Interferon regulatory factor 8 Drives Autoimmune Neuroinflammation in Experimental Autoimmune Encephalomyelitis

Interferon regulatory factor 8 Drives Autoimmune Neuroinflammation in Experimental Autoimmune Encephalomyelitis | 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 Research Article Interferon regulatory factor 8 Drives Autoimmune Neuroinflammation in Experimental Autoimmune Encephalomyelitis Haruto Ojika, Kazuo Kunisawa, Moeka Tanabe, Yuki Kon, Koyo Yoshidomi, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9546988/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. The interferon regulatory factor (IRF) family comprises transcription factors that regulate immune responses; however, their roles in MS pathogenesis remain unclear. In this study, we investigated whether IRFs are involved in MS pathology using experimental autoimmune encephalomyelitis (EAE) model mice. Among the IRFs examined, IRF8 expression was significantly increased in the spinal cord of EAE model mice and predominantly localized to macrophages. Consistently, patients with MS exhibited significantly increased proportions of IRF8-positive myeloid cells in peripheral blood. Notably, IRF8 knockout mice exhibited complete resistance to EAE induction. The mRNA expression levels of neuroinflammatory mediators, including interleukin IL-1β, IL-6, tumor necrosis factor-α, IL-12b, and nitric oxide synthase 2, were significantly reduced in the spinal cord of IRF8 knockout mice. Collectively, these findings suggest that IRF8 is a critical regulator of autoimmune neuroinflammation, highlighting its potential as a therapeutic target for MS. Interferon regulatory factor 8 Multiple sclerosis Macrophages Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Multiple sclerosis (MS), a chronic autoimmune disease characterized by demyelination of the central nervous system (CNS), affects over 2 million people worldwide [ 1 , 2 ]. MS presents with diverse neurological symptoms, including motor dysfunction, sensory disturbances, and autonomic dysfunction, and is pathologically defined by the infiltration of peripheral immune cells into CNS lesions [ 3 , 4 ]. However, the precise molecular mechanisms underlying its pathogenesis remain unclear. MS onset and progression involve complex interactions among diverse immune cell populations, including T cells, B cells, monocytes/macrophages, and dendritic cells [ 5 – 7 ]. Cytokines and chemokines secreted by these immune cells amplify neuroinflammatory cascades and contribute to tissue damage in the CNS [ 8 , 9 ]. Accordingly, MS is widely regarded as a disease resulting from dysregulated immune responses, in which chronic inflammation overwhelms immune homeostasis. Transcriptional regulation is essential for maintaining immune balance, and the interferon regulatory factor (IRF) family plays a crucial role in this process. The IRF family, consisting of nine members (IRF1–9), comprises transcription factors that orchestrate key innate and adaptive immune functions, such as interferon (IFN) signaling, inflammatory responses, immune cell differentiation, and apoptosis [ 10 ]. Each IRF performs distinct and context-dependent biological functions. IRF1 promotes the transcription of inflammatory genes and antigen-presenting molecules, contributing to immune activation in macrophages and dendritic cells [ 11 – 13 ], whereas IRF2 functions primarily as its antagonist [ 14 ]. IRF3 and IRF7 are central regulators of type I IFN (IFN-α/β) production during antiviral immunity [ 15 , 16 ], and IRF9 associates with signal transducer and activator of transcription 1/2 to form the IFN-stimulated gene factor 3 complex, which induces IFN-stimulated gene expression [ 17 ]. IRF4 and IRF8 are critically involved in the development and differentiation of both the lymphoid and myeloid lineages, thereby influencing immune cell fate decisions [ 18 , 19 ]. IRF5 promotes pro-inflammatory macrophage activation and cytokine production [ 20 ], whereas IRF6 is primarily associated with epithelial differentiation and tissue repair [ 21 ], helping to maintain tissue barrier integrity [ 12 , 22 , 23 ]. Considering these diverse immunological functions, aberrant IRF signaling is implicated in several autoimmune and inflammatory diseases, including systemic lupus erythematosus, rheumatoid arthritis, Crohn’s disease, and psoriasis [ 24 , 25 ]. IRF family members may critically modulate the progression of MS, which is characterized by dysregulated IFN responses and excessive myeloid cell activation. However, to date, no study has comprehensively analyzed IRF family members in MS. Therefore, elucidating the expression dynamics and functional contributions of the entire IRF family in the inflamed CNS is essential for a deeper understanding of MS pathogenesis. In this study, we comprehensively analyzed IRF family expression in the spinal cord of experimental autoimmune encephalomyelitis (EAE) model mice, a widely used animal model of MS, to identify key IRF members involved in the regulation of autoimmune neuroinflammation. By assessing the activation patterns of individual IRFs, we identified novel transcriptional regulators and potential therapeutic targets for MS. Materials and Methods Animals C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). IRF8 knockout (KO) mice (C57BL/6 background) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and confirmed via standard polymerase chain reaction (PCR)-based genotyping of genomic DNA. The following primer sequences were used for PCR genotyping: IRF8-forward sense, 5′-GGGCACTGCTGATTTCTCAT-3′, and IRF8-reverse sense, 5′-AGCCAGAGGGAAACCAAAAA-3′. Each mouse (8–10 weeks old) was subjected to EAE induction. The mice were housed in plastic cages and maintained under a 12-h light/dark cycle (lights on at 8:00 a.m.), with food and water provided ad libitum. All experiments adhered to the guidelines of the Japanese Pharmacological Society and Institute for Experimental Animals at Fujita Health University (Aichi, Japan). All protocols were approved by the Ethics Committee of Animal Experiments at the Institute for Experimental Animals, Fujita Health University (Permit Number: APU22059-MD2). Establishment of EAE Model Mice EAE model mice were established as previously described [ 26 ]. Briefly, the myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide MEVGWYRSPFSRVVHLYRNGK (200 µg; Cat# S-PEP; Scrum, Tokyo, Japan) was emulsified in complete Freund’s adjuvant (Cat# D614-0050; RCK, Gilbertsville, PA, USA). The mice were subcutaneously immunized with MOG/complete Freund’s adjuvant at two sites on the dorsal flank, followed by an intraperitoneal injection of pertussis toxin (600 ng; Cat# 516560-50UGCN; Merck, Dusseldorf, Germany) immediately post-immunization and after 48 h. The mice were monitored daily for 28 d post-immunization (dpi) to assess the EAE score (Fig. 1 A). The EAE scores were as follows: 0 = normal, 0.5 = partial drooping of the tail, 1 = complete drooping of the tail, 1.5 = abnormal gait, 2 = abnormal gait with weakness in the hind limbs, 2.5 = paralysis and weakness in one hind limb, 3 = paralysis of both hind limbs, 3.5 = paralysis of both hind limbs in a hunched posture, 4 = paralysis of the hind and fore limbs, and 5 = moribundity or death. If EAE was not induced at 18 dpi, the mice were excluded from analysis. Sample Collection The mice were anesthetized using isoflurane (1 mL/mL; Fujifilm Wako Pure Chemical Co., Osaka, Japan) and transcardially perfused with ice-cold phosphate-buffered saline (PBS). The spinal cords were quickly removed, washed with ice-cold PBS, and immediately frozen on dry ice. All samples were stored at − 80°C until analysis. Quantitative real-time reverse transcription PCR (qRT–PCR) Total RNA was isolated using Sepasol-RNA I Super G (Cat# 09379-84; Nacalai Tesque, Kyoto, Japan), according to the manufacturer’s instructions. All PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT kit (Cat# FSQ-101; Toyobo, Osaka, Japan). For qPCR, THUNDERBIRD Probe qPCR Mix (Cat# QPS-101; Toyobo) was used and subjected to real-time PCR quantification using the StepOne Real-Time PCR System (Cat# 4376357; Life Technologies, Carlsbad, CA, USA). qPCR was performed using the StepOne analyzer (Cat# 4461357; Life Technologies). The PCR program consisted of 40 cycles of 95°C for 30 s and 60°C for 1 min. Melting curve analysis was performed after each PCR to differentiate between specific and non-specific amplifications. β-actin was used as the housekeeping gene to normalize all PCR data. The following primers were used in this study: IRF1 (Mm.PT.58.33516776), IRF2 (Mm.PT.58.11463323), IRF3 (Mm.PT.58.5650168), IRF4 (Mm.PT.58.31041855), IRF5 (Mm.PT.58.6374764), IRF6 (Mm.PT.58.7951480), IRF7 (Mm.PT.58.32394021.g), IRF8 (Mm.PT.58.30819027), IRF9 (Mm.PT.58.31054864), interleukin (IL)-1β (Mm.PT.58.41616450), IL-6 (Mm.58.10005566), tumor necrosis factor (TNF)-α (Mm.PT.58.12575861), IL-12b (Mm.PT.58.12409997), nitric oxide synthase 2 (NOS2; Mm.PT.58.43705194), and β-actin (Mm.PT.39.a.22214843). Western Blotting Analysis Western Blotting Analysis Western blotting was performed as previously described [ 27 ]. The spinal cords were homogenized in ice-cold homogenization buffer (pH 7.4; 50 mM Tris-HCl [pH 8.0] containing 4 mM EGTA, 10 mM EDTA, 150 mM sodium dihydrogen phosphate, and 1% protease inhibitor cocktail (Fujifilm Wako Pure Chemical Co.) via sonication. After centrifugation at 13,000 rpm for 15 min at 4°C, the protein concentration in the supernatant was determined using the Quick Start Bradford 1x Dye Reagent (Cat# 5000205; Bio-Rad, Berkeley, CA, USA) and normalized to 2.0 µg/µL. The respective protein samples were electrophoresed on 10% (w/v) sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to 0.22-mm polyvinylidene difluoride (PVDF) membranes (Cat# GVWP04700; Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 60 min and probed with primary antibodies at 4°C overnight. Then, the PVDF membranes were washed with TBST (thrice for 5 min each) and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. The PVDF membranes were washed again with TBST (thrice for 5 min each) and reacted with the Immobilon Forte Western HRP Substrate (Cat# WBLUF0100; Millipore). The immunoreactive bands were visualized using ATTO LuminoGraphI (Cat# WSE-6100; ATTO., Tokyo, Japan). Band intensities were analyzed using the ImageJ software (National Institute of Mental Health). The following primary antibodies were used: Rabbit anti-IRF8 (1:1000; Cat# 98344; Cell Signaling Technology, Danvers