LPS-induced extracellular AREG triggers macrophages pyroptosis through EGFR/TLR4 signaling pathway

LPS-induced extracellular AREG triggers macrophages pyroptosis through EGFR/TLR4 signaling pathway | 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 LPS-induced extracellular AREG triggers macrophages pyroptosis through EGFR/TLR4 signaling pathway Gang Yuan, Qudi Qiao, Aolin Jiang, Zeihui Jiang, Haihua Luo, Lin Huang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5743694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Amphiregulin (AREG), as another EGF family member, is anchored to the cell surface as a transmembrane protein. In response to external stimulus, its extracellular domain can be release to extracellular matrix in a paracrine or autocrine manner. However, what it plays in septic macrophages pyroptosis remain poorly understood. The role of extracellular AREG was investigated in septic macrophages, mice as well as patients. Here, we found that AREG highly expressed in sepsis increased the expression of IL-6 protein and the expression of Caspase 1, IL-1β, Nlrp3 mRNA, resulting in macrophages pyroptosis. Mechanistically, macrophages pyroptosis was aggravated by extracellular AREG pretreatment and triggered by extracellular AREG and ATP (Adenosine 5'-triphosphate). The neutralizing antibody to AREG reduced LPS-induced EGFR activation, TLR4 expression and pyroptosis. Extracellular AREG-induced macrophages pyroptosis was decreased after applying inhibitions of EGFR and NF-κB as well as knockouts of TLR4 and Myd88. Besides, oxidative extracellular AREG promotes macrophages pyroptosis. In vivo studies reveal that extracellular AREG attenuates systemic inflammation infiltration and delays animal death in septic mouse model. Furthermore, serum AREG was associated with the immune inflammatory mediator, severity and mortality rate of septic patients, and genes of AREG-mediated pyroptosis signaling pathway were highly expressed in severe patients compared normal and general septic patients. Overall, extracellular AREG aggravated or triggered macrophages pyroptosis through EGFR/TLR4/Myd88/NF-κB signaling pathway, which provided promising treatment strategies for sepsis. Amphiregulin EGFR/TLR4 Macrophage Pyroptosis Sepsis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sepsis is a life-threatening pathophysiological condition response to infection, which is characterized by excessive inflammation and immunosuppression [1] . Sepsis caused by infection is still one of the most common diseases among critically ill patients, resulting in the high morbidity, mortality and treatment cost [2] .Therefore, further claifying the cellular and molecular mechanisms underlying this phenomenon is critical. After invading the body, pathogens can usually induce macrophages to secrete a variety of pro-inflammatory cytokines by binding with pattern recognition receptors [3] . These cytokines increase the activation of immune cells through autocrine and parocrine pathways, resulting in the imbalance of immune regulatory networks, and finally initiating cytokine storms [4] . Pyroptosis, also known as inflammatory necrosis of cells, is a newly discovered programmed cell death mode different from apoptosis [5] . In the canonical pyroptosis pathway, endogenous and exogenous stimuli can activate NOD-like receptor family pyrin domain-containing 3 (Nlrp3) inflammasome to control the cleavage and activation of pro-caspase1 [6] .Then, the activated Caspase1 further drives the cleaveage of gasdermin D (GSDMD) and release of its N-terminal fragment, resulting the formation of membrane pore, thereby promoting pyroptosis. Meanwhile, activated caspase-1 induces the maturation and releae of pro-inflammatory cytokines, including IL-1β and IL-18 [7] . Amphiregulin (AREG) belongs to members of the EGF-like family consisting of inactive, membrane-anchored precursor protein. Intracellular AREG funcitons mainly by regulating the cell cycle, proliferation and cytokinesis via yet-unknown mechanisms [8] .Alternatively, AREG processed with metalloproteinase can be released from cell surface to extracellular matrix or bind the EGFR on the surface of neighboring cells in the condition of inflammation, controlling the signaling from the EGFR-family of receptors [9,10] . AREG is significantly expressed in M1 classically polarized macrophages and exert a protective effect after LPS-induced acute lung injury [11, 12] . EGFR (epidermal growth factor receptor), as a transmembrane receptor tyrosine kinase has been considered to be vital to increase TLR4 cell surface expression and signal transduction in LPS-induced macrophages [13] . Other researcher demonstrated EGFR inhibitor Erlotinib protects against LPS-induced Endotoxicity because TLR4 needs EGFR to signal [14] . In addition, recent work has reported that EGFR promoting pyroptosis in intestinal ischemia reperfusion injury [15] .However, whether EGFR participate in macrophages pyroptosis by binding extracellular ligands is unclear. In this study we specifically elucidated the role of extracellular AREG in the LPS-induced macrophages and found extracellular AREG was highly expressed and can aggravate or trigger pyroptosis through EGFR/TLR4 pathway in macrophages. On the other hand, neutralizing LPS-induced extracellular AREG restricted EGFR/TLR4 pathway mediated pyroptosis in macrophages. We revealed the possibility that extracellular AREG exerts a protective effect on the body through pyroptosis in both mouse and human samples and also found genes of AREG-mediated pyroptosis signaling pathway were specifically highly expressed in severe patients. Thus, the regulation of pyroptosis by extracellular AREG may emerge as a potential therapeutic target for in sepsis. Materials And Methods Cell culture and treatment RAW264.7 macrophages were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). All C57BL/6 mice employed were male and 8–12 weeks old. WT C57BL/6 mice were purchased from was acquired from Jackson Laboratory (Bar Harbor, ME, USA). TLR4 -/- , RAGE -/- , TRIF -/- , Myd88 knock out mice were obtained from Kanazawa University (Kanazawa, Japan). These macrophages were cultured by the DMEM culture medium containing 10% fetal bovine serum at 37 °C incubator with 5% CO 2 . RAW264.7 or BMDM was seeded in 6 or 12-well culture plates. RAW264.7 was treated with extracellular AREG for different time point. To determine the effect of extracellular AREG on LPS-induced macrophages pyroptosis, BMDM was divided into four groups, (1) Control group: EGFP (100 ng/mL) stimulated BMDM for 30 min. (2) AREG group: extracellular AREG (100 ng/mL) stimulated BMDM for 30 min. (3) EGFP+LPS+ATP group: EGFP (100 ng/mL) stimulated BMDM for 30 min, followed by LPS (1 μg/mL) stimulation for 2 h, ATP (5 mM) stimulation for 30 min. (4) AREG+LPS+ATP group: extracellular AREG (100 ng/mL) stimulated BMDM for 30 min, followed by LPS (1 μg/mL) stimulation for 2 h, ATP (5 mM) stimulation for 30 min. Besides, BMDM was divided into another three groups, (1) Ctrl group (2) LPS group LPS (1 μg/mL) stimulation for 12 h (3) Anti-AREG+LPS group: neutralizing antibody of AREG (300 ng/mL, bio-techne) stimulated BMDM for 1 h, followed by LPS (1 μg/mL) stimulation for 12 h. (4) LPS+ATP group: LPS (1 μg/mL) stimulation for 2 h, followed by ATP (5 mM) stimulation for 30 min. (5) Anti-AREG+LPS+ATP group: neutralizing antibody of AREG (300 ng/mL) stimulated BMDM for 1 h, followed by LPS (1 μg/mL) stimulation for 2 h, ATP (5 mM) stimulation for 30 min. To determine the effect of extracellular AREG-induced macrophages pyroptosis, BMDM was divided into six groups, (1) Control group: EGFP (100 ng/mL) stimulated BMDM for 30 min. (2) AREG group: extracellular AREG (100 ng/mL) stimulated BMDM for 30min. (3) LPS group: LPS (1 μg/mL) stimulated BMDM for 2 h. (4) EGFP+ATP: EGFP (100 ng/mL) stimulated BMDM for 2.5 h, followed by ATP (5 mM) stimulation for 30 min. (5) AREG+ATP group: extracellular AREG (100 ng/mL) stimulated BMDM for 2.5 h, followed by ATP (5 mM) stimulation for 30 min. (6) LPS+ATP: LPS (1 μg/mL) stimulated BMDM for 2 h, followed by ATP (5 mM) stimulation for 30 min. EGFR inhibitior was obtained from Selleck, and NFκB inhibitior was obtained from MedChemExpress. Western blot BMDM was washed with dulbecco's phosphate buffered solution (DPBS) and lysed with RIPA (Thermo Scientific) on the ice for 30 min. The total protein was extracted to determinate protein concentrations by BCA assay (Thermo Scientific). A total of 20 μg protein was separated by on sodium dodecyl sulfate–polyacrylamide gels. and then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). After being blocked, the membrane was incubated with a primary antibody against rabbit monoclonal anti-EGFR (abclonal), rabbit monoclonal anti-p-EGFR (abclonal), rabbit polyclonal anti-TLR4 (immunoway), rabbit monoclonal anti-p-IκB, IκB, p-P65, P65, Nlrp3, Gapdh (CST), rabbit monoclonal anti-Caspase1 (AdipoGen), rabbit monoclonal anti-GSDMD (Abcam) at 4 ℃ for overnight. After incubation with a secondary antibody (Protein