo;s recommendation. Sample Absorbance values, read at 490nm, were normalized to spontaneous release control (water) and maximum release control (manufacturer supplied lysis buffer) to yield % cytotoxicity values. Values were reported as % Cytotoxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePeptide Synthesis\u003c/h2\u003e \u003cp\u003eThe SOCS1 KIR mimetic peptide also called SOCS1 KIR (53DTHFRTFRSHSDYRRI), SOCS1-KIR dimeric variant also called SOCS1 KIR dimer (DTHFRTFRSHSDYRRIGGGGGDTHFRTFRSHSDYRRI), pJAK2 (1001LPQDKEYYKVKEP), or Scrambled KIR or control peptide (KHRTDSRHSDRIYTFRF) was generated in-house using Applied Biosystems 431a automated peptide synthesizer (Applied Biosystems, Carlsbad, CA) by conventional fluorenylmethylcarbonyl chemical methods as described(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), or purchased from GenScript (Piscataway, NJ) at 95% purity. A palmitoyl-lysine (a lipophilic group) was added to the N-terminus of the peptides during the final step to assist in cell penetration. Peptides were characterized by high-performance liquid chromatography (HPLC) and mass spectrometry. Peptides were dissolved dropwise in DMSO, then suspended to final administration volume in sterile PBS (Sigma Aldrich St. Louis, MO), or dissolved in Nanopure water prior to use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003e4x10\u003csup\u003e5\u003c/sup\u003e PECs, RAW264.7, U3A, or HeLa cells were seeded overnight on 12-well plates in complete DMEM with 10% FBS and 1X antibiotic-antimycotic (ABAM). Next day, cells were washed and treated with human IL-6 (10 or 100 ng/mL), mouse IFNγ (10 ng/mL (R\u0026amp;D Systems cat# 485-MI-100), human IFNγ (R\u0026amp;D systems cat# 285-IF), or Resiquimod (11 \u0026micro;M) (Sigma cat# SML0196-10MG) alongside a 2-hour pre-treatment with 33 \u0026micro;M of SOCS1 mimetics, control peptides, Stattic (Sigma cat# S7947), or AG490 (Sigma cat# T3434-5MG) for 30 minutes. The cells were lysed in RIPA lysis buffer system (Santa Cruz biotechnology cat# sc-24948); thereafter, equal amounts of the lysates were run on 4\u0026ndash;12% polyacrylamide gels followed by transfer to a nitrocellulose membrane. Membranes were probed with the following primary antibodies: anti-phospho S727 Stat1, anti-phospho Y701-Stat1 (Clone 58D6), anti-Stat1 (Clone D1K9Y), anti-phospho Y705-Stat3 (Clone D3A7), or anti-Stat3 (Clone 79D7) antibodies. Goat anti-rabbit HRP (Biorad cat# 1706515) was used for detection. The blots were developed with clarity western ECL substrate (Biorad cat# 1705061) and read on iBright imaging system (ThermoFisher Scientific, MA). Image analysis for relative quantification of protein bands was performed in iBright Analysis Software (ThermoFisher Scientific, MA). All antibodies were purchased from cell signaling technology unless stated otherwise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular staining\u003c/h2\u003e \u003cp\u003eHeLa cells were seeded overnight in complete DMEM on a 12-well plate (4x10\u003csup\u003e5\u003c/sup\u003e Cells/well). On the next day, the cell monolayer was washed with 1X DPBS, supplied with fresh media, and co-treated with human IFNγ (100 ng/mL) in the presence or absence of 33 \u0026micro;M of SOCS1 mimetic peptides, control peptide, or AG490. Cells were incubated for 30min, washed, and fixed in 200 \u0026micro;L warm fixation buffer (BioLegend cat #420801). Fixed cells were washed and permeabilized with TruePhos perm buffer (Biolegend cat# 425401) overnight at -20\u0026deg;C. The cells were incubated with PE anti-STAT1 Phospho (Y701) Antibody (1/20; Biolegend cat# 666404) for 30 minutes in the dark. 20,000 events were acquired by BD Accuri C6. All flowcytometry analysis was performed in FlowJo v10 (Tree Star, San Carlos, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCytokine secretion analysis\u003c/h2\u003e \u003cp\u003eA total of 5x10\u003csup\u003e5\u003c/sup\u003e RAW264.7 or PECs were seeded overnight on a 12-well plate, followed by washing and incubation with 11 or 33 \u0026micro;M of SOCS1 mimetic peptides, control peptide, or AG490 for 24 hours. After incubation, the macrophages were treated with Resiquimod (11 \u0026micro;M) or LPS (2 nM) for 24 hours. Supernatants were collected on Day 3 and assayed for mouse TNFα by ELISA (BD biosciences cat# 555268) or a multiplexed cytokine array (Biolegend LegendPlex Mouse macrophage panel cat# 740845) as per the manufacturer\u0026rsquo;s recommendation. The LegendPlex data was analyzed using the QOGNIT webtool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://legendplex.qognit.com/\u003c/span\u003e\u003cspan address=\"https://legendplex.qognit.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 4x10\u003csup\u003e5\u003c/sup\u003e cells using the MicroPlus kit (Qiagen cat# 74034). First strand cDNA synthesis was done using the iScript gDNA clear cDNA synthesis kit (Biorad cat# 1725035). SSO SYBR Green (Biorad cat# 1725272) and gene-specific primers (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e) were utilized to amplify relative amounts of cDNA on a CFX connect Real Time system (Biorad). The fold change expression was calculated using the value 2\u0026minus;∆∆CT method, with Bio-Rad CFX manager software and RPLP0 as the reference gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eA total of 5x10\u003csup\u003e5\u003c/sup\u003e HeLa (STAT1-Luc) or U3A (STAT3-Luc) reporter cells were cultured for 24 or 4 hours on a 12-well plate under varying treatment conditions (like cytokine secretion assay) and were lysed in passive lysis buffer (Promega) for 15min at room temperature. The luciferase activity of the lysate was measured as per the kit (Promega Cat# E1910) manufacturer\u0026rsquo;s recommendation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism v9 was used to calculate statistical significance using one-way ANOVA or two-way ANOVA coupled with Dunnett\u0026rsquo;s multiple comparison tests with an α of 0.05. p values are indicated within each figure. Bars indicate mean\u0026thinsp;+\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSOCS1 KIR mimetics reduce TLR7-induced inflammatory mediators\u003c/h2\u003e \u003cp\u003eTo gain greater insight on the regulation of TLR7 signaling by the kinase inhibitory region of SOCS1, the RAW264.7 murine macrophage cell line was stimulated with TLR7 agonist Resiquimod (R848) for 24 hours in the presence of SOCS1 KIR peptide mimetics (SOCS1 KIR and SOCS1 KIR dimer 10 \u0026micro;M), or AG490, a known JAK1/2 inhibitor. Additionally, a peptide corresponding to region 1001\u0026ndash;1019 of murine JAK2 (pJAK2(1001\u0026ndash;1019)) was used as a peptide control. Multiplex cytokine analysis showed that while both SOCS1 KIR (p\u0026thinsp;=\u0026thinsp;0.0053) and AG490 (p\u0026thinsp;=\u0026thinsp;0.0006) significantly reduced IL-6 secretion, SOCS1 KIR dimer failed to do so (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The SOCS1 KIR dimer significantly reduced TNFa production (p\u0026thinsp;=\u0026thinsp;0.0013), by 70% on average while TNFa secretion was not significantly impacted by either AG490 or SOCS1 KIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Neither SOCS1 mimetic modulated IL-10 secretion, while AG490 reduced IL-10 to baseline levels (p\u0026thinsp;=\u0026thinsp;0.0002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). pJAK2 (1001\u0026ndash;1019), previously shown to antagonize endogenous SOCS1 function (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), significantly enhanced IL-6 production almost two-fold beyond R848 stimulation but had no additional effect on TNFa or IL-10 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Together, these results suggested regulation of TLR7-mediated effector functions in macrophages by SOCS1 KIR and SOCS1 KIR dimer, which prompted additional experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering differences between the effects of the monomeric and dimeric variants of SOCS1 KIR mimetics on TNFα production, it was likely that dosing of these mimetics had not been optimized. As such, we next ran a dose response experiment with the mimetics, and measured TNFa secretion by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Although SOCS1 KIR failed to inhibit TNFa production initially (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), increasing concentrations of SOCS1 KIR were effective in reducing TNFa production. Consistent with the result in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the SOCS1 KIR dimer had a lower effective half max compared to SOCS1 KIR; however, SOCS1 KIR, SOCS1 KIR dimer, and AG490 all inhibited TNFa in a dose dependent manner reaching maximum effective dose at 33\u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). At the newly defined maximum dose (33\u0026micro;M), SOCS1 KIR [RAW264.7, p\u0026thinsp;=\u0026thinsp;0.0029 and PECs, p\u0026thinsp;=\u0026thinsp;0.0932)] and SOCS1 KIR dimer [RAW264.