MA, USA) and mouse anti-β-actin (1:2000; Cat# A5441; Sigma Aldrich, St. Louis, MO, USA) antibodies. Horseradish peroxidase-linked anti-rabbit and anti-mouse IgG (1:2000 dilution; GE Healthcare, Buckinghamshire, UK) were used the secondary antibodies. Flow Cytometry (FCM) Analysis of the Spinal Cord of EAE Model Mice To prepare immune cells, the spinal cords were harvested from MOG 35–55-immunized mice. The tissues were digested using DNase I (10 U/mL; Cat# DN25; Merck) and 0.25% trypsin (Cat# 15090046; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in the Roswell Park Memorial Institute-1640 with agitation at 250 rpm for 15 min. Single-cell suspensions were obtained by passing the digested tissue through a 70-µm cell strainer (Cat# 352350; Corning Inc., Corning, NY, USA). The cell suspensions were centrifuged at 3000 rpm for 15min in a debris removal solution (gradient solution; Cat# 130-109-398; Miltenyi Biotec, Gaithersburg, MD, USA) to isolate mononuclear cells, and the supernatant was discarded. After thorough washing with PBS, single cells were incubated with an anti-CD16/32 antibody (Cat# 101302; BioLegend, San Jose, CA, USA) to block Fc receptors and prevent non-specific antibody binding. For surface staining experiments, cell viability was assessed using the 7-AAD Viability Staining Solution (Cat# 420404; BioLegend). The cells were stained with fluorochrome-conjugated monoclonal antibodies against CD3-PE/Cy7 (clone 145-2C11; Cat# 100320; BioLegend), CD11b-PE/Cy7 (clone M1/70; Cat# 101216; BioLegend), F4/80-FITC (clone BM8; Cat# 123107; BioLegend), CD8-FITC (clone 53 − 6.7; Cat# 100706; BioLegend), Ly6C-FITC (clone HK1.4; Cat# 128005; BioLegend), CD4-APC/Cy7 (clone GK1.5; Cat# 100414; BioLegend), and Ly6G-APC/Cy7 (clone 1A8; Cat# 127624; BioLegend). For intracellular staining experiments, cell viability was assessed using the Zombie Red Fixable Viability Kit (Cat# 423110; BioLegend), according to the manufacturer’s instructions. The cells were fixed and permeabilized using the Foxp3 Fixation/Permeabilization solution (Cat# 00-5523-00; Thermo Fisher Scientific), followed by staining with anti-mouse IRF8–APC antibody (clone V3GYWCH; Cat# 17-9852-82; Invitrogen Carlsbad, CA, USA) at 4°C for 30 min. After washing with wash buffer, stained cells were analyzed using the CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA), and the data were analyzed using the CytExpert software (Beckman Coulter). Immunohistochemistry For histological analysis, the mice were deeply anesthetized using isoflurane (1 mL/mL; Fujifilm Wako Pure Chemical Co.). Once reflex responses disappeared, the mice were transcardially perfused with 4% paraformaldehyde in PBS. The spinal cords were post-fixed in 4% paraformaldehyde overnight at 4 ℃ The post-fixed tissues were cryoprotected overnight in PBS containing 20% sucrose, embedded in an optimal cutting temperature compound (Cat# 45833; Sakura Finetechnical Co, Tokyo, Japan), and cut into 20-µm sections using a cryostat (Cat# CM1950; Leica, Land Hessen, Germany) for immunohistochemistry. Immunofluorescence staining was performed as previously described [ 28 ]. The tissue sections were incubated in methanol at room temperature for 30 min, followed by incubation in 0.3% Triton X/0.1M Tris-HCl buffer at 37°C for 30 min. The sections were heated using an autoclave machine in 10 mM citrate buffer (pH 6.0) up to 105°C for 2 min. After washing with PBS, the sections were incubated in 1 N HCl at room temperature for 15 min and further incubated with 2 N HCl at 37°C for 20 min. Subsequently, the sections were incubated in 0.1 M borate buffer for 12 min, blocked with 5% normal horse serum (Cat# S-2000; Vector Laboratories, Inc., Burlingame, CA, USA) in PBS for 1 h, and incubated with primary antibodies in PBS at 4°C overnight. After washing with phosphate buffered saline with Tween 20, the sections were incubated with secondary antibodies (1:2000) and Hoechst 33342 (0.1 µg/mL; Cat# 346–07951; Dojindo, Kumamoto, Japan) at room temperature for 3 h. Then, the sections were rinsed with phosphate buffered saline with Tween 20, mounted, covered with glass coverslips, and visualized under a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). The primary antibodies used were rabbit anti-IRF8 (1:500; Cat# 98344; Cell Signaling Technology) and rat anti-F4/80 (1:500; Cat# ab6640; Abcam, Cambridge, UK) antibodies. The secondary antibodies used were Alexa Fluor TM 488 Donkey Anti-Rabbit IgG and Alexa Fluor TM 568 Goat Anti-Rat IgG (Cat# A21206 and A11077, respectively; Invitrogen). The immunohistochemical controls were performed as described above, except for the omission of the primary antibodies. No positively immunostained cells were observed in the controls. The number and density of immunoreactive cells were analyzed using the ImageJ software. Human Blood Samples Human blood samples were collected into EDTA-coated tubes provided by Fujita Laboratory Science with the approval of the Institutional Research Ethics Board (Permit Number: CI124-136). Blood samples were obtained from patients diagnosed with MS, aged 20–60 years, who underwent drug therapy between March and December 2025. Blood samples of controls (HCs) were obtained from healthy volunteers aged 20–60 years. All participants provided written informed consent for enrollment in the study and blood collection. FCM Analysis of Human Blood Samples Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood samples of HCs and patients with MS according to standard FCM protocols [ 29 ]. PBMCs were washed, and red blood cells were lysed with ACK buffer. After thorough washing, the single-cell suspension was incubated with the FcR Blocking Reagent (Cat# 130-059-901; Miltenyi Biotec) to block Fc receptors and prevent non-specific antibody binding. For surface staining, the cells were stained with fluorochrome-conjugated monoclonal antibodies against CD11b-PE/Cy7 (clone M1/70; Cat# 101216; BioLegend), CD3-APC/Cy7 (clone OKT3; Cat# 317342; BioLegend), CD19-APC/Cy7 (clone HIB19; Cat# 302218; BioLegend), and CD235a-APC/Cy7 (clone HI264; Cat# 349116; BioLegend). For intracellular staining, cell viability was assessed using the Zombie Red Fixable Viability Kit (BioLegend), according to the manufacturer’s instructions. The cells were fixed and permeabilized using the Foxp3 Fixation/Permeabilization solution (Thermo Fisher Scientific), according to the manufacturer’s protocol, followed by staining with anti-human IRF8–APC antibody (clone V3GYWCH; Cat# 17-9852-82; Invitrogen) at 4°C for 30 min. After washing with wash buffer (Thermo Fisher Scientific), the stained cells were analyzed using the CytoFLEX Flow Cytometer (Beckman Coulter). Data were analyzed using the CytExpert software (Beckman Coulter). Data Analyses Statistical analyses were conducted using the GraphPad Prism 9.5.1 software (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was assessed via t-test for two groups or repeated-measures analysis of variance for multiple groups. The Tukey–Kramer test was used for post-hoc analyses when F ratios were significant. Statistical significance was set at p < 0.05. All data are expressed as the mean ± standard error of the mean. Results Multiple IRFs are Differentially Expressed in the Spinal Cord during EAE Progression The EAE scores were monitored for 28 dpi (Fig. 1 A), and the mRNA expression levels of IRFs were measured in the spinal cord of EAE model mice at 7, 14, and 28 dpi (Fig. 1 A), corresponding to the onset, active, and chronic phases of EAE, respectively. Expression analysis revealed that the mRNA levels of IRF1, IRF7, IRF8, and IRF9 were significantly upregulated at 14 and 28 dpi (Fig. 1 B, G, H, and I) and those of IRF4 were increased only at 14 dpi (Fig. 1 E). In contrast, IRF2 and IRF3 levels were significantly downregulated at 14 and 28 dpi (Fig. 1 C and D), and IRF6 levels were decreased only at 14 dpi (Fig. 1 F). Notably, IRF5 expression was not detected at any time point (data not shown). Among the IRFs examined, IRF1, IRF7, and IRF8 exhibited the most robust upregulation (Fig. 1 B, G, and H). IRF8 plays critical roles in the differentiation and maturation of immune cells, particularly those in the myeloid lineage [ 30 ]. IRF1, which promotes inflammatory gene expression and antigen presentation [ 31 , 32 ], and IRF7 are primarily associated with IFN signaling [ 16 ]. Considering the central contribution of myeloid cells to neuroinflammatory responses [ 33 , 34 ], we focused on IRF8 as a potential regulator of autoimmune neuroinflammation. Consistent with the transcriptional findings, IRF8 protein levels were significantly increased in the mouse spinal cord at 14 dpi (Fig. 1 J). These results suggest that IRF8 is strongly induced in the spinal cord during EAE progression. IRF8 is Predominantly Expressed in Macrophages in the Spinal Cord of EAE Model Mice To determine the immune cell populations responsible for IRF8 upregulation during EAE, we performed FCM analysis of immune cells isolated from the spinal cord of EAE model mice at 14 dpi, the peak phase of neuroinflammation (Fig. 2 A). IRF8 expression was quantified in monocytes (Ly6-C⁺Ly6-G⁻), neutrophils (Ly6-G⁺Ly-6C⁻), CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), myeloid cells (CD11b + ), and macrophages (CD11b⁺F4/80⁺; Fig. 2 B). The proportion of IRF8-positive cells was significantly increased in monocytes, CD4⁺ T cells, myeloid cells, and macrophages in the spinal cord of EAE model mice (Fig. 2 B and C). Notably, the increase in IRF8 expression was most pronounced in macrophages, and subsequent immunohistochemical analysis confirmed the co-localization of IRF8 with F4/80⁺ macrophages in inflammatory lesions of the spinal cord (Fig. 2 D and E). These results revealed that IRF8 played important roles in macrophage-mediated inflammatory responses in the spinal cord of EAE model mice. IRF8 KO Mice Resist EAE Induction by Suppressing Neuroinflammatory Mediator Expression To assess the functional relevance of IRF8 in EAE pathogenesis, we investigated whether the EAE scores are decreased in IRF8 KO mice. Notably, IRF8 KO mice failed to develop the clinical signs of EAE, demonstrating complete resistance to EAE induction (Fig. 3 A). Although no significant differences were observed in immune cell composition in the spinal cord (Fig. 3 B and C), the expression levels of neuroinflammatory mediators abundantly produced by macrophages, including IL-1β, IL-6, TNF-α, IL-12b, and NOS2, were significantly reduced in the spinal cord of IRF8 KO mice (Fig. 3 D–H). These findings