Tech), the protein bands on the membrane were subsequently visualized with enhanced ECL chemiluminescent substrate (Biosharp). Cell immunofluorescence BMDM was fixed with 4% paraformaldehyde and permeated with 0.2% Triton X-100 ,and blocked with 3% BSA at room temperature. After the reaction, the BMDM was incubated with an primary antibody anti-EGFR (abclonal), TLR4 (immunoway) or ASC (CST), and employed Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Scientific) or Alexa Fluor 594-conjugated anti-rabbit IgG (Thermo Scientific) to bind primary antibody for 1 h in the dark. The nucleus was dyed with 4′,6-Diamidino-2-phenylindole (DAPI). The immunofluorescence was visualized by Nikon confocal microscopy. Experimental data were assessed by function of the ZEN lite. qRT-PCR analysis Total RNA was extracted from RAW264.7 by using Trizol reagent (Thermo Scientific). RNA quantification using Nanodrop (Thermo Scientific), cDNA was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo). The qRT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems) employing SYBR Green PCR reagent kit (Dongsheng Biotech). The specific mouse primer sequences were as follows: Areg 5′-GCAGATACATCGAGAACCTGGAG-3′ and5′-CCTTGTCATCCTCGCTGTGAGT-3′; Nlrp3 5′-TCACAACTCGCCCAAGGAGGAA-3′and5′-AAGAGACCACGGCAGAAGCTAG-3′; Caspase1 5′-CTGGGACCCTCAAGTTTTGC-3′ and 5′-GGCAGGCAGCAAATTCTTTC-3′; Il-1b 5′-CCCAAGCAATACCCAAAGAA-3′ and 5′-GCTTGTGCTCTGCTTGTGAG-3′. The specific human primer sequences were as follows: Areg 5′-GCACCTGGAAGCAGTAACATGC-3′ and 5′-GGCAGCTATGGCTGCTAATGCA-3′; Egfr 5′-AACACCCTGGTCTGGAAGTACG-3′ and 5′-TCGTTGGACAGCCTTCAAGACC-3′; Il-1b 5′-CCACAGACCTTCCAGGAGAATG-3′ and 5′-GTGCAGTTCAGTGATCGTACAGG-3′; Il-18 5′-GATAGCCAGCCTAGAGGTATGG-3′ and 5′-CCTTGATGTTATCAGGAGGATTCA-3′.The expression of these genes was normalized with 18S ribosomal RNA by the relative CT value. ELISA AREG secretion in the cellular supernatant, sera of mouse or sepsis patients were quantitated with ELISA kit from R&D Systems. TNFα and Il-6 in the cellular supernatant were quantitated with ELISA kit from QuantiCyto. IL-1β and IL-18 were quantitated with ELISA kits from Elabscience. These levels of cytokines were detected according to the corresponding manufacturer’ protocols. The concentration of cytokines is calculated according the standard curve (Thermo Scientific). Purification of extracellular AREG In brief, the extracellular segment of AREG without the signal peptide , transmembrane and intracellular segments was constructed in His labeled pet14b vector by subclonal method.The recombinant expression plasmid pET14b-AREG was transformed into competent E.coli BL21 cells to obtain extracellular AREG protein, which was purified with chromatography on Ni-NTA Sepharose Column (Macherey-Nagel). The obtained recombinant protein were identified by SDS-PAGE, and the endotoxin of the recombinant extracellular AREG protein was removed by endotoxin removal gel (Thermo Scientific). Endotoxin assay kit (Pierce) was used to detect endotoxin concentration of extracellular AREG recombinant protein. Electron microscopy image The BMDM was collected into a centrifugal tube and fixed with 2.5% glutaraldehyde after the supernatant was discarded, in the condition of avoiding resuspension and shock. Incubation at room temperature for 1 hour, glutaraldehyde was discarded, DPBS was added and treated in the electron microscope room of the Central Laboratory of Southern Medical University. Isolation of human monocytes Monocytes from normal, general sepsis, and severe sepsis patients were isolated by a specific kit (tbd science, China) Statistical analysis The data were shown as mean ± standard deviation, and the statistical analysis was performed by GraphPad Prism software with more than repeated three times (GraphPad Software). Difference between two groups was analyzed using an unpaired two-tailed t test, considered to be statistically significant in the condition of p values less than 0.05. The restricted cubic splines (RCS) and segmented linear regression were performed using the R software (version 4.2.2), along with MSTATA software (www.mstata.com). Results 1. The dynamic expression of AREG in sepsis. Recent study elucidated the high expression of AREG in M1 classically polarized alveolar macrophages (AMs) and peritoneal macrophages (PMs) [12] .To systematically evaluate the AREG expression in sepsis, we detected AREG mRNA expression in 1, 3, 6, 12, 24 h after LPS stimulation in RAW264.7 by qRT-PCR analysis. AREG rapidly reached its peak at 6 h, and then was down-regulated (Figure1A). Meanwhile, to verify the results of AREG gene expression, we performed ELISA analysis to compared AREG protein expression in culture supernatant of RAW264.7. The AREG protein expression also almost reached its peak in 6 h after LPS stimulation (Figure1B). In addition, higher concentrations of LPS stimulation in RAW264.7 showed that AREG protein was more largely released (Figure1C). The significantly high expression of AREG protein was also detected in serum of septic mouse and BMDM by ELISA and Immunofluorescence analysis (Figure1D-E). 2. EGFR inhibiting and TLR4 silencing impairs extracellular AREG-induced IκB phosphorylation and subsequent NFκB activation in macrophages. EGF family components EGF and TGFα are known to induced activation of NFκB via EFGR and TLR4 [14, 16, 17] . To elucidate whether Areg also promotes NFκB activation, we firstly cloned soluble extracellular AREG without the signal peptide, transmembrane and intracellular segments (Figure2A, B). As shown in (Figure2C-E), extracellular AREG induced significant expressions of TLR4 and phosphorylated EGFR, IκB (inhibitory protein of NF-κB), NFκB P65 in BMDM by Western Blot and Immunofluorescence analysis. This phenomenon was suppressed by inhibition of EGFR kinase activity. As expected, the TLR4 knockout also inhibited the extracellular AREG-induced phosphorylation of IκB and P65 (Figure2F, G). Signaling to extracellular AREG-induced NFκB activation might also another membrane receptor. Ligand-activated RAGE increases inflammation by binding and activating EGFR [18] . However, we observed the ability of extracellular AREG to drive an increase in phosphorylated IκB and P65 is not limited after RAGE knockout (Figure2F, G). These results demonstrate that EGFR/TLR4 is vital for extracellular AREG-induced NFκB activation in macrophages 3. EGFR inhibiting and TLR4 silencing inhibit extracellular AREG-induced macrophages pyroptosis. Another member of the EGF family, TGFα, inhibits microglia pyroptosis in demyelinating diseases through the NF-κB pathway [19] .Therefore, it was worth determining whether extracellular AREG is involved in pyroptosis. We firstly observed that extracellular AREG can rapidly induce the secretion of IL-6 in RAW264.7 (Figure3A), interestingly, without affecting the secretion of TNFα (Not shown). Significant upregulation of mRNA expression of Nlrp3, Caspase1, and IL-1β were also observed (Figure3B). We then detected the effect of extracellular AREG on pyroptosis. As shown in Figure3C and D, treatment of LPS and ATP significantly increased the expression level of p-P65 and GSDMD-N and decreased the expression level of IκB and extracellular AREG pretreatment further up-regulated the expression of p-P65 and GSDMD-N to some extent, without affecting the degradation of IκB. These data indicated that extracellular AREG has an accelerative effect on pyroptosis via the NFκB activation in the LPS and ATP-stimulated inflammatory macrophages. Based on the above results, we speculated that extracellular AREG combined with ATP might induced macrophages pyroptosis via the NFκB signaling module. Pyroptosis can be mediated by the effector molecules GSDMD cleaved by Caspase1 [20] .To confirm our hypothesis, we subsequently used extracellular AREG and ATP to stimulate BMDM. The up-regulation of p-P65, and the down-regulation of IκB were detected in extracellular AREG and ATP-stimulated BMDM. It was also found that the expression of Nlrp3, GSDMD-N, Caspase1-p20 was promoted. However, extracellular AREG alone could not increase expression of Caspase1-p20, GSDMD-N (Figure3E, G). Consistent with the above results, extracellular AREG +ATP or LPS +ATP also contributed to the ASC speck formation in BMDM (Figure3H, I). These data implied that extracellular AREG can induce macrophages pyroptosis by promoting the activation of P65 and degradation of IκB. To demonstrate the involvement of EGFR/TLR4/NFκB pathways in the extracellular AREG as a 1 st signal induced macrophages pyroptosis (Figure3J). We firstly preprocessed extracellular AREG-induced BMDM with inhibitior of the EGFR kinase activity. As shown in Figure3K and L, inhibitior of the EGFR kinase activity decreased the expression of GSDMD-N and Caspase1-p20 in extracellular AREG-induced BMDM, where there was no significant difference on expression levels of Nlrp3 after preprocession of inhibitior EGFR kinase activity. As expected, extracellular AREG-induced the expression of p-P65, Nlrp3, GSDMD-N and Caspase1-p20, the degradation of IκB in BMDM were diminished by TLR4 depletion (Figure3M-O). Besides, we observed that TLR4 depletion in extracellular AREG-induced BMDM pyroptosis significantly reduced the secretion of IL-1β and IL-18 (Figure3P). 