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and PECs, p\u0026thinsp;=\u0026thinsp;0.0518] reduced TNFα secretion induced by R848 in all macrophages to near unstimulated levels. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Considering that previous reports using SOCS1 deficient macrophages demonstrated the importance SOCS1 in the regulation of LPS-induced TNFα (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), we assessed the ability of the SOCS1 KIR mimetics to modulate LPS stimulation. We observed that both SOCS1 KIR mimetics were effective in inhibiting LPS-induced TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), while the control peptide had no effect on R848 (TLR7) or LPS (TLR4) mediated activation of the RAW264.7 cell line or PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). Significantly, minimal cellular toxicity was observed with the SOCS1 mimetic peptides, which contrasted with cellular cytotoxicity by AG490 and Stattic, measured by lactate dehydrogenase (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Together these results showed inhibition of TLR induced TNFa production in a dose-dependent manner by the SOCS1 KIR mimetic peptide, which was comparable to the effect of AG490, but without toxicity.\u003c/p\u003e \u003cp\u003eAlthough chemokine and nitric oxide synthase production upon TLR stimulation play a critical role in the trafficking of leucocytes to the site of infection and subsequent microorganism clearance, overproduction of these molecules is associated with autoimmunity and cancer(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). As such, we next examined the effect of SOCS1 KIR mimetics on TLR-7 mediated chemokine and \u003cem\u003eNos2\u003c/em\u003e transcript profiles in RAW264.7 cells and PECs through qPCR [primers in Table\u0026nbsp;1]. As expected, chemokines \u003cem\u003eccl2, ccl3, ccl5\u003c/em\u003e, \u003cem\u003ecxcl10\u003c/em\u003e, and \u003cem\u003enos2\u003c/em\u003e transcripts were minimally expressed in either RAW264.7 or PECs in the absence of TLR-7 agonist R848 stimulation (as indicated by the white blocks), but their levels were markedly elevated upon R848 administration (as denoted by the conversion of transcriptomic levels to black) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Consistent with Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, R848 stimulation also enhanced \u003cem\u003etnfa\u003c/em\u003e and \u003cem\u003eil6\u003c/em\u003e transcript levels in RAW264.7 cells and murine macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). The administration of either SOCS1 KIR, or SOCS1 KIR dimer, attenuated the TLR-7 agonist mediated transcript levels of \u003cem\u003eccl2, ccl3\u003c/em\u003e, \u003cem\u003ecxcl10, nos2\u003c/em\u003e, and \u003cem\u003etnfa\u003c/em\u003e in both macrophage populations. Also consistent with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cem\u003etnfa\u003c/em\u003e inhibition was more potent with the SOCS1 KIR dimer compared to SOCS1 KIR in both macrophage populations. The quantitative graphical representations show that while chemokine and \u003cem\u003enos2\u003c/em\u003e down-modulation was consistently more pronounced by the SOCS1 KIR dimer within PECs, as compared to SOCS1 KIR, the results were more variable in the RAW264.7 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, and S2A). Indeed, SOCS1 KIR dimer administration reduced \u003cem\u003eccl2, cxcl10\u003c/em\u003e, and \u003cem\u003enos2\u003c/em\u003e transcripts to near baseline in PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). SOCS1 KIR dimer-mediated reduction of R848-induced \u003cem\u003eifnb\u003c/em\u003e and \u003cem\u003eccl5\u003c/em\u003e was also more pronounced in the RAW264.7 cell line compared to SOCS1 KIR, while the level of \u003cem\u003eccl4\u003c/em\u003e reductions were comparable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, and S2B). It is also notable that SOCS1 KIR regulated R848-induced \u003cem\u003eil6\u003c/em\u003e transcript levels in RAW264.7 macrophages, but not in PECs. In contrast, SOCS1 KIR dimer effectively mitigated \u003cem\u003eil6\u003c/em\u003e transcript levels in both the cell line and primary macrophages. As expected, control peptide had no effect on R848-induced transcript up-regulation. Additionally, it should be noted that TLR7 stimulation with R848 had no effect on TATA binding protein (TBP), which served as a control non-TLR inducible gene. (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC\u003c/b\u003e). Taken together, this data indicates that while SOCS1 KIR dimer and SOCS1 KIR both attenuated TLR7-induced inflammatory genes, the SOCS1 KIR dimer demonstrated a higher attenuation efficacy, particularly in primary macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSOCS1 KIR mimetics attenuate STAT1 transcriptional activity\u003c/h2\u003e \u003cp\u003eIFNγ canonically signals through JAK2-mediated phosphorylation of STAT1 and is also a potent activator of macrophage effector functions such as phagocytosis, antigen presentation, and NO synthesis (\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). SOCS1 KIR has been previously shown to bind JAK2 and attenuate STAT1 activation (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) however, the regulation of downstream transcriptional activity by both the monomeric and dimeric variants remains poorly understood. To better understand the effects of the SOCS1 mimetics on STAT1 transcriptional activity, we utilized a HeLa-reporter cell line transfected with a STAT1 responsive luciferase construct. The reporter HeLa cells were treated with IFNγ for 30min and were intracellularly stained for Y701-STAT1 phosphorylation followed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The effect of the SOCS1 mimetic peptides on the inhibition of Y701 phosphorylation was assessed by western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Statistically significant inhibition was observed for SOCS1 KIR dimer (p\u0026thinsp;=\u0026thinsp;0.0263) and AG490 (p\u0026thinsp;=\u0026thinsp;0.0111) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To assess the effect of SOCS1 mimetics on STAT1 transcriptional activity, we treated the HeLa reporter cells with IFNγ for 24 hours and co-treated with inhibitors or control peptide. We observed a dose-dependent inhibitory effect in relative luminescence after treatment with the SOCS1 mimetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with the highest concentration of SOCS1 KIR inhibiting 50% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) luminescence compared to 70% with SOCS1 KIR dimer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results provided an impetus to more closely evaluate the effect of SOCS1 mimetics on the regulation of STAT1-induced genes mediated by IFNg stimulation. We treated RAW264.7 cells and PECs with IFNγ in the presence or absence of SOCS1 KIR dimer and SOCS1 KIR and analyzed selected STAT1 targeted genes. In the absence of IFNγ stimulation, our panel of STAT1 targeted transcripts were minimally activated in RAW264.7 and PECs as denoted by the lightly colored heat map panel which became near black in response to IFNγ signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Although some distinct transcripts were utilized between RAW264.7 macrophages and PECs, based on differential expression levels, the administration of the SOCS1 KIR mimetics reduced the color intensity of the heat map, indicative of reduced transcript levels for multiple targets in both RAW264.7 cells and PECs. The quantitative graphical representations revealed statistically significant reductions in \u003cem\u003eNos2\u003c/em\u003e and \u003cem\u003eCD274\u003c/em\u003e (PDL1) by SOCS1 KIR peptides within both RAW264.7 and primary macrophages. In contrast to the overall results obtained from TLR stimulation, SOCS1 mimetic peptide efficacy varied between primary and RAW264.7 macrophages after IFNg stimulation. While \u003cem\u003eIrf1 and Stat1\u003c/em\u003e were consistently reduced upon administration of the SOCS1 KIR mimetics, statistical significance was only achieved by SOCS1 KIR dimer mediated reduction of \u003cem\u003estat1\u003c/em\u003e transcripts in primary macrophages. Within PECs, the SOCS1 KIR dimer significantly attenuated the expression of C\u003cem\u003excl9, Nos2\u003c/em\u003e, and \u003cem\u003eStat1\u003c/em\u003e. Notably SOCS1 KIR effects were more modest within PECs compared to RAW264.7 cell line; however, a similar trend of inhibition could still be seen. Together, these results highlight the inhibitory effect of the SOCS1 KIR mimetics on STAT1-induced transcription in response to interferon gamma stimulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSOCS1 KIR dimer promotes enhanced inhibition of IFNγ-mediated STAT1 \u0026amp; STAT3 signaling\u003c/h2\u003e \u003cp\u003eGiven our previously published preclinical data showing enhanced reduction of SLE associated pathologies by the SOCS1 KIR dimer compared to SOCS1 KIR,(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) combined with our current data showing enhanced efficacy of the dimer in mitigating R848 and IFNg signaling, we next tested the hypothesis that the attenuation of R848- and IFNγ-induced inflammatory markers in macrophages would be associated with reduced STAT phosphorylation levels. We assessed Y701 and Y705 phosphorylation status on STAT1 and STAT3 respectively in RAW264.7 cells and peritoneal macrophages after co-incubation with IFNγ and the SOCS1 mimics. As can be seen, the SOCS1 KIR dimer, but not SOCS1 KIR, significantly reduced IFNg mediated STAT1 and STAT3 phosphorylation at Y701 and Y705 in the RAW264.7 cell line. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). IFNg-mediated STAT1 and STAT3 phosphorylation at tyrosine Y701 and Y705 was not reduced through SOCS1 KIR administration, while SOCS1 KIR dimer had a trending, though not statistically significant, effect within PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). The control, AG490, caused significant reductions in IFNg mediated STAT1 and STAT3 phosphorylation at Y701and Y705 in both the RAW264.7 cell line and in PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Together these results show that although the SOCS1 KIR and SOCS1 KIR dimer consistently decreased IFNγ-mediated STAT1 activation at Y701, and SOCS1 KIR dimer consistently decreased IFNγ-mediated STAT3 activation at Y705 in RAW264.7 macrophages, only SOCS1 KIR dimer inhibited IFNγ-mediated activation of STAT1 and STAT3 to levels comparable to AG490 in primary peritoneal macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSOCS1 KIR dimer regulates IL-6 mediated STAT1 and STAT3 phosphorylation in macrophages\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIL-6 activates Jak2, promoting the phosphorylation and activation of transcription factors STAT1 and STAT3 (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Since SOCS1 was originally discovered as a regulator of IL-6 signaling (\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e), we next investigated whether the peptide mimics of the KIR region of SOCS1 could inhibit STAT3 activation with respect to IL-6 signaling. To test this, we first treated the U3A reporter cell line, transfected with a STAT3-responsive luciferase construct, with IL-6. SOCS1 KIR (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0027), SOCS1 KIR dimer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0072), and AG490 comparably reduced IL-6 mediated STAT3 transcriptional activity and STAT3 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Stattic, a STAT3 irreversible competitive inhibitor used as a positive control, reduced IL-6 mediated transcriptional levels and STAT3 phosphorylation at Y705 to near background. We also tested the STAT1 transcriptional activity using the HeLa STAT1-luciferase reporter cells. While the luminescence increase was only two-fold with IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), compared to more than 15-fold with IFNγ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), we observed a dose dependent inhibitory effect with SOCS1 KIR dimer, but not with SOCS1 KIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The SOCS1 KIR dimer had a strong and significant effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) on IL-6 induced STAT1 transcriptional activity at the highest concentration tested that was consistently more pronounced than AG490 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Next, we analyzed the inhibitory activity of the SOCS1 mimetics on macrophages treated with IL-6. While the reporter assays suggested regulation of IL-6 mediated STAT3 but not STAT1 activation by SOCS1 KIR, the SOCS1 mimetic monomer failed to inhibit either IL-6 mediated STAT1 or STAT3 phosphorylation in either RAW264.7 cells or PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H). AG490 treatment significantly reduced Y701-STAT1 phosphorylation in PECs but not RAW264.7, while inhibition of Y705-STAT3 failed to reach significance in either cell type. In contrast to SOCS1 KIR, SOCS1 KIR dimer significantly inhibited both IL-6 mediated Y701-pSTAT1 and Y705-pSTAT3 in RAW264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F) and PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H). These data suggest that SOCS1 KIR dimer, and not SOCS1 KIR, effectively inhibited IL-6 mediated Y701-pSTAT1 and Y705-pSTAT3 phosphorylation. The inhibition by SOCS1 KIR dimer was also consistently more potent than Jak inhibitor AG490, while lacking any cytotoxicity observed with AG490 at equivalent concentration (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSOCS1 KIR inhibits TLR7 and combined IFNγ-induced expression of IFNγ gene signature\u003c/h2\u003e \u003cp\u003eWhile phosphorylation of Y701 drives STAT1 nuclear translocation (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), full transcriptional and biological activity requires phosphorylation of S727. Additionally, it has been shown that the induction of effector functions, such as TNFa secretion and Nos2 expression, in RAW264.7 by TLR signaling is dependent on S727-STAT1 phosphorylation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). We next assessed the ability of the SOCS1 mimetics to regulate this process. As such, we stimulated RAW264.7 and murine primary macrophages with either R848 or IFNγ in the presence or absence of SOCS1 KIR, SOCS1 KIR dimer, a control peptide, or AG490. As can be seen within the representative western blots, and graphically compiled from several experiments, treatment of RAW264.7 and PECs with either R848 or IFNγ significantly enhanced the phosphorylation of STAT1 at S727 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). While SOCS1 KIR, SOCS1 KIR dimer, and AG490 significantly reduced R848 induced S727 phosphorylation in RAW264.7 cells, only SOCS1 KIR dimer and AG490 consistently reduced R848-induced S727 phosphorylation in PECS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). Notably IFNγ induced phosphorylation of S727 was unaffected by either SOCS1 mimetic or AG490. As expected, the control peptide had no effect on IFNγ or R848 stimulation in either the RAW264.7 cells or the primary macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that TLR signaling can modulate cellular responsiveness to IFNg in a S727-STAT1 dependent manner (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e), we next assessed the ability of the SOCS1 mimetic peptides to regulate the combinatorial effects of TLR7 and IFNγ signaling within primary murine macrophages. As expected, IFNγ, but not TLR7 stimulation, promoted the phosphorylation of Y701 on STAT1. Additionally, Y701 phosphorylation by the combination of R848 and IFNγ stimulation was statistically indistinct from IFNγ stimulation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In contrast, while both R848 and IFNγ enhanced S727-STAT1 phosphorylation individually, western blot data clearly show enhanced phosphorylation with the combination of R848 and IFNγ treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Notably, murine macrophages cultured in the presence of the combinatorial stimulation consistently had lower levels of Y701 and S727 stimulation in the presence of the SOCS1 KIR mimetic peptides, which reached statistical significance with tyrosine 701 phosphorylation when co-cultured with SOCS1 KIR dimer. While the control peptide had no effect on either Y701 or S727 phosphorylation in response to combinatorial stimulation, AG490 only significantly inhibited S727 phosphorylation. Together these data show that the SOCS1 KIR mimetics, in particular SOCS1 KIR dimer, mediated inhibitory effects on the phosphorylation of S727 within macrophages in response to R848, combined R848 and IFNγ stimulation, but not IFNγ alone.