suggest that IRF8 is essential for EAE development. Moreover, IRF8 deficiency suppresses the acquisition of pathogenic inflammatory functions in macrophages, thereby reducing the production of neuroinflammatory mediators necessary for EAE development. IRF8-Positive Myeloid Cell Proportions are Increased in the Peripheral Blood of Patients with MS To determine whether IRF8 expression is similarly upregulated in patients with MS, we analyzed PBMCs of HCs and patients with MS (Fig. 4 A). FCM analysis revealed a significantly higher proportion of IRF8-positive cells in the peripheral blood of patients with MS than in that of HCs (Fig. 4 B and C). IRF8-positive cells were also predominantly observed among CD11b-positive myeloid cell proportions (Fig. 4 B and D). These results suggest that increased IRF8 expression, particularly in myeloid cells, is a common feature of both EAE and human MS, supporting a potential role of IRF8 in the pathophysiology of MS. Discussion In this study, we performed comprehensive profiling of IRFs in the spinal cord of EAE model mice and identified IRF8 as a key transcriptional regulator associated with autoimmune neuroinflammation. IRF expression analysis revealed that multiple IRFs were differentially regulated during EAE progression, with IRF1, IRF7, and IRF8 showing the most robust induction (Fig. 1 B, G, and H). Among these, IRF8 emerged as particularly notable because of its strong and sustained upregulation (Fig. 1 H). IRF8 is also a key transcription factor regulating myeloid cell differentiation and inflammatory gene programs [ 35 ]. We found that IRF8 is predominantly expressed in macrophages within spinal cord lesions (Fig. 2 ) and is essential for EAE development (Fig. 3 ). Importantly, IRF8-positive myeloid cell proportions were also increased in the peripheral blood of patients with MS (Fig. 4 ), further underscoring the translational relevance of our findings. Time-course analysis revealed coordinated stage-dependent regulation of IRFs beyond IRF8 in the inflamed spinal cord. Several IRFs associated with inflammatory and IFN-related signaling, including IRF1, IRF4, IRF7, and IRF9, were upregulated (Fig. 1 B, E, G, and I), whereas IRF2, IRF3, and IRF6 were downregulated (Fig. 1 C, D, and F) during the active and chronic phases of EAE. This pattern suggests a transcriptional shift toward programs that favor immune activation and inflammatory effector functions. IRF1 promotes inflammatory gene expression and antigen presentation [ 31 , 32 ], whereas IRF2 antagonizes IRF1-dependent transcription [ 36 ], suggesting that reduced IRF2 expression further amplifies pro-inflammatory transcriptional states. Type I IFN-related factors IRF7 and IRF9 were also upregulated, possibly reflecting the activation of IFN-stimulated signaling in the inflammatory environment [ 37 – 39 ]. Because type I IFNs exert context-dependent immunomodulatory effects in EAE [ 40 ], the induction of these factors may represent a parallel inflammatory axis or compensatory regulatory response. IRF4, which contributes to T cell differentiation and inflammatory T cell responses [ 41 ], was also induced. In contrast, IRF3 and IRF6, transcription factors implicated in antiviral innate immunity and tissue homeostasis, respectively [ 21 ], were downregulated. The suppression of these factors possibly reflects a shift away from classical antiviral signaling toward alternative inflammatory transcriptional networks. Collectively, these dynamic expression patterns suggest that each IRF plays distinct regulatory roles in immune cell differentiation, effector function, and tissue adaptation, thereby forming an integrated transcriptional network crucial for EAE pathogenesis. Notably, IRF8 expression in the spinal cord was most prominent in macrophages. FCM and immunohistochemical analyses demonstrated that IRF8-positive cells were enriched in the macrophage lineage and co-localized with F4/80⁺ macrophages in inflammatory lesions (Fig. 2 ). Macrophages are the central drivers of neuroinflammation in EAE, acting as sources of pro-inflammatory cytokines, nitric oxide, and antigen-presenting signals that sustain autoreactive T cell responses [ 42 ]. Our findings strongly suggest that IRF8 contributes to the acquisition of a pathogenic macrophage phenotype during EAE. Interestingly, IRF8 KO mice were resistant to EAE induction (Fig. 3 A). Despite comparable overall composition of immune cells in the spinal cord (Fig. 3 B and C), IRF8 deficiency markedly reduced the mRNA expression levels of macrophage-associated neuroinflammatory mediators, including IL-1β, IL-6, TNF-α, IL-12b, and NOS2 (Fig. 3 D–H) [ 43 – 45 ]. These mediators are key amplifiers of autoimmune neuroinflammation and critical for the propagation of tissue damage and EAE severity [ 46 – 49 ]. The dissociation between immune cell presence and inflammatory output suggests that IRF8 is not required for the infiltration of immune cells but is essential for programming the pathogenic effector functions of myeloid cells once they have infiltrated the CNS. This distinction highlights IRF8 as a regulator of inflammatory competence rather than immune cell abundance, providing important conceptual insights into EAE pathogenesis. Consistent with our murine findings, we observed an increased proportion of IRF8-positive cells in the peripheral blood of patients with MS (Fig. 4 B and C). These cells were predominantly CD11b-positive (Fig. 4 D). Future studies should determine whether IRF8-positive myeloid populations fluctuate with disease course, relapse activity, or therapeutic intervention. IRF8 regulates inflammatory gene expression in myeloid cells in EAE and is involved in immune responses in the spinal cord, spleen, and lymph nodes [ 50 ]. Therefore, in addition to its roles in the CNS, IRF8 may function as a transcription factor that contributes to systemic immune regulation. The present study supports these previous findings and provides several novel insights. First, we demonstrated that IRF8 deficiency markedly reduced the production of inflammatory mediators despite no significant changes in the numbers of immune cells (Fig. 3 ). These results indicate that IRF8 regulates the acquisition of an “inflammatory execution program” rather than cell mobilization or infiltration, suggesting that IRF8 functions as a qualitative rather than quantitative regulator of immune responses. Second, we observed an increased proportion of IRF8-positive CD11b⁺ cells in the peripheral blood of patients with MS (Fig. 4 ). Although this study primarily focused on functional analyses at the mouse tissue level, alterations in IRF8-positive myeloid cells in human circulation have not been fully characterized. Importantly, our study provides translational relevance by integrating mechanistic findings from mouse models with observations from human samples. However, as human analyses were limited to peripheral blood, direct comparison with findings from mouse spinal cord tissue remains a limitation. Importantly, MS is characterized by disruption of the blood–brain barrier, which permits the trafficking of peripheral immune cells into the CNS [ 51 ]. Under such conditions, circulating myeloid cells are not immunologically isolated from the CNS but may actively contribute to lesion formation and propagation. Therefore, the alterations observed in peripheral blood myeloid populations may reflect either cells primed for CNS infiltration or systemic inflammatory programs mechanistically linked to CNS pathology. In this context, the observed changes in CD11b⁺IRF8⁺ myeloid cells may not merely represent peripheral immune fluctuations; rather, these circulating IRF8-positive myeloid cells may be functionally associated with lesion activity and tissue injury in MS. Thus, despite differences between mouse spinal cord and human peripheral blood samples, our findings may reflect pathophysiological mechanisms operating within CNS lesions. This study has several limitations. First, we used a systematic IRF8 KO model. IRF8 is involved in hematopoietic development and immune homeostasis [ 52 ]. Although the immune cell composition in the spinal cord was largely preserved, cell type-specific and inducible deletion strategies are necessary to determine the roles of IRF8 in autoimmune neuroinflammation. Additionally, although we focused on macrophage-associated inflammatory mediators, deeper transcriptomic and epigenetic analyses are needed to investigate the IRF8-dependent gene networks underlying pathogenic myeloid programming. Finally, IRF8 expression during different MS phases could not be determined from the analyzed human samples, as MS is characterized by relapse and progressive stages with fluctuating inflammatory activity. Therefore, longitudinal studies should assess IRF8 expression in relation to its clinical activity to determine whether it serves as a marker of immune activation or plays direct pathogenic roles in human MS. In summary, our findings identified IRF8 as a critical regulator of autoimmune neuroinflammation in EAE and highlighted its relevance in human MS. Rather than regulating immune cell infiltration, IRF8 appeared to govern the acquisition of pathogenic inflammatory functions in myeloid cells, thereby enabling the production of neuroinflammatory mediators required for EAE development. Overall, our results suggest that IRF8 is a crucial transcriptional regulator linking myeloid cell programming to autoimmune MS pathology (Fig. 5 ), highlighting IRF8-dependent pathways as promising therapeutic targets for MS. Abbreviations CNS, central nervous system; dpi, days post-immunization; EAE, experimental autoimmune encephalomyelitis; EDTA, ethylenediaminetetracetic acid; FCM, flow cytometry; HCs, healthy controls; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-12b, interleukin-12 subunit beta; IFN, interferon; IRF, interferon regulatory factor; ISGF3, interferon stimulated gene factor 3; KO, knockout; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NOS2, nitric oxide synthase 2; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PBST, phosphate buffered saline with Tween 20; PTX, pertussis toxin; PVDF, poly vinylidene di fluoride; SLE, systemic lupus erythematosus; TBST, tris buffered saline with Tween 20; TNF-α, tumor necrosis factor-α. Declarations Competing Interests The authors declare no competing interests. Funding This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Grant Numbers: 20K07931, 22J15573, 22K07606, 22K11891, and 23H02843); the Japan Science and Technology Agency (JST) FOREST Program (Grant Number: JPMJFR215H); and the research grant from the Smoking Research Foundation. This study was also supported by a grant from the Education and Research Facility of Animal Models for Human Diseases at Fujita Health University, and by the Fujita Mind-Brain Research & Innovation Center for Drug Generation (Fujita Mind-BRIDGe) under the Japan’s Peak Research Universities (J-PEAKS) Program, funded by JSPS. Data Availability Statement The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request. Clinical trial number not applicable. Acknowledgments The authors thank the Fujita Health University Animal Center for their support and the Fujita Health University Open Facility Center for assistance with fluorescence microscopy imaging. The authors also thank Editage (https://www.editage.jp/) for professional English language editing of the manuscript. Author contributions Haruto Ojika and Kazuo Kunisawa devised the project and main conceptual ideas, conducted all experiments, and wrote the manuscript. Moeka Tanabe, Yuki Kon, Koyo Yoshidomi, Yuta Naruoka, Moeka Ogawa, Mayu Kondo, Shoya Takeuchi, Aimi Sugiyama, Hiroyuki Tezuka, Hiroki Doi, and Tana assisted with the experiments. Toshitaka Nabeshima contributed to the manuscript discussion and revised the manuscript accordingly. Kazuo Kunisawa, Toshitaka Nabeshima, and Akihiro Mouri supervised the study and finalized the manuscript. References Reich, D. S., Lucchinetti, C. F., & Calabresi, P. A. (2018). Multiple Sclerosis. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 May, 2026 Reviewers agreed at journal 05 May, 2026 Reviewers agreed at journal 03 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 30 Apr, 2026 Editor assigned by journal 29 Apr, 2026 Submission checks completed at journal 29 Apr, 2026 First submitted to journal 27 Apr, 2026 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-9546988","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634928471,"identity":"50d86ed3-3c2b-4f32-9ce3-0b80608db56b","order_by":0,"name":"Haruto Ojika","email":"","orcid":"","institution":"Fujita Health University Graduate School of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Haruto","middleName":"","lastName":"Ojika","suffix":""},{"id":634928474,"identity":"c734eeab-2650-420b-8fd2-61cd9fdd184b","order_by":1,"name":"Kazuo Kunisawa","email":"","orcid":"","institution":"Fujita Health University 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01:53:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9546988/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9546988/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108978086,"identity":"2e446624-8479-413d-940b-a8cc3026cb0f","added_by":"auto","created_at":"2026-05-11 11:33:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":279401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple IRFs were differentially expressed in the spinal cords during EAE progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The EAE scores of MOG\u003csub\u003e35-55\u003c/sub\u003e-immunized EAE mice (Two-way ANOVA followed by Tukey’s multiple comparison tests, dpi [F (24, 200) = 109.2, ***p \u0026lt; 0.001], EAE [F (1, 884) = 4187, ***p \u0026lt; 0.001], and dpi x EAE [F (27, 884) = 109.2, ***p \u0026lt; 0.001]; n = 10 mice in each group). Biochemical experiments were conducted at 7, 14, and 28 dpi (Arrow points). (B-I) The mRNA expression of IRF1 (B), IRF2 (C), IRF3 (D), IRF4 (E), IRF6 (F), IRF7 (G), IRF8 (H), and IRF9 (I) in the spinal cord of EAE mice analyzed using qRT-PCR at 7, 14, and 28 dpi (Repeated t-test, IRF1 (7 dpi [t = 0.2642, df = 12, p = 0.7961], 14 dpi [t = 6.641, df = 12, ***p \u0026lt; 0.001], 28 dpi [t = 5.321, df = 11, ***p \u0026lt; 0.001]); IRF2 (7 dpi [t = 0.03834, df = 12, p = 0.9700], 14 dpi [t = 3.058, df = 12, **p \u0026lt; 0.01], 28 dpi [t = 3.762, df = 11, **p \u0026lt; 0.01]); IRF3 (7 dpi [t = 1.742, df = 12, p = 0.1070], 14 dpi [t = 4.599, df = 10, ***p \u0026lt; 0.001], 28 dpi [t = 3.565, df = 11, **p \u0026lt; 0.01]); IRF4 (7 dpi [t = 0.6548, df = 11, p = 0.5261], 14 dpi [t = 2.455, df = 10, *p \u0026lt; 0.01], 28 dpi [t = 1.887, df = 11, p = 0.0858]); IRF6 (7 dpi [t = 0.7593, df = 12, p = 0.4623], 14 dpi [t = 2.310, df = 11, *p \u0026lt; 0.05], 28 dpi [t = 1.957, df = 10, p = 0.0788]); IRF7 (7 dpi [t = 0.01704, df = 12, p = 0.9867], 14 dpi [t = 8.466, df = 12, ***p \u0026lt; 0.001], 28 dpi [t = 4.662, df = 11, ***p = 0.001]); IRF8 (7 dpi [t = 1.199, df = 12, p = 0.2538], 14 dpi [t = 8.108, df = 12, ***p \u0026lt; 0.001], 28 dpi [t = 7.897, df = 11, ***p \u0026lt; 0.001]); IRF9 (7 dpi [t = 0.5683, df = 12, p = 0.5803], 14 dpi [t = 6.832, df = 12, ***p \u0026lt; 0.001], 28 dpi [t = 2.690, df = 11, *p \u0026lt; 0.0210]); *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, n = 5-7 mice in each group). (J) Representative Western blots and quantification of IRF8 and β-actin in spinal cord from Control and EAE mice (Student’s t-test, t = 2.280, df = 9, *p \u0026lt; 0.05; n = 5,6 mice in each group). The data are expressed as mean ± SEM\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9546988/v1/85fc62e6b09cc593783eed1f.png"},{"id":108970602,"identity":"31f1dd48-e256-44da-98a5-4933a25de280","added_by":"auto","created_at":"2026-05-11 10:17:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":519788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIRF8 is predominantly expressed in macrophages in the spinal cord of EAE mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schema of FCM analysis for the immune cells isolated from the spinal cords of control and EAE mice at 14 dpi. The spinal cords were harvested, dissociated into single-cell suspensions, and analyzed by FCM. (B) Representative FCM plots of IRF8-positive monocytes (Ly-6C\u003csup\u003e+\u003c/sup\u003eLy-6G\u003csup\u003e-\u003c/sup\u003e; red), neutrophils (Ly-6G\u003csup\u003e+-\u003c/sup\u003eLy-6C\u003csup\u003e-\u003c/sup\u003e; orange), CD4\u003csup\u003e+ \u003c/sup\u003eT cells (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e; green), CD8\u003csup\u003e+ \u003c/sup\u003eT cells (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e; purple), myeloid cells (CD11b\u003csup\u003e+\u003c/sup\u003e; yellow), and macrophages (CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e; blue). (C) The percentage of IRF8-positive monocytes, neutrophils, CD4\u003csup\u003e+ \u003c/sup\u003eT cells, CD8\u003csup\u003e+ \u003c/sup\u003eT cells, and macrophages (Repeated t-test, monocytes [t = 2.433, df = 10, *p \u0026lt; 0.05]; neutrophils [t = 1.426, df = 10, p = 0.1844]; CD4\u003csup\u003e+ \u003c/sup\u003eT cells [t = 2.570, df = 10, *p \u0026lt; 0.05]; CD8\u003csup\u003e+ \u003c/sup\u003eT cells [t = 1.467, df = 10, p = 0.1730]; myeloid cells [t = 2.662, df = 8, *p \u0026lt; 0.05]; macrophages [t = 2.429, df = 8, *p \u0026lt; 0.05]; n = 5-6 mice in each group). (D) Representative images of staining with anti-IRF8 (green), anti-F4/80 (red) and Hoechst (blue) in the spinal cord of control and EAE mice at 14 dpi. Arrowheads indicate double positive cells for both IRF8 and F4/80. Scale bar: 100 μm. (E) The number of IRF8\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e cells was quantified in the spinal cord (Student’s t-test, t = 2.426, df = 9, *p \u0026lt; 0.05; n = 4,7 mice in each group). The data are expressed as mean ± SEM\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9546988/v1/a9d6ea0327101f7c71e582f7.png"},{"id":108970604,"identity":"711a7683-64a2-439d-bd7c-e57bd0bed427","added_by":"auto","created_at":"2026-05-11 10:17:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":413380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIRF8 KO confers resistance to EAE induction with suppressing neuroinflammatory mediators.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The EAE score of wild-type and IRF8 KO mice following immunization with MOG\u003csub\u003e35-55\u003c/sub\u003e (Two-way ANOVA followed by Tukey’s multiple comparison tests, dpi [F (24,200) = 5.512, ***p \u0026lt; 0.001], EAE [F (1,200) = 194.0, ***p \u0026lt; 0.001], and dpi x EAE [F (24,200) = 5.512, ***p \u0026lt; 0.001]; n = 6 mice in each group). (B) Representative FCM plots of monocytes (Ly-6C\u003csup\u003e+\u003c/sup\u003eLy-6G\u003csup\u003e-\u003c/sup\u003e; red), neutrophils (Ly-6G\u003csup\u003e+-\u003c/sup\u003eLy-6C\u003csup\u003e-\u003c/sup\u003e; orange), CD4\u003csup\u003e+ \u003c/sup\u003eT cells (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e; green), CD8\u003csup\u003e+ \u003c/sup\u003eT cells (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e; purple), myeloid cells (CD11b\u003csup\u003e+\u003c/sup\u003e; yellow), and macrophages (CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e; blue) in the spinal cord and of wild-type and IRF8\u003csup\u003e \u003c/sup\u003eKO at the active phase of EAE. (C) The percentage of IRF8-positive monocytes, neutrophils, CD4\u003csup\u003e+ \u003c/sup\u003eT cells, CD8\u003csup\u003e+ \u003c/sup\u003eT cells, and macrophages in the spinal cords of wild-type and IRF8 KO mice (Repeated t-test, monocytes [t = 0.1430, df = 12, p = 0.8886]; neutrophils [t = 0.8719, df = 12, p = 0.4004]; CD4\u003csup\u003e+ \u003c/sup\u003eT cells [t = 0.8635, df = 11, p = 0.4063]; CD8\u003csup\u003e+ \u003c/sup\u003eT cells [t = 0.2045, df = 12, p = 0.8414]; myeloid cells [t = 1.002, df = 12, p = 0.3359]; macrophages [t = 1.082, df = 12, p = 0.3006]; n = 5-8 mice in each group). (D-H) The mRNA expression of IL-1β (D), IL-6 (E), TNF-α (F), IL-12b (G), and NOS2 (H) in the spinal cord of IRF8 KO mice analyzed using qRT-PCR at the active phase of EAE (Student’s t-test, IL-1β (t = 2.338, df = 12, *p \u0026lt; 0.05); IL-6 (t = 3.552, df = 11, **p \u0026lt; 0.01); TNF-α (t = 2.386, df = 12, *p \u0026lt; 0.05); IL-12b (t = 3.632, df = 12, **p \u0026lt; 0.01); NOS2 (t = 2.268, df = 11, *p \u0026lt; 0.05); *p \u0026lt; 0.05, **p \u0026lt; 0.01, n = 6-7 mice in each group). The data are expressed as mean ± SEM\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9546988/v1/9e70495ed382446bdaf51955.png"},{"id":108970605,"identity":"0dbe0110-d3e6-4e58-9958-3d00fa3c4c57","added_by":"auto","created_at":"2026-05-11 10:17:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":270156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIRF8-positive myeloid cells were also increased in the peripheral blood of MS patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schema of FCM analysis of IRF8-positive cells and IRF8-positive myeloid cells in PBMCs from