4. Neutralizing extracellular AREG decreases LPS-induced TLR4 expression and pyroptosis in macrophages. We next addressed whether extracellular AREG was involved in LPS-mediated activation of EGFR tyrosine kinase activity and expression of TLR4. We found that neutralizing antibody of AREG reduced expression of p-EGFR and TLR4 by West Blot and Immunofluorescence (Figure4A, C, E), which strongly suggested that the reduction in EGFR tyrosine kinase activity and TLR4 expression in LPS-induced macrophages is likely to be an explanation at least, in part, by reducing production of AREG. Therefore, we investigated whether extracellular AREG may influence LPS-induced macrophages pyroptosis. Expression of GSDMD-N, oligomerization of ASC and formation of pyrosome were significant reduced after applying neutralizing antibody of AREG in LPS-induced macrophages pyroptosis by West Blot , Immunofluorescence and Transmission Electron Microscope (Figure4B, D, F-H). These compelling evidence also manifested that extracellular AREG links the TLR4 pathway with activation of EGFR in macrophages pyroptosis. 5. Myd88 silencing and NFκB inhibiting restrain extracellular AREG-induced macrophages pyroptosis. TLR4 signalling activates translocation of the transcription factor NF-κB into the nucleus through the Myd88 and Trif-dependent pathway [21] . We further investigated the effect of TLR4 downstream signaling in extracellular AREG-induced macrophages pyroptosis by West Blot, the results showed that Myd88 knockout, but not Trif, significantly decreased the expression of Nlrp3, GSDMD-N and Caspase1-p20 in extracellular AREG-induced BMDM pyroptosis (Figure5A, B). We also subsequently found that the NFκB inhibitors down-regulated the expression of Nlrp3, GSDMD-N and Caspase1-p20 in extracellular AREG-induced or LPS-induced BMDM pyroptosis (Figure5C, D). 6. Oxidative extracellular AREG promotes macrophages pyroptosis. The inner ring structure of the disulfide bond in the member domain of the EGF family is the receptor binding region necessary for the composition of biological activity, and extracellular AREG is synthesized in membrane-bound form, which is proteolytic and then releases the soluble EGF domain including disulfide bond to play its role outside environment [22] . Therefore, we employed extracellular AREG or LPS pretreated with reducing agent (DTT) or oxidizing agent (H 2 O 2 ) to stimulate BMDM and then induced BMDM with ATP. The results showed LPS pretreated with DTT stimulated BMDM, and then ATP induced BMDM, significantly increased expression of GSDMD-N and Caspase1-p20, which obviously inhibited after LPS pretreated with H 2 O 2 stimulated BMDM, and then ATP induced BMDM (Figure6A, B). Interestingly, it was found that stimulation of extracellular AREG pretreated with DTT or H 2 O 2 , and then induction of ATP, down-regulated the expression of GSDMD-N and Caspase1-p20. In addition, neither DTT nor H 2 O 2 treatment of AREG or LPS affected the expression of Nlrp3 protein. In addition, DTT or H 2 O 2 treatment of AREG or LPS did not affect the expression of Nlrp3. Thus, these results confirmed that DTT may specifically oxidize the disulfide bond of AREG extracellular domain to inhibit AREG extracellular-induced macrophages pyroptosis. 7. Serum AREG mediates sepsis in mice and is associated with the severity and mortality rate of septic patients. To evaluate the significance of serum AREG in patients with sepsis, we first examined the expression of AREG in LPS-stimulated THP1 and septic patients. As expected, it was observed that AREG was highly expressed in LPS-stimulated THP1 culture supernant and the serum of septic patients (Figure7A). The most widely utilized and accepted model of sepsis is the cecal ligation and puncture (CLP), which is considered to have significant compatibility with human sepsis and suitable for potential mechanistic and therapies. Previous studies have reported that Areg can alleviate LPS-induced acute lung injury [12] . Based on this, we explored the effect of extracellular AREG on the progression of sepsis. The administration of extracellular AREG pretreatment lowered CLP-induced mortality (Figure7B). On the other hand, we conducted a focused study of 54 patients respectively diagnosed with general sepsis, severe sepsis, or septic shock, in which we employed restricted cubic splines (RCS) in the context of a clinical study to explore association of serum AREG level among concentration of serum CRP (C reaction protein), severity or mortality rate of septic patients. The analysis of RCS represented significant overall correlation among serum AREG lever, mortality rate or severity of septic patients. It could be seen that risk of exacerbation of sepsis alleviated with the increase of AREG concentration was observed (Figure7C). AREG concentration is less than 113 pg/mL, CRP level decreased with the increase of AREG concentration, and greater than 113 pg/mL, CRP level did not increase significantly with the increase of AREG concentration (Figure7D, E). In addition, when AREG concentration was below 64 pg/mL, the risk of death rapidly decreased with the increase of AREG concentration, but there was no significant change in the risk of death when AREG concentration was above 64 pg/mL (Figure7F, G). These findings provide further evidence to support the involvement of cellular AREG in the pathogenesis of septic patients. Histopathological analysis of lung, kidney, and liver indicated that inflammatory cell infiltration was inhibited following the administration of extracellular AREG (Figure7H, I). More importantly, we further explored the possibility of extracellular AREG promote the tissue restoration and survival of the body in sepsis through macrophages pyroptosis. We found the Areg, EGFR, Il1b and Il18 were highly expressed in monocytes of patients with severe sepsis compared normal and general septic patients (Figure7J). Discussion There is growing evidence indicates that the employment of LPS as an endotoxin model to explore the mechanisms of inflammatory response in various diseases, such as acute liver [23] or lung injury [24] , angiocardiopathy [25, 26] , as well as intestinal damage [27, 28] . Hui Liang et al. reported that LPS-primed BMDM could facilitate inflammation and oxidative stress, which contributed to accelerate acute lung injury [29] . In addition, LPS- primed BMDM serves as an in vitro cell model to induce lung inflammation and injury [30] . Pyroptosis is a gasdermin-mediated programmed cell death [31] . Although it is well known that pyroptosis plays crucial role in the innate immune defense, the regulation effects and molecular mechanisms of extracellular AREG on pyroptosis are remain unclear. In the present study, in vitro LPS or extracellular AREG-stimulated BMDM was employed to explore the regulatory mechanism of extracellular AREG in macrophages pyroptosis. Our research demonstrated that extracellular AREG exacerbates pyroptosis in LPS-treated macrophages. But to our surprise, we found that extracellular AREG combined with ATP induces macrophages pyroptosis via EGFR/TLR4/NFκB signal pathway. AREG is initially described as an epithelial cell-derived factor and mainly involved in cell proliferation, differentiation, apoptosis and autophagy in several diseases [32, 33] . On the other hand, AREG is also expressed on the surface of alveolar macrophages and peritoneal macrophages as a type I transmembrane protein precursor (proAR) [34-36] . When the body is stimulated by inflammatory mediators, extracellular AREG can be released into the extracellular matrix or bind to EGFR on the surface of neighboring cells and activate the EGFR signaling pathway [37] . As a critical intracellular nuclear transcription factor, NF-κB is mainly involved in inflammatory and immune response, regulation of cell death etc [38, 39] .NFκB can be activated by EGF/EGFR pathway in aggravation of the inflammatory process and cancer [40-42] . Therefore, understanding the causes of NFκB activation in sepsis is an important issue. Herein, we found that secretion of AREG, a member of the EGF family, was increased in LPS-stimulated RAW264.7, and then extracellular AREG induced IκB phosphorylation and subsequent NFκB activation in BMDM. We further show that inhibition of EGFR phosphorylation and knockout of TLR4 impairs extracellular AREG-induced NFκB activation in BMDM, and inhibition of EGFR phosphorylation also down-regulates the expression of TLR4. We revealed that there is a close connection between TLR4 and EGFR in extracellular AREG-induced NFκB activation in BMDM. As far as we know, this is the first report showing that extracellular AREG-induced NFκB activation through EGFR/TLR4 signaling. Consequently, inhibition EGFR phosphorylation and knockout of EGFR dramatically decreased in LPS-induced TLR4 phosphorylation at Y674A and Y680A [43] . Tyrosine phosphorylation of TLR4 is essential for downstream signal transduction, and the TLR4 mutants at Y674A and Y680A of TIR domain suppresses LPS-dependent activation of NFκB [44] . Therefore, it is deserved to further verify whether extracellular AREG can promote TLR4 tyrosine phosphorylation through combination with EGFR. In the process of determining the underlying effect of extracellular AREG on macrophages pyroptosis, we surprisingly found extracellular AREG pretreatment remarkably enhanced LPS+ATP-induced increased NFκB activation and macrophages pyroptosis, which indicated extracellular AREG promotes macrophages pyroptosis most likely through activation of NF-κB. Function of extracellular AREG is different with other EGFR ligands in pyroptosis, for which the explanation may be that EGFR exert different cellular responses through direct combination of its ligand, which depends on specific ligand or cell type and pathological condition [45] . Different from other EGFR ligands, AREG has a low affinity with EGFR, which contributes to continuously inducing downstream signal instead of leading to receptor internalization, degradation, and negative feedback loops [46] . Thus, we speculate that extracellular AREG incessantly activates NF-κB signal and then exacerbate macrophages pyroptosis. In addition, extracellular AREG rapidly induced the secretion of IL-6 in RAW264.7 without inducing the secretion of TNF-α. Interestingly, neither exogenous recombinant AREG nor intercepting endogenous secreted AREG could affect expression of TNF-α, IL-6 and GM-CSF in classically activated macrophages [14] .Similarly, some previous studies also reported that AREG plays a pro-inflammatory role by mediating the production of cytokines, including IL-6, IL-8, and GM-CSF in epithelial cell [47, 48] .We further confirmed that extracellular AREG alone upregulated transcription of Nlrp3, Caspase1 and IL-1b associated with pyroptosis initiation step, the addition of ATP further activates Nlrp3, Caspase1, and IL-1β, leading to the assembly of the Nlrp3 inflammasome and the activation of Caspase1, the cleavage of GSDMD and the release of the active amino terminal fragment of GSDMD, and ultimately resulting in pyroptosis, which provided a partial explanation that why extracellular AREG has the ability to induce macrophages pyroptosis. AREG signaling decided by the processing and trafficking of the protein can be triggered following manners: autocrine, juxtacrine, paracrine, by intracellular nuclear translocation, and inclusion in exosome [49-51] . The biological effects of extracellular AREG are also exhibited by EGFR-mediated introcellular signaling pathways, including Ras/MAPK, PI3K/AKT, mTOR, STAT and PLCγ, which are involved in the regulation of gene expression and elicit multiple cellular responses such as survival, proliferation, angiogenesis, motility and invasiveness [52-54] . It has also been reported that AREG played an important role in LPS-induced macrophages activation [37]. However, the effect and mechanism of AREG in LPS-induced macrophages pyroptosis is still unclear. Taken together, our date showed that extracellular AREG-induced macrophages pyroptosis through EGFR/TLR4/Myd88/NF-κB axis. Our study discovered association of AREG level of serum among CRP level, the mortality and severity of septic patients. These finding are consistent with a previous report that the expression of serum AREG was correlated with disease severity in pulmonary fibrosis patients [55] . Most patients with severe COVID-19 (78%) met sepsis 3.0 criteria, meaning sepsis with acute respiratory distress syndrome (ARDS) was the most common organ dysfunction (88%) [56] . Peripheral blood monocyte (PBMC) pyroptosis increases in patients with sepsis, and the degree of pyroptosis is related to the mortality of patients [57] . Monocytes are blood-resident phagocytes of bone marrow origin that are recruited and differentiate into macrophages during bacterial or viral senses to protect organisms from invading pathogens and help effectively eliminate inflammation [58] . Single-cell transcriptome analysis of monocytes from patients with COVID-19 found that the cell subset with high expression of Areg and IL-18 related to pyroptosis and enrichment of EGFR signaling pathway were specifically present in severely septic patients [59] .In addition, single-cell transcriptome analysis of antigen-presenting cells (including monocytes and a few dendritic cells) from COVID-19 patients also showed that Areg and IL1β associated with pyroptosis were highly expressed in the antigen-presenting cells of severely septic patients compared with normal objects and moderate septic patients [60] . These studies that have been reported further support our finding that genes of Areg-mediated pyroptosis signaling pathway were highly expressed in severe patients compared normal and general septic patients. Our study firstly reveals that the molecular mechanisms of extracellular AREG triggers macrophages pyroptosis through EGFR/TLR4/Myd88/NF-κB signaling pathway and underscores the possibility of sustained activation of AREG/EGFR signaling pathway-mediated pyroptosis can promote the tissue restoration and survival of the body in sepsis, which may involve in the release of inflammatory molecules and metabolites released during Areg-induced macrophages pyroptosis, which may be serve as a potential treatment strategy for septic patients with ARDS and provides a new way to better understand the pathogenesis of sepsis combined with ARDS. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University, Guangzhou, China (No.2020028) and was performed in accordance with the ethical standards of the responsible committee on human experimentation. Serum samples were was obtained from the septic patients and healthy donors in the study. Consent for publication All authors read and approved the final manuscript. Availability of data and Materials Not applicable. Statement of Competing Interest All authors declare that they have no competing interests. Funding This study was supported by grants from the National Natural Science Foundation of China (82130063 and 82241061), Guang Dong Basic and Applied Basic Research Foundation (2022B1515120024). Author’s Contributions Gang Yuan: conceptualization, validation, writing-original draft. Qudi Qiao, Aolin Jiang, Zeihui Jiang: validation. Haihua Luo: conceptualization, methodology. Lin Huang: clinical sample collection. Jieyan Wang: conceptualization. Acknowledgements We would like to acknowledge the service provided by School of Basic Medical Sciences, Southern Medical University. References Bullock B, Benham MD. Bacterial sepsis. 2024 . van der Poll T, Shankar-Hari M, Wiersinga WJ. 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Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells. J Biol Chem 2006 , 281(49): 37728-37737. Fang L, Sun YP, Cheng JC. The role of amphiregulin in ovarian function and disease. Cell Mol Life Sci 2023 , 80(3): 60. Raimondo S, Saieva L, Vicario E, Pucci M, Toscani D, Manno M, Raccosta S, Giuliani N, Alessandro R. Multiple myeloma-derived exosomes are enriched of amphiregulin (areg) and activate the epidermal growth factor pathway in the bone microenvironment leading to osteoclastogenesis. J Hematol Oncol 2019 , 12(1): 2. Berasain C, Castillo J, Perugorria MJ, Prieto J, Avila MA. Amphiregulin: a new growth factor in hepatocarcinogenesis. Cancer Lett 2007 , 254(1): 30-41. Busser B, Sancey L, Brambilla E, Coll JL, Hurbin A. The multiple roles of amphiregulin in human cancer. Biochim Biophys Acta 2011 , 1816(2): 119-131. Wang L, Wang L, Zhang H, Lu J, Zhang Z, Wu H, Liang Z. Areg mediates the epithelial‑mesenchymal transition in pancreatic cancer cells via the egfr/erk/nf‑kappab signalling pathway. Oncol Rep 2020 , 43(5): 1558-1568. Zhao R, Wang Z, Wang G, Geng J, Wu H, Liu X, Bin E, Sui J, Dai H, Tang N. Sustained amphiregulin expression in intermediate alveolar stem cells drives progressive fibrosis. Cell Stem Cell 2024 . Herminghaus A, Osuchowski MF. How sepsis parallels and differs from covid-19. EBioMedicine 2022 , 86: 104355. Zhao X, Xie J, Duan C, Wang L, Si Y, Liu S, Wang Q, Wu D, Wang Y, Yin W, Zhuang R, Li J. Adar1 protects pulmonary macrophages from sepsis-induced pyroptosis and lung injury through mir-21/a20 signaling. Int J Biol Sci 2024 , 20(2): 464-485. Knoll R, Schultze JL, Schulte-Schrepping J. Monocytes and macrophages in covid-19. Front Immunol 2021 , 12: 720109. Zhang Y, Wang S, Xia H, Guo J, He K, Huang C, Luo R, Chen Y, Xu K, Gao H, Sheng J, Li L. Identification of monocytes associated with severe covid-19 in the pbmcs of severely infected patients through single-cell transcriptome sequencing. Engineering (Beijing) 2022 , 17: 161-169. Saichi M, Ladjemi MZ, Korniotis S, Rousseau C, Ait HZ, Massenet-Regad L, Amblard E, Noel F, Marie Y, Bouteiller D, Medvedovic J, Pene F, Soumelis V. Single-cell rna sequencing of blood antigen-presenting cells in severe covid-19 reveals multi-process defects in antiviral immunity. Nat Cell Biol 2021 , 23(5): 538-551. Additional Declarations No competing interests reported. Supplementary Files Sourcedataforgelsandblots.