\u003c/p\u003e \u003cp\u003eWe next assessed the ability of the SOCS1 mimetic peptides to modulate transcriptional activity induced by combined R848 and IFNγ stimulation by designing a panel of responsive transcripts (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). Baseline transcript levels for S\u003cem\u003etat1, Nos2, Cd274 (PDL1), Socs1\u003c/em\u003e, and \u003cem\u003eSocs3\u003c/em\u003e were more elevated in primary macrophages than the RAW264.7 macrophage cell line, as can be seen with darker shading of grey within the PECs compared to the RAW264.7 cells. Except for \u003cem\u003eSocs3\u003c/em\u003e transcript levels within primary macrophages, the addition of 30 \u0026micro;M R848 minimally affected other transcripts. The addition of 1ng/ml of IFNg alone induced transcript changes within RAW264.7 cells after 24 hours of culture. However, the combined treatment of 1ng/ml IFNγ and 30 \u0026micro;M R848 mediated statistically significant up-regulation of \u003cem\u003eStat1, Nos2, Cd274, Socs1\u003c/em\u003e, and \u003cem\u003eSocs3\u003c/em\u003e in both primary macrophages and the RAW264.7 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). The enhanced transcriptional activity was consistent with the increased S727-STAT1 phosphorylation in response to the combined treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), suggesting S727-STAT1 dependent enhancement of IFNg responsiveness by TLR signaling(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). The administration of SOCS1 KIR dimer, but not the monomer consistently reduced \u003cem\u003eStat1\u003c/em\u003e transcription in both the RAW264.7 cell line and primary macrophages. \u003cem\u003eNos2, Cd274, Socs1\u003c/em\u003e, and \u003cem\u003eSocs3\u003c/em\u003e gene transcription was significantly reduced in the macrophage cell line and consistently reduced in PECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). As expected, the control peptide had no effect on the transcription activity mediated by the combinatorial stimulation. Notably, the reduction in transcription by SOCS1 KIR dimer was comparable to the reduction mediated by AG490. However, unlike AG490, the transcriptional inhibition mediated by SOCS1 KIR dimer was at a concentration that did not promote cellular toxicity. To dissect the role of the S727 residue on STAT1 in this dual signal-enhanced transcription, we transfected RAW264.7 cells with a S727A-STAT1 plasmid and evaluated transcript levels in response to R848 and IFNγ co-treatment. An approximate 50% reduction in all transcript levels except PDL1 was noted, highlighting at least a partial role of the S727-STAT1 residue in mediating R848 and IFNγ-mediated transcription (\u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). Together, these data show that SOCS1 KIR dimer administration ameliorated transcriptional activity promoted by TLR7/IFNg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eRecent advances in RNA sequencing technologies have confirmed the results of numerous rodent studies, and preliminary studies involving human samples (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e), demonstrating the importance of SOCS1 in the regulation of human diseases (\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Indeed, early seminal studies, conducted shortly after the discovery of SOCS1, demonstrated the critical role of SOCS1 in regulating the magnitude and duration of macrophage effector functions (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). It is therefore no surprise that SOCS1 deficiencies, and/or aberrations in the magnitude and duration macrophage effector functions have been associated with autoimmunity, asthma, and cancer (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). While it is well established that the SOCS box region of SOCS1 is crucial to the regulation of TLR and cytokine signaling, the role of the kinase inhibitory region (KIR) is less well understood (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). In this study we have expanded upon previous work by our group and others, by exploring specific macrophage signaling pathways that are regulated by peptide mimics of the SOCS1 kinase inhibitory region and potential mechanisms of action. We show that peptide mimetics of SOCS1 KIR attenuated TLR7, IFNg, and TLR7-enhanced IFNg induced gene expression in murine macrophages. Mechanistically we show that the SOCS1 KIR dimer variant attenuated IFNg and IL-6 mediated phosphorylation of STAT1 Y701 and STAT3 Y705 more potently than the SOCS1 KIR monomeric variant. Additionally, the SOCS1 KIR dimer reduced TLR7, but not IFNg induced phosphorylation of the S727 transactivating domain of STAT1.\u003c/p\u003e \u003cp\u003eMacrophage recognition of viral ssRNA by TLR7 is critical for the induction of an antiviral state within an individual cell, and the killing of the internalized pathogen. TLR7 is widely utilized as an endosomal sensor for ssRNA and 2\u0026rsquo;,3\u0026rsquo;-cGMP to activate the innate immune system, which includes the induction of type 1 interferons. The TLR7 agonist imiquimod is currently used to treat certain infections and cancers in immunodeficient individuals (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). Additionally, recent works have suggested that stimulation of the TLR7 pathway could enhance immune activation against hepatitis B virus (HBV) infections and to combat SARS CoV-2 (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). Conversely, pre-clinical work and human studies have indicated that dysregulated TLR7 signaling likely has important relevance in the autoimmune diseases lupus and psoriasis. It has been shown recently that a TLR7 gain-of-function genetic variation enhanced aberrant survival of activated B lymphocytes and caused human lupus (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In this study, we have shown that SOCS1 KIR mimetics reduce TLR7-associated inflammatory markers in primary macrophages with a dose-dependent effect on TNFα at both protein and transcript levels. Our results show that AG490 inhibited the production of both IL-6 and IL-10, consistent with another JAK inhibitor Ruxolitinib (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). Meanwhile, both SOCS1 KIR mimetic peptides inhibited IL-6 production but left IL-10 unaffected. While IL-6 is intimately associated with the progression of inflammatory processes, IL-10 is involved in the restoration of tolerance. Notably, it has been previously shown that the differential regulation of IL-6 and IL-10 production by macrophages can be mediated in a SOCS1 dependent manner. Several PRR pathways signal through MyD88 to induce transcription of cytokines such as IL-6 and IL-10 and while it is known that SOCS1 KIR domain modulates MyD88 signaling through MAL degradation, evidence suggests that LPS-mediated IL-10 production is not completely MyD88 dependent which could provide insight into the differential regulation seen with SOCS1 KIR mimetics (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). It is tempting to speculate that therapeutic targeting of the SOCS1 kinase inhibitory region may exert a biological effect that is distinct from commercially available small molecule targets of janus kinases given their ability to maintain IL-10 levels.\u003c/p\u003e \u003cp\u003eIn this study, we have shown that SOCS1 KIR mimetics reduce TLR7-associated inflammatory markers in primary macrophages with a dose-dependent effect on TNFα at both protein and transcript levels. This has potential biological relevance as macrophages are the major cellular producer of TNFα. Although TLR-mediated production of TNFα is critical in maintaining proper immune functions, uncontrolled TNFα production is associated with autoimmune/auto-inflammatory diseases including rheumatoid arthritis, Crohn\u0026rsquo;s disease, and psoriasis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Increased expression of IFNγ and TNFα in the liver can also mediate chronic hepatitis (\u003cspan additionalcitationids=\"CR80 CR81\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). Notably, it has been recently shown that PEDGA gel-encapsulated SOCS1 KIR peptide reduced M1 macrophage associated markers such as TNFα and Nos2 in the RAW264.7 macrophage cell line (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e). Our results are consistent with SOCS1 conditional knockout experiments in macrophages which led to increased sensitivity to LPS and incessant production of TNFα, IL-6, and CCL2 (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). We have previously shown that SOCS1-KIR mimetics can reduce TNFα production by both CD4 and CD8 T lymphocytes, suggesting that SOCS1 KIR regulation of TNFα may extend to multiple cell types. Although additional, definitive studies are necessary to establish the mechanism(s) by which SOCS1 KIR mimetics inhibit TLR7 mediated TNFα production, numerous reports support the notion that the regulation may be a secondary effect of interferon beta regulation (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). TLR7 possesses Toll/interleukin-1 receptor domain, which serves as a docking site for MyD88, which recruits serine/threonine kinases that drive the cascade culminating in the activation of transcription factors such as NF-kB, activating protein-1(AP-1), and interferon regulatory factors (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). In turn, the transcription of NF-kB responsive genes such as TNFα ensues, as well as interferon regulatory factors IRF7 and IRF3 contributing to the production of IFNβ. IFNβ promotes the up-regulation of IRF7, in a Jak/STAT dependent manner, forming a feed forward loop to produce more IFNβ. In addition to driving IFNβ production, IRF7 forms a complex with MyD88 and other molecules to drive inflammatory mediators such as TNFα (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). Notably, the SOCS1-KIR mimetics also inhibited IFNβ production. As such, it is quite possible that SOCS1-KIR mimic peptide may inhibit TNFα production by inhibiting IFNβ mediated production of IRF7 by targeting the Jak/STAT pathway.