HCs and MS patients. (B) Representative FCM plots of IRF8-positive cells and IRF8-positive myeloid cells in PBMCs from HCs and MS patients. The boxed areas represent the IRF8-positive cells (red) and IRF8-positive myeloid cells (blue). (C) The percentage of IRF8-positive cells in human PBMCs (Student’s t-test, t = 2.550, df = 19, *p \u0026lt; 0.05; n = 8-13 in each group). (D) The percentage of IRF8-positive myeloid cells (CD11b\u003csup\u003e+\u003c/sup\u003eIRF8\u003csup\u003e+\u003c/sup\u003e cells) in the human PBMCs (Student’s t-test, t = 2.138, df = 19, *p \u0026lt; 0.05; n = 8-13 in each group). The data are expressed as mean ± SEM\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9546988/v1/85ff534a955e47e25f09c822.png"},{"id":108970606,"identity":"b262e3a6-e29d-467c-801c-a547960d67e1","added_by":"auto","created_at":"2026-05-11 10:17:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism of IRF8 action in EAE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of the proposed role of IRF8 in EAE. During EAE development, IRF8 is predominantly expressed in myeloid cells and regulates the acquisition of pathogenic inflammatory functions. Increased IRF8 expression promotes the production of pro-inflammatory cytokines, leading to enhanced inflammatory responses in the spinal cord and contributing to EAE pathogenesis. In contrast, in IRF8 KO mice, the pathogenic inflammatory program in myeloid cells is insufficiently induced, resulting in reduced production of inflammatory mediators. Consequently, inflammatory responses in the spinal cord are attenuated, and EAE development is suppressed.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9546988/v1/6a2f239875ef4d37bbbcdf21.png"},{"id":108979957,"identity":"bfc269ca-3fc4-46f9-a950-affcd7e4ab69","added_by":"auto","created_at":"2026-05-11 12:02:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2002983,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9546988/v1/9e3fc3cc-d647-4aae-8727-2ec8800c7e72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eInterferon regulatory factor 8 Drives Autoimmune Neuroinflammation in Experimental Autoimmune Encephalomyelitis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple sclerosis (MS), a chronic autoimmune disease characterized by demyelination of the central nervous system (CNS), affects over 2\u0026nbsp;million people worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MS presents with diverse neurological symptoms, including motor dysfunction, sensory disturbances, and autonomic dysfunction, and is pathologically defined by the infiltration of peripheral immune cells into CNS lesions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the precise molecular mechanisms underlying its pathogenesis remain unclear.\u003c/p\u003e \u003cp\u003eMS onset and progression involve complex interactions among diverse immune cell populations, including T cells, B cells, monocytes/macrophages, and dendritic cells [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Cytokines and chemokines secreted by these immune cells amplify neuroinflammatory cascades and contribute to tissue damage in the CNS [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Accordingly, MS is widely regarded as a disease resulting from dysregulated immune responses, in which chronic inflammation overwhelms immune homeostasis.\u003c/p\u003e \u003cp\u003eTranscriptional regulation is essential for maintaining immune balance, and the interferon regulatory factor (IRF) family plays a crucial role in this process. The IRF family, consisting of nine members (IRF1\u0026ndash;9), comprises transcription factors that orchestrate key innate and adaptive immune functions, such as interferon (IFN) signaling, inflammatory responses, immune cell differentiation, and apoptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEach IRF performs distinct and context-dependent biological functions. IRF1 promotes the transcription of inflammatory genes and antigen-presenting molecules, contributing to immune activation in macrophages and dendritic cells [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], whereas IRF2 functions primarily as its antagonist [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. IRF3 and IRF7 are central regulators of type I IFN (IFN-α/β) production during antiviral immunity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and IRF9 associates with signal transducer and activator of transcription 1/2 to form the IFN-stimulated gene factor 3 complex, which induces IFN-stimulated gene expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. IRF4 and IRF8 are critically involved in the development and differentiation of both the lymphoid and myeloid lineages, thereby influencing immune cell fate decisions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. IRF5 promotes pro-inflammatory macrophage activation and cytokine production [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], whereas IRF6 is primarily associated with epithelial differentiation and tissue repair [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], helping to maintain tissue barrier integrity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering these diverse immunological functions, aberrant IRF signaling is implicated in several autoimmune and inflammatory diseases, including systemic lupus erythematosus, rheumatoid arthritis, Crohn\u0026rsquo;s disease, and psoriasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. IRF family members may critically modulate the progression of MS, which is characterized by dysregulated IFN responses and excessive myeloid cell activation. However, to date, no study has comprehensively analyzed IRF family members in MS. Therefore, elucidating the expression dynamics and functional contributions of the entire IRF family in the inflamed CNS is essential for a deeper understanding of MS pathogenesis.\u003c/p\u003e \u003cp\u003eIn this study, we comprehensively analyzed IRF family expression in the spinal cord of experimental autoimmune encephalomyelitis (EAE) model mice, a widely used animal model of MS, to identify key IRF members involved in the regulation of autoimmune neuroinflammation. By assessing the activation patterns of individual IRFs, we identified novel transcriptional regulators and potential therapeutic targets for MS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). IRF8 knockout (KO) mice (C57BL/6 background) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and confirmed via standard polymerase chain reaction (PCR)-based genotyping of genomic DNA. The following primer sequences were used for PCR genotyping: IRF8-forward sense, 5\u0026prime;-GGGCACTGCTGATTTCTCAT-3\u0026prime;, and IRF8-reverse sense, 5\u0026prime;-AGCCAGAGGGAAACCAAAAA-3\u0026prime;. Each mouse (8\u0026ndash;10 weeks old) was subjected to EAE induction. The mice were housed in plastic cages and maintained under a 12-h light/dark cycle (lights on at 8:00 a.m.), with food and water provided ad libitum. All experiments adhered to the guidelines of the Japanese Pharmacological Society and Institute for Experimental Animals at Fujita Health University (Aichi, Japan). All protocols were approved by the Ethics Committee of Animal Experiments at the Institute for Experimental Animals, Fujita Health University (Permit Number: APU22059-MD2).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEstablishment of EAE Model Mice\u003c/h3\u003e\n\u003cp\u003eEAE model mice were established as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, the myelin oligodendrocyte glycoprotein (MOG) 35\u0026ndash;55 peptide MEVGWYRSPFSRVVHLYRNGK (200 \u0026micro;g; Cat# S-PEP; Scrum, Tokyo, Japan) was emulsified in complete Freund\u0026rsquo;s adjuvant (Cat# D614-0050; RCK, Gilbertsville, PA, USA). The mice were subcutaneously immunized with MOG/complete Freund\u0026rsquo;s adjuvant at two sites on the dorsal flank, followed by an intraperitoneal injection of pertussis toxin (600 ng; Cat# 516560-50UGCN; Merck, Dusseldorf, Germany) immediately post-immunization and after 48 h. The mice were monitored daily for 28 d post-immunization (dpi) to assess the EAE score (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The EAE scores were as follows: 0\u0026thinsp;=\u0026thinsp;normal, 0.5\u0026thinsp;=\u0026thinsp;partial drooping of the tail, 1\u0026thinsp;=\u0026thinsp;complete drooping of the tail, 1.5\u0026thinsp;=\u0026thinsp;abnormal gait, 2\u0026thinsp;=\u0026thinsp;abnormal gait with weakness in the hind limbs, 2.5\u0026thinsp;=\u0026thinsp;paralysis and weakness in one hind limb, 3\u0026thinsp;=\u0026thinsp;paralysis of both hind limbs, 3.5\u0026thinsp;=\u0026thinsp;paralysis of both hind limbs in a hunched posture, 4\u0026thinsp;=\u0026thinsp;paralysis of the hind and fore limbs, and 5\u0026thinsp;=\u0026thinsp;moribundity or death. If EAE was not induced at 18 dpi, the mice were excluded from analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eThe mice were anesthetized using isoflurane (1 mL/mL; Fujifilm Wako Pure Chemical Co., Osaka, Japan) and transcardially perfused with ice-cold phosphate-buffered saline (PBS). The spinal cords were quickly removed, washed with ice-cold PBS, and immediately frozen on dry ice. All samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time reverse transcription PCR (qRT–PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using Sepasol-RNA I Super G (Cat# 09379-84; Nacalai Tesque, Kyoto, Japan), according to the manufacturer\u0026rsquo;s instructions. All PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT kit (Cat# FSQ-101; Toyobo, Osaka, Japan). For qPCR, THUNDERBIRD Probe qPCR Mix (Cat# QPS-101; Toyobo) was used and subjected to real-time PCR quantification using the StepOne Real-Time PCR System (Cat# 4376357; Life Technologies, Carlsbad, CA, USA). qPCR was performed using the StepOne analyzer (Cat# 4461357; Life Technologies). The PCR program consisted of 40 cycles of 95\u0026deg;C for 30 s and 60\u0026deg;C for 1 min. Melting curve analysis was performed after each PCR to differentiate between specific and non-specific amplifications. β-actin was used as the housekeeping gene to normalize all PCR data.\u003c/p\u003e \u003cp\u003eThe following primers were used in this study: IRF1 (Mm.PT.58.33516776), IRF2 (Mm.PT.58.11463323), IRF3 (Mm.PT.58.5650168), IRF4 (Mm.PT.58.31041855), IRF5 (Mm.PT.58.6374764), IRF6 (Mm.PT.58.7951480), IRF7 (Mm.PT.58.32394021.g), IRF8 (Mm.PT.58.30819027), IRF9 (Mm.PT.58.31054864), interleukin (IL)-1β (Mm.PT.58.41616450), IL-6 (Mm.58.10005566), tumor necrosis factor (TNF)-α (Mm.PT.58.12575861), IL-12b (Mm.PT.58.12409997), nitric oxide synthase 2 (NOS2; Mm.PT.58.43705194), and β-actin (Mm.PT.39.a.22214843).