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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cellular and molecular mechanisms underlying this phenomenon is critical.\u0026nbsp;After invading the body, pathogens can usually induce macrophages to secrete a variety of pro-inflammatory cytokines by binding with pattern recognition receptors\u003csup\u003e[3]\u003c/sup\u003e. These cytokines increase the activation of immune cells through autocrine and parocrine pathways, resulting in the imbalance of immune regulatory networks, and finally initiating cytokine storms\u003csup\u003e[4]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePyroptosis, also known as inflammatory necrosis of cells, is a newly discovered programmed cell death mode different from apoptosis\u003csup\u003e[5]\u003c/sup\u003e. In the canonical pyroptosis pathway, endogenous and exogenous stimuli can activate NOD-like receptor family pyrin domain-containing 3 (Nlrp3) inflammasome to control the cleavage and activation of pro-caspase1\u003csup\u003e[6]\u003c/sup\u003e.Then,\u0026nbsp;the activated Caspase1 further drives the cleaveage of gasdermin D (GSDMD) and release of its N-terminal fragment, resulting the formation of membrane pore, thereby promoting pyroptosis. Meanwhile, activated caspase-1 induces the maturation and releae of pro-inflammatory cytokines, including IL-1\u0026beta; and IL-18\u0026nbsp;\u003csup\u003e[7]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmphiregulin (AREG) belongs to members of the EGF-like family consisting of inactive, membrane-anchored precursor protein. Intracellular AREG funcitons mainly by regulating the cell cycle, proliferation and cytokinesis via yet-unknown mechanisms\u003csup\u003e[8]\u003c/sup\u003e.Alternatively, AREG processed with metalloproteinase can be released from cell surface to extracellular matrix or bind the EGFR on the surface of neighboring cells in the condition of inflammation, controlling the signaling from the EGFR-family of receptors\u003csup\u003e[9,10]\u003c/sup\u003e.\u0026nbsp;AREG is significantly expressed in M1 classically polarized macrophages and exert a protective effect after LPS-induced acute lung injury\u003csup\u003e[11, 12]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEGFR (epidermal growth factor receptor), as a transmembrane receptor tyrosine kinase has been considered to be vital to increase TLR4 cell surface expression and signal transduction in LPS-induced macrophages\u003csup\u003e[13]\u003c/sup\u003e. Other researcher demonstrated EGFR inhibitor Erlotinib protects against LPS-induced Endotoxicity because TLR4 needs EGFR to signal\u003csup\u003e[14]\u003c/sup\u003e. In addition, recent work has reported that EGFR promoting pyroptosis in intestinal ischemia reperfusion injury\u003csup\u003e[15]\u003c/sup\u003e.However, whether EGFR participate in macrophages pyroptosis by binding extracellular ligands is unclear.\u003c/p\u003e\n\u003cp\u003eIn this study we specifically elucidated the role of extracellular AREG in the LPS-induced macrophages and found extracellular AREG was highly expressed and can aggravate or trigger pyroptosis through EGFR/TLR4 pathway in macrophages. On the other hand, neutralizing LPS-induced extracellular AREG restricted EGFR/TLR4 pathway mediated pyroptosis in macrophages. We revealed the possibility that extracellular AREG exerts a protective effect on the body through pyroptosis in both mouse and human samples and also found genes of AREG-mediated pyroptosis signaling pathway were specifically highly expressed in severe patients. Thus, the regulation of pyroptosis by extracellular AREG may emerge as a potential therapeutic target for in sepsis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 macrophages were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).\u0026nbsp;All C57BL/6 mice employed were male and 8\u0026ndash;12 weeks old. WT C57BL/6 mice were purchased from was acquired from Jackson Laboratory (Bar Harbor, ME, USA). TLR4\u003csup\u003e-/-\u003c/sup\u003e, RAGE\u003csup\u003e-/-\u003c/sup\u003e, TRIF\u003csup\u003e-/-\u003c/sup\u003e, Myd88 knock out mice were obtained from Kanazawa University (Kanazawa, Japan). These macrophages were cultured by the DMEM culture medium containing 10% fetal bovine serum at 37 \u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. RAW264.7 or BMDM was seeded in 6 or 12-well culture plates. RAW264.7 was treated with extracellular AREG for different time point.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine the effect of extracellular AREG on LPS-induced macrophages pyroptosis, BMDM was divided into four groups, (1) Control group: EGFP (100 ng/mL) stimulated BMDM for 30 min. (2) AREG group: extracellular AREG (100 ng/mL) stimulated BMDM for 30 min. (3) EGFP+LPS+ATP group: EGFP (100 ng/mL) stimulated BMDM for 30 min, followed by LPS (1 \u0026mu;g/mL) stimulation for 2 h, ATP (5 mM) stimulation for 30 min. (4) AREG+LPS+ATP group: extracellular AREG (100 ng/mL) stimulated BMDM for 30 min, followed by LPS (1 \u0026mu;g/mL) stimulation for 2 h, ATP (5 mM) stimulation for 30 min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides, BMDM was divided into another three groups, (1) Ctrl group (2) LPS group LPS (1 \u0026mu;g/mL) stimulation for 12 h (3) Anti-AREG+LPS group: neutralizing antibody of AREG (300 ng/mL, bio-techne) stimulated BMDM for 1 h, followed by LPS (1 \u0026mu;g/mL) stimulation for 12 h. (4) LPS+ATP group: LPS (1 \u0026mu;g/mL) stimulation for 2 h, followed by ATP (5 mM) stimulation for 30 min. (5) Anti-AREG+LPS+ATP group: neutralizing antibody of AREG (300 ng/mL) stimulated BMDM for 1 h, followed by LPS (1 \u0026mu;g/mL) stimulation for 2 h, ATP (5 mM) stimulation for 30 min.\u003c/p\u003e\n\u003cp\u003eTo determine the effect of extracellular AREG-induced macrophages pyroptosis, BMDM was divided into six groups, (1) Control group: EGFP (100 ng/mL) stimulated BMDM for 30 min. (2) AREG group: extracellular AREG (100 ng/mL) stimulated BMDM for 30min. (3) LPS group: LPS (1 \u0026mu;g/mL) stimulated BMDM for 2 h. (4) EGFP+ATP: EGFP (100 ng/mL) stimulated BMDM for 2.5 h, followed by ATP (5 mM) stimulation for 30 min. (5) AREG+ATP group: extracellular AREG (100 ng/mL) stimulated BMDM for 2.5 h, followed by ATP (5 mM) stimulation for 30 min. (6) LPS+ATP: LPS (1 \u0026mu;g/mL) stimulated BMDM for 2 h, followed by ATP (5 mM) stimulation for 30 min. EGFR inhibitior was obtained from Selleck, and NF\u0026kappa;B inhibitior was obtained from MedChemExpress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDM was washed with dulbecco\u0026apos;s phosphate buffered solution (DPBS) and lysed with RIPA (Thermo Scientific) on the ice for 30 min. The total protein was extracted to determinate protein concentrations by BCA assay (Thermo Scientific).\u0026nbsp;A total of 20 \u0026mu;g protein was separated by on sodium dodecyl sulfate\u0026ndash;polyacrylamide gels. and then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). After being blocked, the membrane was incubated with a primary antibody against rabbit monoclonal anti-EGFR (abclonal),\u0026nbsp;rabbit monoclonal anti-p-EGFR (abclonal), rabbit polyclonal anti-TLR4 (immunoway), rabbit monoclonal anti-p-I\u0026kappa;B, I\u0026kappa;B, p-P65, P65, Nlrp3, Gapdh (CST),\u0026nbsp;rabbit monoclonal anti-Caspase1 (AdipoGen), rabbit monoclonal anti-GSDMD (Abcam) at 4 ℃ for overnight.\u0026nbsp;After incubation with a secondary antibody (Protein\u0026nbsp;Tech), the protein bands on the membrane were subsequently visualized with enhanced ECL chemiluminescent substrate (Biosharp).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDM was fixed with 4% paraformaldehyde and permeated with 0.2% Triton X-100 ,and blocked with 3% BSA at room temperature.\u0026nbsp;After the reaction, the BMDM was incubated with an primary antibody anti-EGFR (abclonal), TLR4 (immunoway) or ASC (CST), and employed Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Scientific) or Alexa Fluor 594-conjugated anti-rabbit IgG (Thermo Scientific) to bind primary antibody for 1 h in the dark.\u0026nbsp;The nucleus was dyed with 4\u0026prime;,6-Diamidino-2-phenylindole (DAPI).\u0026nbsp;The immunofluorescence was\u0026nbsp;visualized\u0026nbsp;by Nikon confocal microscopy.\u0026nbsp;Experimental data were assessed by function of the ZEN lite.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from RAW264.7 by using Trizol reagent (Thermo Scientific). RNA quantification using Nanodrop (Thermo Scientific), cDNA was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo).\u0026nbsp;The qRT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems) employing SYBR Green PCR reagent kit (Dongsheng Biotech).