\u003c/p\u003e \u003cp\u003e TLR7 stimulation also promotes the generation of a localized inflammatory response which drives the trafficking of immune cells to the area of microbial insult and elicitation of effector functions necessary for clearance. In this study we show that the administration of SOCS1 KIR and SOCS1 KIR dimer attenuated transcript levels of \u003cem\u003eccl2, ccl3, ccl5\u003c/em\u003e, and \u003cem\u003ecxcl10.\u003c/em\u003e It has been previously shown that the administration of the SOCS1 KIR monomer reduced chemokine expression in the retinae in a rat model of uveitis (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Intriguingly, it has been observed that the inhibition of miR155, a post transcriptional regulator of SOCS1, in microglia reduces LPS-induced Nos2, TNFα, IL-6, CCL2, CXCL10, and CXCL9 expression (\u003cspan additionalcitationids=\"CR87\" citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). In our study, we have shown that SOCS1 mimetics reduced these gene products under both TLR7- and IFNγ-stimulation conditions in peritoneal macrophages. The results observed fit well with our current understanding that SOCS1 deficiency can cause anterior uveitis, marked by increased immune cell infiltration where chemokines play a marked role in the pathology. Interestingly, SOCS1 can reduce chemokine expression, under homeostasis and in response to pathogenic threats and therefore, may be an essential factor in maintaining the ocular immune privilege (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). Interferon-inducible CXCR3-responsive chemokines such as CXCL9 and CXCL10 have been implicated in inflammatory endocrine, neuronal, and ocular disorders (\u003cspan additionalcitationids=\"CR91 CR92\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e). Moreover, monocyte chemotactic factors such as CCL2-5 are potent recruiters of dendritic cells and macrophages (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e). Therefore, chemokine regulation could be an alternative approach to ameliorating chronic inflammation.\u003c/p\u003e \u003cp\u003eWe and others have previously demonstrated that peritoneal, and even topical administration, of SOCS1 KIR was efficacious in prolonging the survival of SOCS1 deficient mice, reducing paralysis in a murine model of multiple sclerosis, and mitigating uveitis that was induced in rodent models (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). In translational studies, we have recently demonstrated that the administration of topical eye drops containing SOCS1 KIR reduced clinical pathology in horses bearing equine recurrent uveitis, one of the leading causes of equine blindness (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e). It is well established that combination of bio-active peptide regions, using peptide linkers, can generate molecules with increased therapeutic efficacy. Peptide linkers often consist of glycines and/or serines as their flexibility and hydrophilic properties are often beneficial and not inhibitory to the main protein domains. We generated a dimeric SOCS1 KIR mimic (KIR dimer), using a glycine repeat linker, and found that KIR dimer possessed increased efficacy in comparison to SOCS1 KIR in a murine model of lupus (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In this study we show that while SOCS1 KIR statically inhibited IL-6 and IFNγ, KIR dimer was more efficacious in the inhibition of TNFα and chemokine mRNA production in PECs. Although both SOCS1 KIR and SOCS1 KIR dimer reduced IFNγ mediated Y701 phosphorylation of STAT1 and Y705 of STAT3, SOCS1 KIR dimer was a more effective inhibitor. KIR dimer was also more effective in the inhibition of IL-6 mediated phosphorylation of STAT1 and STAT3 tyrosine phosphorylation. We hypothesize that the dimer has increased efficacy through enhanced STAT1 regulation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCL: CC chemokine ligand\u003c/p\u003e\n\u003cp\u003eCCR: CC chemokine receptor\u003c/p\u003e\n\u003cp\u003ecGMP: Cyclic guanosine monophosphate\u003c/p\u003e\n\u003cp\u003eCNS: Central nervous system\u003c/p\u003e\n\u003cp\u003eCXCL: C-X-C motif chemokine ligand\u003c/p\u003e\n\u003cp\u003eCXCR: C-X-C motif chemokine receptor\u003c/p\u003e\n\u003cp\u003eHBV: Hepatitis B virus\u003c/p\u003e\n\u003cp\u003eIFN: Interferon\u003c/p\u003e\n\u003cp\u003eIL: Interleukin\u003c/p\u003e\n\u003cp\u003eJAK: Janus kinase\u003c/p\u003e\n\u003cp\u003eKIR: Kinase inhibitory region\u003c/p\u003e\n\u003cp\u003eLPS: Lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eMAL: MyD88-adapter-like\u003c/p\u003e\n\u003cp\u003eMAPK: Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003ePDL1: Programmed death-ligand 1\u003c/p\u003e\n\u003cp\u003ePEC: Peritoneal exudate cells\u003c/p\u003e\n\u003cp\u003ePRR: Pattern recognition receptor\u003c/p\u003e\n\u003cp\u003eR848: Resiquimod\u003c/p\u003e\n\u003cp\u003eSLE: Systemic lupus erythematosus\u003c/p\u003e\n\u003cp\u003eSOCS1: Suppressor of cytokine signaling-1\u003c/p\u003e\n\u003cp\u003essRNA: Single-stranded ribonucleic acid\u003c/p\u003e\n\u003cp\u003eSTAT: Signal transducer and activator of transcription\u003c/p\u003e\n\u003cp\u003eT1D: Type I diabetes\u003c/p\u003e\n\u003cp\u003eTAD: Transactivation domain\u003c/p\u003e\n\u003cp\u003eTem: Effector memory T cells\u003c/p\u003e\n\u003cp\u003eTLR: Toll-like receptor\u003c/p\u003e\n\u003cp\u003eTNF: Tumor necrosis factor\u003c/p\u003e\n\u003cp\u003eTreg: Regulatory T cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJL3 was partially supported by a grant from The Grayson Jockey Research Foundation, Private Funding, and the University of Florida.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.S., P.E.K., and J.L.3. designed the experiments. J.S. and J.L.3. conducted experiments and performed data analysis. J.S. and J.L.3. wrote the manuscript. L.S., W.C.S., P.E.K., and J.L.3. edited the paper. V.V., and T.O.R. assisted in experiments. All authors approve the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOCS mimetic technology is protected by patents 15/113,725 and 11,603,387. The research was supported, in part, by a company currently licensing the technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding support\u003c/strong\u003e: University of Florida, The Grayson Jockey Research Foundation, and private funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHirayama D, Iida T, Nakase H. The phagocytic function of macrophage-enforcing innate immunity and tissue homeostasis. Vol. 19, International Journal of Molecular Sciences. 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedzhitov R, Preston-Hurlburt P, Janeway CA. A human homologue of the Drosophila toll protein signals activation of adaptive immunity. Nature. 1997;388(6640).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol. 1999;162(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999;11(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Broser M, Rom WN. Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-κB. Proc Natl Acad Sci U S A. 1994;91(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Vol. 13, Annual Review of Immunology. 1995.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoyle SE, O\u0026rsquo;Connell RM, Miranda GA, Vaidya SA, Chow EK, Liu PT, et al. Toll-like Receptors Induce a Phagocytic Gene Program through p38. Journal of Experimental Medicine. 2004;199(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-Specific Recognition of Single-Stranded RNA via Till-like Receptor 7 and 8. Science (1979). 2004;303(5663).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWirth JR, Molano I, Ruiz P, Coutermarsh-Ott S, Cunningham MA. TLR7 Agonism Accelerates Disease and Causes a Fatal Myeloproliferative Disorder in NZM 2410 Lupus Mice. Front Immunol. 2020;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJenks SA, Cashman KS, Zumaquero E, Marigorta UM, Patel A V., Wang X, et al. Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus. Immunity. 2018;49(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePawar RD, Ramanjaneyulu A, Kulkarni OP, Lech M, Segerer S, Anders HJ. Inhibition of Toll-like receptor-7 (TLR-7) or TLR-7 plus TLR-9 attenuates glomerulonephritis and lung injury in experimental lupus. Journal of the American Society of Nephrology. 