\u003c/p\u003e\n\u003ch3\u003eWestern Blotting Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blotting Analysis\u003c/div\u003e \u003cp\u003eWestern blotting was performed as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The spinal cords were homogenized in ice-cold homogenization buffer (pH 7.4; 50 mM Tris-HCl [pH 8.0] containing 4 mM EGTA, 10 mM EDTA, 150 mM sodium dihydrogen phosphate, and 1% protease inhibitor cocktail (Fujifilm Wako Pure Chemical Co.) via sonication. After centrifugation at 13,000 rpm for 15 min at 4\u0026deg;C, the protein concentration in the supernatant was determined using the Quick Start Bradford 1x Dye Reagent (Cat# 5000205; Bio-Rad, Berkeley, CA, USA) and normalized to 2.0 \u0026micro;g/\u0026micro;L. The respective protein samples were electrophoresed on 10% (w/v) sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to 0.22-mm polyvinylidene difluoride (PVDF) membranes (Cat# GVWP04700; Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 60 min and probed with primary antibodies at 4\u0026deg;C overnight. Then, the PVDF membranes were washed with TBST (thrice for 5 min each) and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. The PVDF membranes were washed again with TBST (thrice for 5 min each) and reacted with the Immobilon Forte Western HRP Substrate (Cat# WBLUF0100; Millipore). The immunoreactive bands were visualized using ATTO LuminoGraphI (Cat# WSE-6100; ATTO., Tokyo, Japan). Band intensities were analyzed using the ImageJ software (National Institute of Mental Health). The following primary antibodies were used: Rabbit anti-IRF8 (1:1000; Cat# 98344; Cell Signaling Technology, Danvers MA, USA) and mouse anti-β-actin (1:2000; Cat# A5441; Sigma Aldrich, St. Louis, MO, USA) antibodies. Horseradish peroxidase-linked anti-rabbit and anti-mouse IgG (1:2000 dilution; GE Healthcare, Buckinghamshire, UK) were used the secondary antibodies.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry (FCM) Analysis of the Spinal Cord of EAE Model Mice\u003c/h2\u003e \u003cp\u003eTo prepare immune cells, the spinal cords were harvested from MOG 35\u0026ndash;55-immunized mice. The tissues were digested using DNase I (10 U/mL; Cat# DN25; Merck) and 0.25% trypsin (Cat# 15090046; Thermo Fisher Scientific, Waltham, MA, USA) at 37\u0026deg;C in the Roswell Park Memorial Institute-1640 with agitation at 250 rpm for 15 min. Single-cell suspensions were obtained by passing the digested tissue through a 70-\u0026micro;m cell strainer (Cat# 352350; Corning Inc., Corning, NY, USA). The cell suspensions were centrifuged at 3000 rpm for 15min in a debris removal solution (gradient solution; Cat# 130-109-398; Miltenyi Biotec, Gaithersburg, MD, USA) to isolate mononuclear cells, and the supernatant was discarded. After thorough washing with PBS, single cells were incubated with an anti-CD16/32 antibody (Cat# 101302; BioLegend, San Jose, CA, USA) to block Fc receptors and prevent non-specific antibody binding.\u003c/p\u003e \u003cp\u003eFor surface staining experiments, cell viability was assessed using the 7-AAD Viability Staining Solution (Cat# 420404; BioLegend). The cells were stained with fluorochrome-conjugated monoclonal antibodies against CD3-PE/Cy7 (clone 145-2C11; Cat# 100320; BioLegend), CD11b-PE/Cy7 (clone M1/70; Cat# 101216; BioLegend), F4/80-FITC (clone BM8; Cat# 123107; BioLegend), CD8-FITC (clone 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7; Cat# 100706; BioLegend), Ly6C-FITC (clone HK1.4; Cat# 128005; BioLegend), CD4-APC/Cy7 (clone GK1.5; Cat# 100414; BioLegend), and Ly6G-APC/Cy7 (clone 1A8; Cat# 127624; BioLegend).\u003c/p\u003e \u003cp\u003e For intracellular staining experiments, cell viability was assessed using the Zombie Red Fixable Viability Kit (Cat# 423110; BioLegend), according to the manufacturer\u0026rsquo;s instructions. The cells were fixed and permeabilized using the Foxp3 Fixation/Permeabilization solution (Cat# 00-5523-00; Thermo Fisher Scientific), followed by staining with anti-mouse IRF8\u0026ndash;APC antibody (clone V3GYWCH; Cat# 17-9852-82; Invitrogen Carlsbad, CA, USA) at 4\u0026deg;C for 30 min. After washing with wash buffer, stained cells were analyzed using the CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA), and the data were analyzed using the CytExpert software (Beckman Coulter).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFor histological analysis, the mice were deeply anesthetized using isoflurane (1 mL/mL; Fujifilm Wako Pure Chemical Co.). Once reflex responses disappeared, the mice were transcardially perfused with 4% paraformaldehyde in PBS. The spinal cords were post-fixed in 4% paraformaldehyde overnight at 4 ℃ The post-fixed tissues were cryoprotected overnight in PBS containing 20% sucrose, embedded in an optimal cutting temperature compound (Cat# 45833; Sakura Finetechnical Co, Tokyo, Japan), and cut into 20-\u0026micro;m sections using a cryostat (Cat# CM1950; Leica, Land Hessen, Germany) for immunohistochemistry. Immunofluorescence staining was performed as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The tissue sections were incubated in methanol at room temperature for 30 min, followed by incubation in 0.3% Triton X/0.1M Tris-HCl buffer at 37\u0026deg;C for 30 min. The sections were heated using an autoclave machine in 10 mM citrate buffer (pH 6.0) up to 105\u0026deg;C for 2 min. After washing with PBS, the sections were incubated in 1 N HCl at room temperature for 15 min and further incubated with 2 N HCl at 37\u0026deg;C for 20 min. Subsequently, the sections were incubated in 0.1 M borate buffer for 12 min, blocked with 5% normal horse serum (Cat# S-2000; Vector Laboratories, Inc., Burlingame, CA, USA) in PBS for 1 h, and incubated with primary antibodies in PBS at 4\u0026deg;C overnight. After washing with phosphate buffered saline with Tween 20, the sections were incubated with secondary antibodies (1:2000) and Hoechst 33342 (0.1 \u0026micro;g/mL; Cat# 346\u0026ndash;07951; Dojindo, Kumamoto, Japan) at room temperature for 3 h. Then, the sections were rinsed with phosphate buffered saline with Tween 20, mounted, covered with glass coverslips, and visualized under a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). The primary antibodies used were rabbit anti-IRF8 (1:500; Cat# 98344; Cell Signaling Technology) and rat anti-F4/80 (1:500; Cat# ab6640; Abcam, Cambridge, UK) antibodies. The secondary antibodies used were Alexa Fluor TM 488 Donkey Anti-Rabbit IgG and Alexa Fluor TM 568 Goat Anti-Rat IgG (Cat# A21206 and A11077, respectively; Invitrogen). The immunohistochemical controls were performed as described above, except for the omission of the primary antibodies. No positively immunostained cells were observed in the controls. The number and density of immunoreactive cells were analyzed using the ImageJ software.\u003c/p\u003e\n\u003ch3\u003eHuman Blood Samples\u003c/h3\u003e\n\u003cp\u003eHuman blood samples were collected into EDTA-coated tubes provided by Fujita Laboratory Science with the approval of the Institutional Research Ethics Board (Permit Number: CI124-136). Blood samples were obtained from patients diagnosed with MS, aged 20\u0026ndash;60 years, who underwent drug therapy between March and December 2025. Blood samples of controls (HCs) were obtained from healthy volunteers aged 20\u0026ndash;60 years. All participants provided written informed consent for enrollment in the study and blood collection.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFCM Analysis of Human Blood Samples\u003c/h2\u003e \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood samples of HCs and patients with MS according to standard FCM protocols [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. PBMCs were washed, and red blood cells were lysed with ACK buffer. After thorough washing, the single-cell suspension was incubated with the FcR Blocking Reagent (Cat# 130-059-901; Miltenyi Biotec) to block Fc receptors and prevent non-specific antibody binding.\u003c/p\u003e \u003cp\u003eFor surface staining, the cells were stained with fluorochrome-conjugated monoclonal antibodies against CD11b-PE/Cy7 (clone M1/70; Cat# 101216; BioLegend), CD3-APC/Cy7 (clone OKT3; Cat# 317342; BioLegend), CD19-APC/Cy7 (clone HIB19; Cat# 302218; BioLegend), and CD235a-APC/Cy7 (clone HI264; Cat# 349116; BioLegend).\u003c/p\u003e \u003cp\u003eFor intracellular staining, cell viability was assessed using the Zombie Red Fixable Viability Kit (BioLegend), according to the manufacturer\u0026rsquo;s instructions. The cells were fixed and permeabilized using the Foxp3 Fixation/Permeabilization solution (Thermo Fisher Scientific), according to the manufacturer\u0026rsquo;s protocol, followed by staining with anti-human IRF8\u0026ndash;APC antibody (clone V3GYWCH; Cat# 17-9852-82; Invitrogen) at 4\u0026deg;C for 30 min. After washing with wash buffer (Thermo Fisher Scientific), the stained cells were analyzed using the CytoFLEX Flow Cytometer (Beckman Coulter). Data were analyzed using the CytExpert software (Beckman Coulter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData Analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using the GraphPad Prism 9.5.1 software (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was assessed via t-test for two groups or repeated-measures analysis of variance for multiple groups. The Tukey\u0026ndash;Kramer test was used for post-hoc analyses when F ratios were significant. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMultiple IRFs are Differentially Expressed in the Spinal Cord during EAE Progression\u003c/h2\u003e \u003cp\u003eThe EAE scores were monitored for 28 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the mRNA expression levels of IRFs were measured in the spinal cord of EAE model mice at 7, 14, and 28 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), corresponding to the onset, active, and chronic phases of EAE, respectively. Expression analysis revealed that the mRNA levels of IRF1, IRF7, IRF8, and IRF9 were significantly upregulated at 14 and 28 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G, H, and I) and those of IRF4 were increased only at 14 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In contrast, IRF2 and IRF3 levels were significantly downregulated at 14 and 28 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D), and IRF6 levels were decreased only at 14 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Notably, IRF5 expression was not detected at any time point (data not shown).