\u0026nbsp;The specific mouse primer sequences were as follows: \u003cem\u003eAreg\u0026nbsp;\u003c/em\u003e5\u0026prime;-GCAGATACATCGAGAACCTGGAG-3\u0026prime; and5\u0026prime;-CCTTGTCATCCTCGCTGTGAGT-3\u0026prime;; \u003cem\u003eNlrp3\u003c/em\u003e 5\u0026prime;-TCACAACTCGCCCAAGGAGGAA-3\u0026prime;and5\u0026prime;-AAGAGACCACGGCAGAAGCTAG-3\u0026prime;; \u003cem\u003eCaspase1\u003c/em\u003e 5\u0026prime;-CTGGGACCCTCAAGTTTTGC-3\u0026prime; and 5\u0026prime;-GGCAGGCAGCAAATTCTTTC-3\u0026prime;; \u003cem\u003eIl-1b\u003c/em\u003e 5\u0026prime;-CCCAAGCAATACCCAAAGAA-3\u0026prime; and 5\u0026prime;-GCTTGTGCTCTGCTTGTGAG-3\u0026prime;.\u0026nbsp;The specific human primer sequences were as follows: \u003cem\u003eAreg\u003c/em\u003e 5\u0026prime;-GCACCTGGAAGCAGTAACATGC-3\u0026prime; and 5\u0026prime;-GGCAGCTATGGCTGCTAATGCA-3\u0026prime;; \u003cem\u003eEgfr\u0026nbsp;\u003c/em\u003e5\u0026prime;-AACACCCTGGTCTGGAAGTACG-3\u0026prime; and 5\u0026prime;-TCGTTGGACAGCCTTCAAGACC-3\u0026prime;; \u003cem\u003eIl-1b\u003c/em\u003e 5\u0026prime;-CCACAGACCTTCCAGGAGAATG-3\u0026prime; and 5\u0026prime;-GTGCAGTTCAGTGATCGTACAGG-3\u0026prime;; \u003cem\u003eIl-18\u003c/em\u003e 5\u0026prime;-GATAGCCAGCCTAGAGGTATGG-3\u0026prime; and 5\u0026prime;-CCTTGATGTTATCAGGAGGATTCA-3\u0026prime;.The expression of these genes was normalized with 18S ribosomal RNA by the relative CT value.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAREG secretion in the cellular supernatant, sera of mouse or\u0026nbsp;sepsis patients\u0026nbsp;were quantitated with ELISA kit from R\u0026amp;D Systems. TNF\u0026alpha; and Il-6 in the cellular supernatant were quantitated with ELISA kit from QuantiCyto.\u0026nbsp;IL-1\u0026beta; and IL-18 were quantitated with ELISA kits from Elabscience.\u0026nbsp;These levels of cytokines were detected according to the corresponding manufacturer\u0026rsquo; protocols.\u0026nbsp;The concentration of cytokines is calculated according the standard curve (Thermo Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of extracellular AREG\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn brief, the extracellular segment of AREG without the signal peptide , transmembrane and intracellular segments was constructed in His labeled pet14b vector by subclonal method.The recombinant expression plasmid pET14b-AREG was transformed into competent E.coli BL21 cells to obtain extracellular AREG protein, which was purified with chromatography on Ni-NTA Sepharose Column (Macherey-Nagel).\u0026nbsp;The obtained recombinant protein were identified by SDS-PAGE, and the endotoxin of the recombinant extracellular AREG protein was removed by endotoxin removal gel (Thermo Scientific).\u0026nbsp;Endotoxin assay kit (Pierce) was used to detect endotoxin concentration of extracellular AREG recombinant protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectron microscopy image\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BMDM was collected into a centrifugal tube and fixed with 2.5% glutaraldehyde after the supernatant was discarded, in the condition of avoiding resuspension and shock. Incubation at room temperature for 1 hour, glutaraldehyde was discarded, DPBS was added and treated in the electron microscope room of the Central Laboratory of Southern Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of human monocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMonocytes from normal, general sepsis, and severe sepsis patients were isolated by a specific kit (tbd science, China)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were shown as mean \u0026plusmn; standard deviation, and the statistical analysis was performed by GraphPad Prism software with more than repeated three times (GraphPad Software). Difference between two groups was analyzed using an unpaired two-tailed t test, considered to be statistically significant in the condition of p values less than 0.05. The restricted cubic splines (RCS) and segmented linear regression were performed using the R software (version 4.2.2), along with MSTATA software (www.mstata.com).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. The dynamic expression of AREG in sepsis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecent study elucidated the high expression of AREG in M1 classically polarized alveolar macrophages (AMs) and peritoneal macrophages (PMs)\u003csup\u003e[12]\u003c/sup\u003e.To systematically evaluate the AREG expression in sepsis, we detected AREG mRNA expression in 1, 3, 6, 12, 24 h after LPS stimulation in RAW264.7 by qRT-PCR analysis. AREG rapidly reached its peak at 6 h, and then was down-regulated (Figure1A).\u0026nbsp;Meanwhile,\u0026nbsp;to verify the results of AREG gene expression, we performed ELISA analysis to compared AREG protein expression in culture supernatant of RAW264.7.\u0026nbsp;The\u0026nbsp;AREG protein expression also almost reached its peak in 6 h after LPS stimulation (Figure1B).\u0026nbsp;In addition, higher concentrations of LPS stimulation in RAW264.7 showed that AREG protein was more largely released (Figure1C).\u0026nbsp;The significantly high expression of AREG protein was also detected in serum of septic mouse and BMDM by ELISA and Immunofluorescence analysis (Figure1D-E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. EGFR inhibiting and TLR4 silencing impairs extracellular AREG-induced I\u0026kappa;B phosphorylation and subsequent NF\u0026kappa;B activation in macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEGF family components EGF and TGF\u0026alpha; are known to induced activation of NF\u0026kappa;B via EFGR and TLR4\u003csup\u003e[14, 16, 17]\u003c/sup\u003e.\u0026nbsp;To elucidate whether Areg also promotes NF\u0026kappa;B activation, we firstly cloned soluble extracellular AREG without the signal peptide, transmembrane and intracellular segments (Figure2A, B).\u0026nbsp;As shown in (Figure2C-E), extracellular AREG induced significant expressions of TLR4 and phosphorylated EGFR, I\u0026kappa;B (inhibitory protein of NF-\u0026kappa;B), NF\u0026kappa;B P65 in BMDM by Western Blot and Immunofluorescence analysis. This phenomenon was suppressed by inhibition of EGFR kinase activity. As expected, the TLR4 knockout also inhibited the extracellular AREG-induced phosphorylation of I\u0026kappa;B and P65 (Figure2F, G).\u0026nbsp;Signaling to extracellular AREG-induced NF\u0026kappa;B activation might also another membrane receptor.\u0026nbsp;Ligand-activated RAGE increases inflammation by binding and activating EGFR\u003csup\u003e[18]\u003c/sup\u003e. However, we observed the ability of extracellular AREG to drive an increase in phosphorylated I\u0026kappa;B and P65 is not limited after RAGE knockout (Figure2F, G).\u0026nbsp;These results demonstrate that EGFR/TLR4 is vital for extracellular AREG-induced NF\u0026kappa;B activation in macrophages\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. EGFR inhibiting and TLR4 silencing inhibit extracellular AREG-induced macrophages pyroptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnother member of the EGF family, TGF\u0026alpha;, inhibits microglia pyroptosis in demyelinating diseases through the NF-\u0026kappa;B pathway\u003csup\u003e[19]\u003c/sup\u003e.Therefore, it was worth determining whether extracellular AREG is involved in pyroptosis. We firstly observed that extracellular AREG can rapidly induce the secretion of IL-6 in RAW264.7 (Figure3A), interestingly, without affecting the secretion of TNF\u0026alpha; (Not shown).\u0026nbsp;Significant upregulation of mRNA expression of Nlrp3, Caspase1, and IL-1\u0026beta; were also observed (Figure3B).\u0026nbsp;We then detected the effect of extracellular AREG on pyroptosis. As shown in Figure3C and D,\u0026nbsp;treatment of LPS and ATP significantly increased the expression level of p-P65 and GSDMD-N and decreased the expression level of I\u0026kappa;B and extracellular AREG pretreatment further up-regulated the expression of p-P65 and GSDMD-N to some extent,\u0026nbsp;without affecting the degradation of I\u0026kappa;B. These data indicated that extracellular AREG has an accelerative effect on pyroptosis via the NF\u0026kappa;B activation in the LPS and ATP-stimulated inflammatory macrophages.\u003c/p\u003e\n\u003cp\u003eBased on the above results, we speculated that extracellular AREG combined with ATP might induced macrophages pyroptosis via the NF\u0026kappa;B signaling module. Pyroptosis can be mediated by the effector molecules GSDMD cleaved by Caspase1\u003csup\u003e[20]\u003c/sup\u003e.To confirm our hypothesis,\u0026nbsp;we subsequently used extracellular AREG and ATP to stimulate BMDM. The up-regulation of p-P65, and the down-regulation of I\u0026kappa;B were detected in extracellular AREG and ATP-stimulated BMDM. It was also found that the expression of Nlrp3, GSDMD-N, Caspase1-p20 was promoted. However, extracellular AREG alone could not increase expression of Caspase1-p20, GSDMD-N (Figure3E, G). Consistent with the above results, extracellular AREG +ATP or LPS +ATP also contributed to the ASC speck formation in BMDM (Figure3H, I).\u0026nbsp;These data implied that extracellular AREG can induce macrophages pyroptosis by promoting the activation of P65 and degradation of I\u0026kappa;B.