2007;18(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePisitkun P. Autoreactive B Cell Responses to RNA-Related Antigens Due to TLR7 Gene Duplication. Science (1979) [Internet]. 2006;312(5780):1669\u0026ndash;72. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sciencemag.org/cgi/doi/\u003c/span\u003e\u003cspan address=\"http://www.sciencemag.org/cgi/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1124978\u003c/span\u003e\u003cspan address=\"10.1126/science.1124978\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohan C, Putterman C. Genetics and pathogenesis of systemic lupus erythematosus and lupus nephritis. Vol. 11, Nature Reviews Nephrology. 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee YH, Choi SJ, Ji JD, Song GG. Association between toll-like receptor polymorphisms and systemic lupus erythematosus: A meta-analysis update. Lupus. 2016;25(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown GJ, Ca\u0026ntilde;ete PF, Wang H, Medhavy A, Bones J, Roco JA, et al. TLR7 gain-of-function genetic variation causes human lupus. Nature [Internet]. 2022;605(7909):349\u0026ndash;56. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-022-04642-z\u003c/span\u003e\u003cspan address=\"10.1038/s41586-022-04642-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RAA, Shlomchik MJ. Toll-like Receptor 7 and TLR9 Dictate Autoantibody Specificity and Have Opposing Inflammatory and Regulatory Roles in a Murine Model of Lupus. Immunity. 2006;25(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAringer M, Smolen JS. The role of tumor necrosis factor-alpha in systemic lupus erythematosus. Vol. 10, Arthritis Research and Therapy. 2008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManara M, Sinigaglia L. Bone and TNF in rheumatoid arthritis: Clinical implications. Vol. 1, RMD Open. 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee LF, Xu B, Michie SA, Beilhack GF, Warganich T, Turley S, et al. The role of TNF-α in the pathogenesis of type 1 diabetes in the nonobese diabetic mouse: Analysis of dendritic cell maturation. Proc Natl Acad Sci U S A. 2005;102(44).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuarez-Pinzon WL, Mabley JG, Strynadka K, Power RF, Szab\u0026oacute; C, Rabinovitch A. An inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite prevents diabetes development in NOD mice. J Autoimmun. 2001;16(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan L, Yang S, Wang J, Xu M, Wang S, Yi H. Inducible nitric oxide synthase and systemic lupus erythematosus: A systematic review and meta-analysis. Vol. 21, BMC Immunology. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenaglio P, Zhu H, Okino ML, Yan J, Elgamal R, Nariai N, et al. Type 1 diabetes risk genes mediate pancreatic beta cell survival in response to proinflammatory cytokines. Cell Genomics. 2022;2(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagy G, Perl A. The role of nitric oxide in abnormal T cell signal transduction in systemic lupus erythematosus. Vol. 118, Clinical Immunology. 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagy G, Koncz A, Phillips P, Perl A. Mitochondrial Signal Transduction Abnormalities in Systemic Lupus Erythematosus. Curr Immunol Rev. 2005;1(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed CMI, Larkin J, Johnson HM. SOCS1 mimetics and antagonists: A complementary approach to positive and negative regulation of immune function. Vol. 6, Frontiers in Immunology. 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChmielewski S, Olejnik A, Sikorski K, Pelisek J, Błaszczyk K, Aoqui C, et al. STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis. PLoS One. 2014;9(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, et al. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol. 2002;3(1529\u0026ndash;2908).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNajjar I, Fagard R. STAT1 and pathogens, not a friendly relationship. Vol. 92, Biochimie. 2010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindler C, Fu XY, Improta T, Aebersold R, Darnell JE. Proteins of transcription factor ISGF-3: One gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon α. Proc Natl Acad Sci U S A. 1992;89(16).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuu K, Greenhill CJ, Majoros A, Decker T, Jenkins BJ, Mansell A. STAT1 plays a role in TLR signal transduction and inflammatory responses. Immunol Cell Biol. 2014;92(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarinou L, Ramsauer K, Karaghiosoff M, Kolbe T, Pfeffer K, M\u0026uuml;ller M, et al. Phosphorylation of the Stat1 Transactivation Domain Is Required for Full-Fledged IFN-γ-Dependent Innate Immunity. Immunity. 2003;19(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovarik P, Stoiber D, Eyers PA, Menghini R, Neininger A, Gaestel M, et al. Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway. Proc Natl Acad Sci U S A. 1999;96(24).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChodisetti SB, Fike AJ, Domeier PP, Singh H, Choi NM, Corradetti C, et al. Type II but Not Type I IFN Signaling Is Indispensable for TLR7-Promoted Development of Autoreactive B Cells and Systemic Autoimmunity. The Journal of Immunology. 2020;204(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma J, Larkin J. Therapeutic implication of SOCS1 modulation in the treatment of autoimmunity and cancer. Frontiers in Pharmacology. 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiau NPD, Laktyushin A, Lucet IS, Murphy JM, Yao S, Whitlock E, et al. The molecular basis of JAK/STAT inhibition by SOCS1. Nat Commun [Internet]. 2018;9(1):1558. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/articles/s41467-018-04013-1\u003c/span\u003e\u003cspan address=\"http://www.nature.com/articles/s41467-018-04013-1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoggett K, Keating N, Dehkhoda F, Bidgood GM, Meza Guzman LG, Leong E, et al. The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo. Cytokine. 2023;165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang JG, Farley A, Nicholson SE, Willson TA, Zugaro LM, Simpson RJ, et al. The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proceedings of the National Academy of Sciences [Internet]. 1999;96(5):2071\u0026ndash;6. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.pnas.org/cgi/doi/\u003c/span\u003e\u003cspan address=\"http://www.pnas.org/cgi/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.96.5.2071\u003c/span\u003e\u003cspan address=\"10.1073/pnas.96.5.2071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitz O, Guiter C, Dorsch K, Dusanter-Fourt I, Wegener S, Jouault H, et al. STAT6 activity is regulated by SOCS-1 and modulates BCL-XL expression in primary mediastinal B-Cell lymphoma. Leukemia [Internet]. 2008;22(11):2106\u0026ndash;10. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/articles/leu200885\u003c/span\u003e\u003cspan address=\"http://www.nature.com/articles/leu200885\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalpke AH, Opper S, Zimmermann S, Heeg K. Suppressors of Cytokine Signaling (SOCS)-1 and SOCS-3 Are Induced by CpG-DNA and Modulate Cytokine Responses in APCs. The Journal of Immunology. 2001;166(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePi\u0026ntilde;eros Alvarez AR, Glosson-Byers N, Brandt S, Wang S, Wong H, Sturgeon S, et al. SOCS1 is a negative regulator of metabolic reprogramming during sepsis. JCI Insight. 2017;2(13).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu CF, Peng WM, Schlee M, Barchet W, Eis-H\u0026uuml;binger AM, Kolanus W, et al. SOCS1 and SOCS3 Target IRF7 Degradation To Suppress TLR7-Mediated Type I IFN Production of Human Plasmacytoid Dendritic Cells. The Journal of Immunology. 2018;200(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X, Liu Z, Cheng X, Zheng Y, Zeng F, He Y. Socs1 and Socs3 degrades Traf6 via polyubiquitination in LPS-induced acute necrotizing pancreatitis. Cell Death Dis [Internet]. 2015;6(12):e2012. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/doifinder/\u003c/span\u003e\u003cspan address=\"http://www.nature.com/doifinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/cddis.2015.342\u003c/span\u003e\u003cspan address=\"10.1038/cddis.2015.342\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrebovsky J, Walker P, Lang R, Dalpke AH. Suppressor of cytokine signaling 1 (SOCS1) limits NFκB signaling by decreasing p65 stability within the cell nucleus. The FASEB Journal [Internet]. 2011;25(3):863\u0026ndash;74. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.fasebj.org\u003c/span\u003e\u003cspan address=\"http://www.fasebj.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/doi/10.1096/fj.10-170597\u003c/span\u003e\u003cspan address=\"/doi/10.1096/fj.10-170597\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePr\u0026ecirc;le CM, Woodward EA, Bisley J, Keith-Magee A, Nicholson SE, Hart PH. SOCS1 Regulates the IFN but Not NFκB Pathway in TLR-Stimulated Human Monocytes and Macrophages. The Journal of Immunology. 