\u003c/p\u003e \u003cp\u003eAmong the IRFs examined, IRF1, IRF7, and IRF8 exhibited the most robust upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G, and H). IRF8 plays critical roles in the differentiation and maturation of immune cells, particularly those in the myeloid lineage [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. IRF1, which promotes inflammatory gene expression and antigen presentation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and IRF7 are primarily associated with IFN signaling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Considering the central contribution of myeloid cells to neuroinflammatory responses [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we focused on IRF8 as a potential regulator of autoimmune neuroinflammation. Consistent with the transcriptional findings, IRF8 protein levels were significantly increased in the mouse spinal cord at 14 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eThese results suggest that IRF8 is strongly induced in the spinal cord during EAE progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIRF8 is Predominantly Expressed in Macrophages in the Spinal Cord of EAE Model Mice\u003c/h2\u003e \u003cp\u003eTo determine the immune cell populations responsible for IRF8 upregulation during EAE, we performed FCM analysis of immune cells isolated from the spinal cord of EAE model mice at 14 dpi, the peak phase of neuroinflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). IRF8 expression was quantified in monocytes (Ly6-C⁺Ly6-G⁻), neutrophils (Ly6-G⁺Ly-6C⁻), CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), myeloid cells (CD11b\u003csup\u003e+\u003c/sup\u003e), and macrophages (CD11b⁺F4/80⁺; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The proportion of IRF8-positive cells was significantly increased in monocytes, CD4⁺ T cells, myeloid cells, and macrophages in the spinal cord of EAE model mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). Notably, the increase in IRF8 expression was most pronounced in macrophages, and subsequent immunohistochemical analysis confirmed the co-localization of IRF8 with F4/80⁺ macrophages in inflammatory lesions of the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results revealed that IRF8 played important roles in macrophage-mediated inflammatory responses in the spinal cord of EAE model mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIRF8 KO Mice Resist EAE Induction by Suppressing Neuroinflammatory Mediator Expression\u003c/h2\u003e \u003cp\u003eTo assess the functional relevance of IRF8 in EAE pathogenesis, we investigated whether the EAE scores are decreased in IRF8 KO mice. Notably, IRF8 KO mice failed to develop the clinical signs of EAE, demonstrating complete resistance to EAE induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Although no significant differences were observed in immune cell composition in the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C), the expression levels of neuroinflammatory mediators abundantly produced by macrophages, including IL-1β, IL-6, TNF-α, IL-12b, and NOS2, were significantly reduced in the spinal cord of IRF8 KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings suggest that IRF8 is essential for EAE development. Moreover, IRF8 deficiency suppresses the acquisition of pathogenic inflammatory functions in macrophages, thereby reducing the production of neuroinflammatory mediators necessary for EAE development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIRF8-Positive Myeloid Cell Proportions are Increased in the Peripheral Blood of Patients with MS\u003c/h2\u003e \u003cp\u003eTo determine whether IRF8 expression is similarly upregulated in patients with MS, we analyzed PBMCs of HCs and patients with MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). FCM analysis revealed a significantly higher proportion of IRF8-positive cells in the peripheral blood of patients with MS than in that of HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). IRF8-positive cells were also predominantly observed among CD11b-positive myeloid cell proportions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results suggest that increased IRF8 expression, particularly in myeloid cells, is a common feature of both EAE and human MS, supporting a potential role of IRF8 in the pathophysiology of MS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we performed comprehensive profiling of IRFs in the spinal cord of EAE model mice and identified IRF8 as a key transcriptional regulator associated with autoimmune neuroinflammation. IRF expression analysis revealed that multiple IRFs were differentially regulated during EAE progression, with IRF1, IRF7, and IRF8 showing the most robust induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G, and H). Among these, IRF8 emerged as particularly notable because of its strong and sustained upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). IRF8 is also a key transcription factor regulating myeloid cell differentiation and inflammatory gene programs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We found that IRF8 is predominantly expressed in macrophages within spinal cord lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and is essential for EAE development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Importantly, IRF8-positive myeloid cell proportions were also increased in the peripheral blood of patients with MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), further underscoring the translational relevance of our findings.\u003c/p\u003e \u003cp\u003eTime-course analysis revealed coordinated stage-dependent regulation of IRFs beyond IRF8 in the inflamed spinal cord. Several IRFs associated with inflammatory and IFN-related signaling, including IRF1, IRF4, IRF7, and IRF9, were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, E, G, and I), whereas IRF2, IRF3, and IRF6 were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, and F) during the active and chronic phases of EAE. This pattern suggests a transcriptional shift toward programs that favor immune activation and inflammatory effector functions. IRF1 promotes inflammatory gene expression and antigen presentation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], whereas IRF2 antagonizes IRF1-dependent transcription [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], suggesting that reduced IRF2 expression further amplifies pro-inflammatory transcriptional states. Type I IFN-related factors IRF7 and IRF9 were also upregulated, possibly reflecting the activation of IFN-stimulated signaling in the inflammatory environment [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Because type I IFNs exert context-dependent immunomodulatory effects in EAE [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], the induction of these factors may represent a parallel inflammatory axis or compensatory regulatory response. IRF4, which contributes to T cell differentiation and inflammatory T cell responses [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], was also induced. In contrast, IRF3 and IRF6, transcription factors implicated in antiviral innate immunity and tissue homeostasis, respectively [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], were downregulated. The suppression of these factors possibly reflects a shift away from classical antiviral signaling toward alternative inflammatory transcriptional networks. Collectively, these dynamic expression patterns suggest that each IRF plays distinct regulatory roles in immune cell differentiation, effector function, and tissue adaptation, thereby forming an integrated transcriptional network crucial for EAE pathogenesis.\u003c/p\u003e \u003cp\u003eNotably, IRF8 expression in the spinal cord was most prominent in macrophages. FCM and immunohistochemical analyses demonstrated that IRF8-positive cells were enriched in the macrophage lineage and co-localized with F4/80⁺ macrophages in inflammatory lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Macrophages are the central drivers of neuroinflammation in EAE, acting as sources of pro-inflammatory cytokines, nitric oxide, and antigen-presenting signals that sustain autoreactive T cell responses [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our findings strongly suggest that IRF8 contributes to the acquisition of a pathogenic macrophage phenotype during EAE.\u003c/p\u003e \u003cp\u003eInterestingly, IRF8 KO mice were resistant to EAE induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Despite comparable overall composition of immune cells in the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C), IRF8 deficiency markedly reduced the mRNA expression levels of macrophage-associated neuroinflammatory mediators, including IL-1β, IL-6, TNF-α, IL-12b, and NOS2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;H) [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These mediators are key amplifiers of autoimmune neuroinflammation and critical for the propagation of tissue damage and EAE severity [\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The dissociation between immune cell presence and inflammatory output suggests that IRF8 is not required for the infiltration of immune cells but is essential for programming the pathogenic effector functions of myeloid cells once they have infiltrated the CNS. This distinction highlights IRF8 as a regulator of inflammatory competence rather than immune cell abundance, providing important conceptual insights into EAE pathogenesis.\u003c/p\u003e \u003cp\u003eConsistent with our murine findings, we observed an increased proportion of IRF8-positive cells in the peripheral blood of patients with MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). These cells were predominantly CD11b-positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Future studies should determine whether IRF8-positive myeloid populations fluctuate with disease course, relapse activity, or therapeutic intervention.