\u003c/p\u003e\n\u003cp\u003eTo demonstrate the involvement of EGFR/TLR4/NF\u0026kappa;B pathways in the extracellular AREG as a 1\u003csup\u003est\u003c/sup\u003e signal induced macrophages pyroptosis (Figure3J). We firstly preprocessed extracellular AREG-induced BMDM with inhibitior of the EGFR kinase activity.\u0026nbsp;As shown in Figure3K and L, inhibitior of the EGFR kinase activity decreased the expression of GSDMD-N and Caspase1-p20 in extracellular AREG-induced BMDM, where there was no significant difference on expression levels of Nlrp3 after preprocession of inhibitior EGFR kinase activity.\u0026nbsp;As expected, extracellular AREG-induced the expression of p-P65, Nlrp3, GSDMD-N and Caspase1-p20, the degradation of I\u0026kappa;B in BMDM were diminished by TLR4 depletion (Figure3M-O). Besides, we observed that TLR4 depletion in extracellular AREG-induced BMDM pyroptosis significantly reduced the secretion of IL-1\u0026beta; and IL-18 (Figure3P).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Neutralizing extracellular AREG decreases LPS-induced TLR4 expression and pyroptosis in macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next addressed whether extracellular AREG was involved in LPS-mediated activation of EGFR tyrosine kinase activity and expression of TLR4.\u0026nbsp;We found that neutralizing antibody of AREG reduced expression of p-EGFR and TLR4 by West Blot and Immunofluorescence (Figure4A, C, E), which strongly suggested that the reduction in EGFR tyrosine kinase activity and TLR4 expression in LPS-induced macrophages is likely to be an explanation at least, in part, by reducing production of AREG. Therefore, we investigated whether extracellular AREG may influence LPS-induced macrophages pyroptosis. Expression of GSDMD-N, oligomerization of ASC and formation of pyrosome were significant reduced after applying neutralizing antibody of AREG in LPS-induced macrophages pyroptosis by West Blot , Immunofluorescence and Transmission Electron Microscope (Figure4B, D, F-H).\u0026nbsp;These compelling evidence also manifested that extracellular AREG links the TLR4 pathway with activation of EGFR in macrophages pyroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Myd88 silencing and NF\u0026kappa;B inhibiting restrain extracellular AREG-induced macrophages pyroptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTLR4 signalling activates translocation of the transcription factor NF-\u0026kappa;B into the nucleus through the Myd88 and Trif-dependent pathway\u003csup\u003e[21]\u003c/sup\u003e.\u0026nbsp;We further investigated the effect of TLR4 downstream signaling in extracellular AREG-induced macrophages pyroptosis by West Blot,\u0026nbsp;the results showed that Myd88 knockout, but not Trif, significantly decreased the expression of Nlrp3, GSDMD-N and Caspase1-p20 in extracellular AREG-induced BMDM pyroptosis (Figure5A, B). We also subsequently found that the NF\u0026kappa;B inhibitors down-regulated the expression of Nlrp3, GSDMD-N and Caspase1-p20 in extracellular AREG-induced or LPS-induced BMDM pyroptosis (Figure5C, D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Oxidative extracellular AREG promotes macrophages pyroptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inner ring structure of the disulfide bond in the member domain of the EGF family is the receptor binding region necessary for the composition of biological activity, and extracellular AREG is synthesized in membrane-bound form, which is proteolytic and then releases the soluble EGF domain including disulfide bond to play its role outside environment\u003csup\u003e[22]\u003c/sup\u003e.\u0026nbsp;Therefore, we employed extracellular AREG or LPS pretreated with reducing agent (DTT) or oxidizing agent (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to stimulate BMDM and then induced BMDM with ATP.\u0026nbsp;The results showed LPS pretreated with DTT stimulated BMDM, and then ATP induced BMDM, significantly increased expression of GSDMD-N and Caspase1-p20, which obviously inhibited after LPS pretreated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulated BMDM, and then ATP induced BMDM (Figure6A, B).\u0026nbsp;Interestingly, it was found that stimulation of extracellular AREG pretreated with DTT or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and then induction of ATP, down-regulated the expression of GSDMD-N and Caspase1-p20.\u0026nbsp;In addition, neither DTT nor H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment of AREG or LPS affected the expression of Nlrp3 protein.\u0026nbsp;In addition, DTT or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment of AREG or LPS did not affect the expression of Nlrp3.\u0026nbsp;Thus, these results confirmed that DTT may specifically oxidize the disulfide bond of AREG extracellular domain to inhibit AREG extracellular-induced macrophages pyroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Serum AREG mediates sepsis in mice and is associated with the severity and mortality rate of septic patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the significance of serum AREG in patients with sepsis, we first examined the expression of AREG in LPS-stimulated THP1 and septic patients. As expected, it was observed that AREG was highly expressed in LPS-stimulated THP1 culture supernant and the serum of septic patients (Figure7A). The most widely utilized and accepted model of sepsis is the cecal ligation and puncture (CLP), which is considered to have significant compatibility with human sepsis and suitable for potential mechanistic and therapies.\u0026nbsp;Previous studies have reported that Areg can alleviate LPS-induced acute lung injury\u003csup\u003e[12]\u003c/sup\u003e.\u0026nbsp;Based on this, we explored the effect of extracellular AREG on the progression of sepsis. The administration of extracellular AREG pretreatment lowered CLP-induced mortality (Figure7B). On the other hand, we conducted a focused study of 54 patients respectively diagnosed with general sepsis, severe sepsis, or septic shock, in which we employed restricted cubic splines (RCS) in the context of a clinical study to explore association of serum AREG level among concentration of serum CRP (C reaction protein), severity or mortality rate of septic patients.\u003c/p\u003e\n\u003cp\u003eThe analysis of RCS represented significant overall correlation among serum AREG lever, mortality rate or severity of septic patients. It could be seen that risk of exacerbation of sepsis alleviated with the increase of AREG concentration was observed (Figure7C). AREG concentration is less than 113 pg/mL, CRP level decreased with the increase of AREG concentration, and greater than 113 pg/mL, CRP level did not increase significantly with the increase of AREG concentration (Figure7D, E).\u0026nbsp;In addition, when AREG concentration was below 64 pg/mL, the risk of death rapidly decreased with the increase of AREG concentration, but there was no significant change in the risk of death when AREG concentration was above 64 pg/mL (Figure7F, G). These findings provide further evidence to support the involvement of cellular AREG in the pathogenesis of septic patients. Histopathological analysis of lung, kidney, and liver indicated that inflammatory cell infiltration was inhibited following the administration of extracellular AREG (Figure7H, I). More importantly,\u0026nbsp;we further explored the possibility of extracellular AREG promote the tissue restoration and survival of the body in sepsis through macrophages pyroptosis. We found the\u0026nbsp;\u003cem\u003eAreg, EGFR, Il1b and Il18\u003c/em\u003e were highly expressed in monocytes of patients with severe sepsis compared normal and general septic patients (Figure7J).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is growing evidence indicates that the employment of LPS as an endotoxin model to explore the mechanisms of inflammatory response in various diseases, such as acute liver\u003csup\u003e[23]\u003c/sup\u003e or lung injury\u003csup\u003e[24]\u003c/sup\u003e,\u0026nbsp;angiocardiopathy\u003csup\u003e[25, 26]\u003c/sup\u003e, as well as intestinal damage\u0026nbsp;\u003csup\u003e[27, 28]\u003c/sup\u003e.\u0026nbsp;Hui Liang et al. reported that LPS-primed BMDM could facilitate inflammation and oxidative stress, which contributed to accelerate acute lung injury\u003csup\u003e[29]\u003c/sup\u003e.\u0026nbsp;In addition, LPS- primed BMDM serves as an in vitro cell model to induce lung inflammation and injury\u003csup\u003e[30]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePyroptosis is a gasdermin-mediated programmed cell death\u003csup\u003e[31]\u003c/sup\u003e. Although it is well known that pyroptosis plays crucial role in the innate immune defense, the regulation effects and molecular mechanisms of extracellular AREG on pyroptosis are remain unclear.\u0026nbsp;In the present study,\u0026nbsp;in vitro LPS or extracellular AREG-stimulated BMDM was employed to explore the regulatory mechanism of extracellular AREG in macrophages pyroptosis. Our research demonstrated that extracellular AREG exacerbates pyroptosis in LPS-treated macrophages. But to our surprise, we found that extracellular AREG combined with ATP induces macrophages pyroptosis via EGFR/TLR4/NF\u0026kappa;B signal pathway.