2008;181(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollins EL, Jager LD, Dabelic R, Benitez P, Holdstein K, Lau K, et al. Inhibition of SOCS1 \u0026ndash;/\u0026ndash; Lethal Autoinflammatory Disease Correlated to Enhanced Peripheral Foxp3\u0026thinsp;+\u0026thinsp;Regulatory T Cell Homeostasis. The Journal of Immunology. 2011;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJager LD, Dabelic R, Waiboci LW, Lau K, Haider MS, Ahmed CMI, et al. The kinase inhibitory region of SOCS-1 is sufficient to inhibit T-helper 17 and other immune functions in experimental allergic encephalomyelitis. J Neuroimmunol [Internet]. 2011;232(1\u0026ndash;2):108\u0026ndash;18. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://linkinghub.elsevier.com/retrieve/pii/S0165572810004686\u003c/span\u003e\u003cspan address=\"https://linkinghub.elsevier.com/retrieve/pii/S0165572810004686\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe C, Yu CR, Mattapallil MJ, Sun L, Larkin J, Egwuagu CE. SOCS1 Mimetic Peptide Suppresses Chronic Intraocular Inflammatory Disease (Uveitis). Mediators Inflamm. 2016;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThiam K, Loing E, Verwaerde C, Auriault C, Gras-Masse H. IFN-γ-derived lipopeptides: Influence of lipid modification on the conformation and the ability to induce MHC class II expression on murine and human cells. J Med Chem. 1999;42(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzente BE, Soos JM, Johnson HM. The C-Terminus of IFN-γ Is Sufficient for Intracellular Function. Biochem Biophys Res Commun. 1994;203(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma J, Collins TD, Roach T, Mishra S, Lam BK, Mohamed ZS, et al. Suppressor of cytokine signaling-1 mimetic peptides attenuate lymphocyte activation in the MRL/lpr mouse autoimmune model. Sci Rep. 2021;11(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed CMI, Dabelic R, Martin JP, Jager LD, Haider SM, Johnson HM. Enhancement of Antiviral Immunity by Small Molecule Antagonist of Suppressor of Cytokine Signaling. The Journal of Immunology. 2010;185(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakagawa R, Naka T, Tsutsui H, Fujimoto M, Kimura A, Abe T, et al. SOCS-1 Participates in Negative Regulation of LPS Responses. Immunity [Internet]. 2002;17(5):677\u0026ndash;87. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://linkinghub.elsevier.com/retrieve/pii/S1074761302004491\u003c/span\u003e\u003cspan address=\"https://linkinghub.elsevier.com/retrieve/pii/S1074761302004491\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKinjyo I, Hanada T, Inagaki-Ohara K, Mori H, Aki D, Ohishi M, et al. SOCS1/JAB Is a Negative Regulator of LPS-Induced Macrophage Activation. Immunity [Internet]. 2002;17(5):583\u0026ndash;91. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://linkinghub.elsevier.com/retrieve/pii/S1074761302004466\u003c/span\u003e\u003cspan address=\"https://linkinghub.elsevier.com/retrieve/pii/S1074761302004466\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhafouri-Fard S, Shahir M, Taheri M, Salimi A. A review on the role of chemokines in the pathogenesis of systemic lupus erythematosus. Vol. 146, Cytokine. 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbst S, Schaible UE, Schneider BE. Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis. PLoS One. 2011;6(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoseDasgupta S, Pieters J. Inflammatory Stimuli Reprogram Macrophage Phagocytosis to Macropinocytosis for the Rapid Elimination of Pathogens. PLoS Pathog. 2014;10(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Vol. 8, Nature Reviews Immunology. 2008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlummer CE, Polk T, Sharma J, Bae SS, Barr O, Jones A, et al. Open label safety and efficacy pilot to study mitigation of equine recurrent uveitis through topical suppressor of cytokine signaling-1 mimetic peptide. Sci Rep [Internet]. 2022;12(1):7177. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-11338-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-11338-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNarazaki M, Witthuhn BA, Yoshida K, Silvennoinen O, Yasukawa K, Ihle JN, et al. Activation of JAK2 kinase mediated by the interleukin 6 signal transducer gp130. Proc Natl Acad Sci U S A. 1994;91(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature [Internet]. 1997;387(6636):924\u0026ndash;9. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/articles/43219\u003c/span\u003e\u003cspan address=\"http://www.nature.com/articles/43219\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStarr R, Willson TA, Viney EM, Murray LJL, Rayner JR, Jenkins BJ, et al. A family of cytokine-inducible inhibitors of signalling. Nature [Internet]. 1997;387(6636):917\u0026ndash;21. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/articles/43206\u003c/span\u003e\u003cspan address=\"http://www.nature.com/articles/43206\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEndo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature [Internet]. 1997;387(6636):921\u0026ndash;4. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/articles/43213\u003c/span\u003e\u003cspan address=\"http://www.nature.com/articles/43213\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadzak I, Schiff M, Gattermeier I, Glinitzer R, Sauer I, Saalm\u0026uuml;ller A, et al. Recruitment of Stat1 to chromatin is required for interferon-induced serine phosphorylation of Stat1 transactivation domain. Proc Natl Acad Sci U S A. 2008;105(26).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarinou L, Ramsauer K, Karaghiosoff M, Kolbe T, Pfeffer K, Muller M, et al. Phosphorylation of the STAT1 transactivation domain is required for full-fledged IFN-gamma-dependent innate immunity. In: Epitheorese Klinikes Farmakologias kai Farmakokinetikes. 2005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalpke AH, Eckerle S, Frey M, Heeg K. Triggering of toll-like receptors modulates IFN signalling: Involvement of serine 727 STAT1 phosphorylation and suppressors of cytokine signaling. Eur J Immunol. 2003;33(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimura A, Suzuki M, Sakaguchi R, Hanada T, Yasukawa H. SOCS, inflammation, and autoimmunity. Vol. 3, Frontiers in Immunology. 2012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaka T, Fujimoto M. SOCS1, a negative regulator of cytokine signals and TLR responses, in human liver diseases. Gastroenterology Research and Practice. 2010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHadjadj J, Castro CN, Tusseau M, Stolzenberg MC, Mazerolles F, Aladjidi N, et al. Early-onset autoimmunity associated with SOCS1 haploinsufficiency. Nat Commun. 2020;11(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSachithanandan N, Graham KL, Galic S, Honeyman JE, Fynch SL, Hewitt KA, et al. Macrophage Deletion of SOCS1 Increases Sensitivity to LPS and Palmitic Acid and Results in Systemic Inflammation and Hepatic Insulin Resistance. Diabetes [Internet]. 2011;60(8):2023\u0026ndash;31. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://diabetes.diabetesjournals.org/cgi/doi/\u003c/span\u003e\u003cspan address=\"http://diabetes.diabetesjournals.org/cgi/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2337/db11-0259\u003c/span\u003e\u003cspan address=\"10.2337/db11-0259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Li R, Wang M, Hou Y, Liu S, Peng T, et al. Multiomics Analysis Identifies SOCS1 as Restraining T Cell Activation and Preventing Graft-Versus-Host Disease. Advanced Science [Internet]. 2022;n/a(n/a):2200978. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/advs.202200978\u003c/span\u003e\u003cspan address=\"10.1002/advs.202200978\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMisharin A V., Morales-Nebreda L, Reyfman PA, Cuda CM, Walter JM, McQuattie-Pimentel AC, et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. Journal of Experimental Medicine. 2017;214(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBubna A. Imiquimod-Its role in the treatment of cutaneous malignancies. Vol. 47, Indian Journal of Pharmacology. 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdwards L, Ferenczy A, Eron L, Baker D, Owens ML, Fox TL, et al. Self-administered topical 5% imiquimod cream for external anogenital warts. Arch Dermatol. 1998;134(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoulas K, Farsalinos K, Zanidis C. Activation of TLR7 and Innate Immunity as an Efficient Method Against COVID-19 Pandemic: Imiquimod as a Potential Therapy. Vol. 11, Frontiers in Immunology. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas R, Guan P, Sprague L, Verbist K, Tedrick P, An QA, et al. Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis. Blood. 2016;127(13).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerezani CH, Lewis C, Jancar S, Peters-Golden M. Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression. Journal of Clinical Investigation. 2011;121(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMansell A, Smith R, Doyle SL, Gray P, Fenner JE, Crack PJ, et al. Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation. Nat Immunol [Internet]. 2006;7(2):148\u0026ndash;55. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nature.com/articles/ni1299\u003c/span\u003e\u003cspan address=\"http://www.nature.com/articles/ni1299\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolbrook J, Lara-Reyna S, Jarosz-Griffiths H, McDermott MF. Tumour necrosis factor signalling in health and disease. F1000Res. 2019;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFederici M, Giustizieri ML, Scarponi C, Girolomoni G, Albanesi C. Impaired IFN- -Dependent Inflammatory Responses in Human Keratinocytes Overexpressing the Suppressor of Cytokine Signaling 1. The Journal of Immunology [Internet]. 2002;169(1):434\u0026ndash;42. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.jimmunol.org/cgi/doi/\u003c/span\u003e\u003cspan address=\"http://www.jimmunol.org/cgi/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.169.1.434\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.169.1.434\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadonna S, Scarponi C, Doti N, Carbone T, Cavani A, Scognamiglio PL, et al. Therapeutical potential of a peptide mimicking the SOCS1 kinase inhibitory region in skin immune responses. Eur J Immunol. 2013;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkamoto T, Furuya M, Yamakawa T, Yamamura K ichi, Hino O. TNF-α gene expression in the liver of the IFN-γ transgenic mouse with chronic active hepatitis. Biochem Biophys Res Commun. 1996;226(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyonaga T, Hino O, Sugai S, Wakasugi S, Abe K, Shichiri M, et al. Chronic active hepatitis in transgenic mice expressing interferon-γ in the liver. Proc Natl Acad Sci U S A. 1994;91(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJha A, Larkin J, Moore E. SOCS1-KIR peptide in PEDGA Hydrogels Reduces Pro‐Inflammatory Macrophage Activation. Macromol Biosci. 2023;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaetz A, Frey M, Heeg K, Dalpke AH. Suppressor of cytokine signaling (SOCS) proteins indirectly regulate Toll-like receptor signaling in innate immune cells. Journal of Biological Chemistry. 2004;279(52).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawasaki T, Kawai T. Toll-like receptor signaling pathways. Vol. 5, Frontiers in Immunology. 2014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardoso AL, Guedes JR, Pereira de Almeida L, Pedroso de Lima MC. miR-155 modulates microglia-mediated immune response by down-regulating SOCS-1 and promoting cytokine and nitric oxide production. Immunology. 2012;135(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePathak S, Grillo AR, Scarpa M, Brun P, D\u0026rsquo;Inc\u0026agrave; R, Nai L, et al. MiR-155 modulates the inflammatory phenotype of intestinal myofibroblasts by targeting SOCS1 in ulcerative colitis. Exp Mol Med. 2015;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe J, Guo R, Shi Y, Qi F, Guo C, Yang L. MiR-155 Regulated Inflammation Response by the SOCS1-STAT3-PDCD4 Axis in Atherogenesis. Mediators Inflamm. 2016;2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu CR, Hayashi K, Lee YS, Mahdi RM, Shen DF, Chan CC, et al. Suppressor of Cytokine Signaling 1 (SOCS1) Mitigates Anterior Uveitis and Confers Protection Against Ocular HSV-1 Infection. Inflammation. 2015;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFallahi P, Ferrari SM, Ragusa F, Ruffilli I, Elia G, Paparo SR, et al. Th1 chemokines in autoimmune endocrine disorders. Vol. 105, Journal of Clinical Endocrinology and Metabolism. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoward OMZ, Hui FD, Shao BS, Caspi RR, Chen X, Plotz P, et al. Autoantigens signal through chemokine receptors: Uveitis antigens induce CXCR3- and CXCR5-expressing lymphocytes and immature dendritic cells to migrate. Vol. 105, Blood. 2005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorjini N, Fern\u0026aacute;ndez M, Giardino L, Calz\u0026agrave; L. Cytokine and chemokine alterations in tissue, CSF, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (EAE), in a rat model of multiple sclerosis. J Neuroinflammation. 2016;13(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristen U, Kimmel R. Chemokines as Drivers of the Autoimmune Destruction in Type 1 Diabetes: Opportunity for Therapeutic Intervention in Consideration of an Optimal Treatment Schedule. Vol. 11, Frontiers in Endocrinology. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreaves DR, Schall TJ. Chemokines and myeloid cell recruitment. Vol. 2, Microbes and Infection. 2000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe C, Yu CR, Sun L, Mahdi RM, Larkin J, Egwuagu CE. Topical administration of a suppressor of cytokine signaling-1 (SOCS1) mimetic peptide inhibits ocular inflammation and mitigates ocular pathology during mouse uveitis. J Autoimmun [Internet]. 2015;62:31\u0026ndash;8. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sciencedirect.com/science/article/pii/S0896841115000827\u003c/span\u003e\u003cspan address=\"https://www.sciencedirect.com/science/article/pii/S0896841115000827\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlummer C, Polk T, Sharma J, Bae S, Barr O, Jones A, et al. Mitigation of Equine Recurrent Uveitis Through Topical Suppressor of Cytokine Signaling-1 Mimetic Peptide: Open Label Safety and Efficacy Pilot Study [Internet]. Research Square; 2022. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://europepmc.org/abstract/PPR/PPR442089\u003c/span\u003e\u003cspan address=\"http://europepmc.org/abstract/PPR/PPR442089\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"therapeutic, cytokine, SOCS mimetic, translation, jak/stat, signal transduction, chemokine","lastPublishedDoi":"10.21203/rs.3.rs-3925558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3925558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough it is known that SOCS1 can modulate JAK/STAT signaling through both its kinase inhibitory region (KIR) and SOCS box domain, and limit toll-like receptor (TLR) induced inflammation via the SOCS1 box domain, the relative contribution of the KIR domain to TLR regulation is not well understood. In this study, we utilized peptide mimics of SOCS1 KIR to study the effect of the KIR domain in modulating TLR7 and interferon γ (IFNγ) signaling in murine primary macrophages and cell lines. We found that SOCS1 KIR mimetics were able to inhibit, by up to 50%, the inflammatory signatures associated with TLR7 stimulation, IFNγ stimulation, and the enhanced IFNγ-induced gene signature, mediated by TLR7 and IFNγ co-treatment. While inhibition of IFNg mediated activation correlated with reduced Y701 phosphorylation on STAT1 and Y705 phosphorylation on STAT3, the inhibition of TLR7-induced inflammation and the TLR7-enhanced IFNγ-induced gene signature coincided with a reduction in both Y701 and S727 phosphorylation on the STAT1 transactivation domain. Altogether, we report for the first time a novel role of the SOCS1 KIR domain in regulating TLR7-mediated, and TLR7-enhanced IFNγ-mediated, inflammation.\u003c/p\u003e","manuscriptTitle":"SOCS1 kinase inhibitory region peptide mimics regulate interferon gamma and TLR7-induced inflammatory signatures in murine macrophages.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 21:15:01","doi":"10.21203/rs.3.rs-3925558/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6022a0e3-a356-4fe8-a1eb-99e20a6a83f2","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28976937,"name":"Biological sciences/Cell biology/Cell growth"},{"id":28976938,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":28976939,"name":"Biological sciences/Cell biology"},{"id":28976940,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages"},{"id":28976941,"name":"Biological sciences/Immunology"},{"id":28976942,"name":"Biological sciences/Immunology/Chemokines"},{"id":28976943,"name":"Biological sciences/Immunology/Cytokines"},{"id":28976944,"name":"Biological sciences/Immunology/Inflammation"},{"id":28976945,"name":"Biological sciences/Immunology/Innate immune cells"},{"id":28976946,"name":"Biological sciences/Immunology/Signal transduction"}],"tags":[],"updatedAt":"2025-06-06T17:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 21:15:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3925558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3925558","identity":"rs-3925558","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}