\u003c/p\u003e \u003cp\u003eIRF8 regulates inflammatory gene expression in myeloid cells in EAE and is involved in immune responses in the spinal cord, spleen, and lymph nodes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therefore, in addition to its roles in the CNS, IRF8 may function as a transcription factor that contributes to systemic immune regulation. The present study supports these previous findings and provides several novel insights. First, we demonstrated that IRF8 deficiency markedly reduced the production of inflammatory mediators despite no significant changes in the numbers of immune cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicate that IRF8 regulates the acquisition of an \u0026ldquo;inflammatory execution program\u0026rdquo; rather than cell mobilization or infiltration, suggesting that IRF8 functions as a qualitative rather than quantitative regulator of immune responses. Second, we observed an increased proportion of IRF8-positive CD11b⁺ cells in the peripheral blood of patients with MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although this study primarily focused on functional analyses at the mouse tissue level, alterations in IRF8-positive myeloid cells in human circulation have not been fully characterized. Importantly, our study provides translational relevance by integrating mechanistic findings from mouse models with observations from human samples. However, as human analyses were limited to peripheral blood, direct comparison with findings from mouse spinal cord tissue remains a limitation. Importantly, MS is characterized by disruption of the blood\u0026ndash;brain barrier, which permits the trafficking of peripheral immune cells into the CNS [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Under such conditions, circulating myeloid cells are not immunologically isolated from the CNS but may actively contribute to lesion formation and propagation. Therefore, the alterations observed in peripheral blood myeloid populations may reflect either cells primed for CNS infiltration or systemic inflammatory programs mechanistically linked to CNS pathology. In this context, the observed changes in CD11b⁺IRF8⁺ myeloid cells may not merely represent peripheral immune fluctuations; rather, these circulating IRF8-positive myeloid cells may be functionally associated with lesion activity and tissue injury in MS. Thus, despite differences between mouse spinal cord and human peripheral blood samples, our findings may reflect pathophysiological mechanisms operating within CNS lesions.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, we used a systematic IRF8 KO model. IRF8 is involved in hematopoietic development and immune homeostasis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Although the immune cell composition in the spinal cord was largely preserved, cell type-specific and inducible deletion strategies are necessary to determine the roles of IRF8 in autoimmune neuroinflammation. Additionally, although we focused on macrophage-associated inflammatory mediators, deeper transcriptomic and epigenetic analyses are needed to investigate the IRF8-dependent gene networks underlying pathogenic myeloid programming. Finally, IRF8 expression during different MS phases could not be determined from the analyzed human samples, as MS is characterized by relapse and progressive stages with fluctuating inflammatory activity. Therefore, longitudinal studies should assess IRF8 expression in relation to its clinical activity to determine whether it serves as a marker of immune activation or plays direct pathogenic roles in human MS.\u003c/p\u003e \u003cp\u003eIn summary, our findings identified IRF8 as a critical regulator of autoimmune neuroinflammation in EAE and highlighted its relevance in human MS. Rather than regulating immune cell infiltration, IRF8 appeared to govern the acquisition of pathogenic inflammatory functions in myeloid cells, thereby enabling the production of neuroinflammatory mediators required for EAE development. Overall, our results suggest that IRF8 is a crucial transcriptional regulator linking myeloid cell programming to autoimmune MS pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), highlighting IRF8-dependent pathways as promising therapeutic targets for MS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCNS, central nervous system; dpi, days post-immunization; EAE, experimental autoimmune encephalomyelitis; EDTA, ethylenediaminetetracetic acid; FCM, flow cytometry; HCs, healthy controls; IL-1\u0026beta;, interleukin-1\u0026beta;; IL-6, interleukin-6; IL-12b, interleukin-12 subunit beta; IFN, interferon; IRF, interferon regulatory factor; ISGF3, interferon stimulated gene factor 3; KO, knockout; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NOS2, nitric oxide synthase 2; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PBST, phosphate buffered saline with Tween 20; PTX, pertussis toxin; PVDF, poly vinylidene di fluoride; SLE, systemic lupus erythematosus; TBST, tris buffered saline with Tween 20; TNF-\u0026alpha;, tumor necrosis factor-\u0026alpha;.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Grant Numbers: 20K07931, 22J15573, 22K07606, 22K11891, and 23H02843); the Japan Science and Technology Agency (JST) FOREST Program (Grant Number: JPMJFR215H); and the research grant from the Smoking Research Foundation. This study was also supported by a grant from the Education and Research Facility of Animal Models for Human Diseases at Fujita Health University, and by the Fujita Mind-Brain Research \u0026amp; Innovation Center for Drug Generation (Fujita Mind-BRIDGe) under the Japan\u0026rsquo;s Peak Research Universities (J-PEAKS) Program, funded by JSPS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Fujita Health University Animal Center for their support and the Fujita Health University Open Facility Center for assistance with fluorescence microscopy imaging. The authors also thank Editage (https://www.editage.jp/) for professional English language editing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaruto Ojika and Kazuo Kunisawa devised the project and main conceptual ideas, conducted all experiments, and wrote the manuscript. Moeka Tanabe, Yuki Kon, Koyo Yoshidomi, Yuta Naruoka, Moeka Ogawa, Mayu Kondo, Shoya Takeuchi, Aimi Sugiyama, Hiroyuki Tezuka, Hiroki Doi, and Tana assisted with the experiments. Toshitaka Nabeshima contributed to the manuscript discussion and revised the manuscript accordingly. Kazuo Kunisawa, Toshitaka Nabeshima, and Akihiro Mouri supervised the study and finalized the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReich, D. S., Lucchinetti, C. F., \u0026amp; Calabresi, P. A. (2018). 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IRF8: Mechanism of Action and Health Implications. \u003cem\u003eCells\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(17). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cells11172630\u003c/span\u003e\u003cspan address=\"10.3390/cells11172630\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"neuromolecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nemm","sideBox":"Learn more about [NeuroMolecular Medicine](http://link.springer.com/journal/12017)","snPcode":"12017","submissionUrl":"https://submission.nature.com/new-submission/12017/3","title":"NeuroMolecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Interferon regulatory factor 8, Multiple sclerosis, Macrophages, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-9546988/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9546988/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. The interferon regulatory factor (IRF) family comprises transcription factors that regulate immune responses; however, their roles in MS pathogenesis remain unclear. In this study, we investigated whether IRFs are involved in MS pathology using experimental autoimmune encephalomyelitis (EAE) model mice. Among the IRFs examined, IRF8 expression was significantly increased in the spinal cord of EAE model mice and predominantly localized to macrophages. Consistently, patients with MS exhibited significantly increased proportions of IRF8-positive myeloid cells in peripheral blood. Notably, IRF8 knockout mice exhibited complete resistance to EAE induction. The mRNA expression levels of neuroinflammatory mediators, including interleukin IL-1β, IL-6, tumor necrosis factor-α, IL-12b, and nitric oxide synthase 2, were significantly reduced in the spinal cord of IRF8 knockout mice. Collectively, these findings suggest that IRF8 is a critical regulator of autoimmune neuroinflammation, highlighting its potential as a therapeutic target for MS.\u003c/p\u003e","manuscriptTitle":"Interferon regulatory factor 8 Drives Autoimmune Neuroinflammation in Experimental Autoimmune Encephalomyelitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 10:17:45","doi":"10.21203/rs.3.rs-9546988/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T18:22:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286922893539901667307506682017296114334","date":"2026-05-05T15:27:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121517747876305077686812741386835151421","date":"2026-05-04T02:42:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279831274136054221963732662836482616927","date":"2026-04-30T17:27:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-30T15:16:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T10:41:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T10:40:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"NeuroMolecular Medicine","date":"2026-04-28T01:36:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neuromolecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nemm","sideBox":"Learn more about [NeuroMolecular Medicine](http://link.springer.com/journal/12017)","snPcode":"12017","submissionUrl":"https://submission.nature.com/new-submission/12017/3","title":"NeuroMolecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"937254d3-b7d6-464c-955f-bdfd0a87f4c9","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T18:22:26+00:00","index":19,"fulltext":""},{"type":"reviewerAgreed","content":"286922893539901667307506682017296114334","date":"2026-05-05T15:27:56+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"121517747876305077686812741386835151421","date":"2026-05-04T02:42:29+00:00","index":17,"fulltext":""},{"type":"reviewerAgreed","content":"279831274136054221963732662836482616927","date":"2026-04-30T17:27:09+00:00","index":15,"fulltext":""},{"type":"reviewersInvited","content":"5","date":"2026-04-30T15:16:04+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T10:17:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 10:17:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9546988","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9546988","identity":"rs-9546988","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}