\u003c/p\u003e\n\u003cp\u003eAREG is initially described as an epithelial cell-derived factor and mainly involved in cell proliferation, differentiation,\u0026nbsp;apoptosis and autophagy in several diseases\u0026nbsp;\u003csup\u003e[32, 33]\u003c/sup\u003e.\u0026nbsp;On the other hand, AREG is also expressed on the surface of alveolar macrophages and peritoneal macrophages as a type I transmembrane protein precursor (proAR)\u003csup\u003e[34-36]\u003c/sup\u003e.\u0026nbsp;When the body is stimulated by inflammatory mediators, extracellular AREG can be released into the extracellular matrix or bind to EGFR on the surface of neighboring cells and activate the EGFR signaling pathway\u003csup\u003e[37]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs a critical intracellular nuclear transcription factor, NF-\u0026kappa;B is mainly involved in inflammatory and immune response, regulation of cell death etc\u003csup\u003e[38, 39]\u003c/sup\u003e.NF\u0026kappa;B can be activated by EGF/EGFR pathway in aggravation of the inflammatory process and cancer\u003csup\u003e[40-42]\u003c/sup\u003e. Therefore, understanding the causes of NF\u0026kappa;B activation in sepsis is an important issue. Herein, we found that\u0026nbsp;secretion of AREG, a member of the EGF family, was increased in LPS-stimulated RAW264.7, and then extracellular AREG induced I\u0026kappa;B phosphorylation and subsequent NF\u0026kappa;B activation in BMDM.\u0026nbsp;We further show that inhibition of EGFR phosphorylation and knockout of TLR4 impairs extracellular AREG-induced NF\u0026kappa;B activation in BMDM, and inhibition of EGFR phosphorylation also down-regulates the expression of TLR4. We revealed that there is a close connection between TLR4 and EGFR in extracellular AREG-induced NF\u0026kappa;B activation in BMDM.\u0026nbsp;As far as we know, this is the first report showing that extracellular AREG-induced NF\u0026kappa;B activation through EGFR/TLR4 signaling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsequently, inhibition EGFR phosphorylation and knockout of EGFR dramatically decreased in LPS-induced TLR4 phosphorylation at Y674A and Y680A\u003csup\u003e[43]\u003c/sup\u003e.\u0026nbsp;Tyrosine phosphorylation of TLR4 is essential for downstream signal transduction, and the TLR4 mutants at Y674A and Y680A of TIR domain suppresses LPS-dependent activation of NF\u0026kappa;B\u003csup\u003e[44]\u003c/sup\u003e.\u0026nbsp;Therefore, it is deserved to further verify whether extracellular AREG can promote TLR4 tyrosine phosphorylation through combination with EGFR.\u003c/p\u003e\n\u003cp\u003eIn the process of determining the underlying effect of extracellular AREG on macrophages pyroptosis, we surprisingly found extracellular AREG pretreatment remarkably enhanced LPS+ATP-induced increased NF\u0026kappa;B activation and macrophages pyroptosis, which indicated extracellular AREG promotes macrophages pyroptosis most likely through activation of NF-\u0026kappa;B. Function of extracellular AREG is different with other EGFR ligands in pyroptosis, for which the explanation may be that EGFR exert different cellular responses through direct combination of its ligand, which depends on specific ligand or cell type and pathological condition\u003csup\u003e[45]\u003c/sup\u003e.\u0026nbsp;Different from\u0026nbsp;other EGFR ligands, AREG has a low affinity with EGFR, which contributes to continuously inducing downstream signal instead of leading to receptor internalization, degradation, and negative feedback loops\u003csup\u003e[46]\u003c/sup\u003e. Thus, we speculate that extracellular AREG incessantly activates NF-\u0026kappa;B signal and then exacerbate macrophages pyroptosis.\u003c/p\u003e\n\u003cp\u003eIn addition, extracellular AREG rapidly induced the secretion of IL-6 in RAW264.7 without inducing the secretion of TNF-\u0026alpha;.\u0026nbsp;Interestingly, neither exogenous recombinant AREG nor intercepting endogenous secreted AREG could affect expression of TNF-\u0026alpha;, IL-6 and GM-CSF in classically activated macrophages\u003csup\u003e[14]\u003c/sup\u003e.Similarly, some previous studies also reported that AREG plays a pro-inflammatory role by mediating the production of cytokines, including IL-6, IL-8, and GM-CSF in epithelial cell\u0026nbsp;\u003csup\u003e[47, 48]\u003c/sup\u003e.We further confirmed that extracellular AREG alone upregulated transcription of Nlrp3, Caspase1 and IL-1b associated with pyroptosis initiation step, the addition of ATP further activates Nlrp3, Caspase1, and IL-1\u0026beta;, leading to the assembly of the Nlrp3 inflammasome and the activation of Caspase1, the cleavage of GSDMD and the release of the active amino terminal fragment of GSDMD, and ultimately resulting in pyroptosis, which provided a partial explanation that why extracellular AREG has the ability to induce macrophages pyroptosis.\u003c/p\u003e\n\u003cp\u003eAREG signaling decided by the processing and trafficking of the protein can be triggered following manners: autocrine, juxtacrine, paracrine, by intracellular nuclear translocation, and inclusion in exosome\u003csup\u003e[49-51]\u003c/sup\u003e. The biological effects of extracellular AREG are also exhibited by EGFR-mediated introcellular signaling pathways, including Ras/MAPK, PI3K/AKT, mTOR, STAT and PLC\u0026gamma;, which are involved in the regulation of gene expression and elicit multiple cellular responses such as survival, proliferation, angiogenesis, motility and invasiveness\u003csup\u003e[52-54]\u003c/sup\u003e.\u0026nbsp;It has also been reported that AREG played an important role in LPS-induced macrophages activation\u003csup\u003e[37].\u003c/sup\u003e However, the effect and mechanism of AREG in LPS-induced macrophages pyroptosis is still unclear. Taken together, our date showed that extracellular AREG-induced macrophages pyroptosis through EGFR/TLR4/Myd88/NF-\u0026kappa;B axis. Our study discovered association of AREG level of serum among CRP level, the mortality and severity of septic patients.\u0026nbsp;These finding are consistent with a previous report that the expression of serum AREG was correlated with disease severity in pulmonary fibrosis patients\u003csup\u003e[55]\u003c/sup\u003e.\u0026nbsp;Most patients with severe COVID-19 (78%) met sepsis 3.0 criteria, meaning sepsis with acute respiratory distress syndrome (ARDS) was the most common organ dysfunction (88%)\u003csup\u003e[56]\u003c/sup\u003e.\u0026nbsp;Peripheral blood monocyte (PBMC) pyroptosis increases in patients with sepsis, and the degree of pyroptosis is related to the mortality of patients\u003csup\u003e[57]\u003c/sup\u003e.\u0026nbsp;Monocytes are blood-resident phagocytes of bone marrow origin that are recruited and differentiate into macrophages during bacterial or viral senses to protect organisms from invading pathogens and help effectively eliminate inflammation\u003csup\u003e[58]\u003c/sup\u003e.\u0026nbsp;Single-cell transcriptome analysis of monocytes from patients with COVID-19 found that the cell subset with high expression of \u003cem\u003eAreg\u003c/em\u003e and IL-18 related to pyroptosis and enrichment of EGFR signaling pathway were specifically present in severely septic patients\u003csup\u003e[59]\u003c/sup\u003e.In addition,\u0026nbsp;single-cell transcriptome analysis of antigen-presenting cells (including monocytes and a few dendritic cells) from COVID-19 patients also showed that \u003cem\u003eAreg\u003c/em\u003e and IL1\u0026beta; associated with pyroptosis were highly expressed in the antigen-presenting cells of severely septic patients compared with normal objects and moderate septic patients\u003csup\u003e[60]\u003c/sup\u003e. These studies that have been reported further support our finding that\u003c/p\u003e\n\u003cp\u003egenes of Areg-mediated pyroptosis signaling pathway were highly expressed in severe patients compared normal and general septic patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study firstly reveals that the molecular mechanisms of extracellular AREG triggers macrophages pyroptosis through EGFR/TLR4/Myd88/NF-\u0026kappa;B signaling pathway and underscores the possibility of sustained activation of AREG/EGFR signaling pathway-mediated pyroptosis can promote the tissue restoration and survival of the body in sepsis, which may involve in the release of inflammatory molecules and metabolites released during Areg-induced macrophages pyroptosis, which may be serve as a potential treatment strategy for septic patients with ARDS and provides a new way to better understand the pathogenesis of sepsis combined with ARDS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University, Guangzhou, China (No.2020028) and was performed in accordance with the ethical standards of the responsible committee on human experimentation. Serum samples were was obtained from the septic patients and healthy donors in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (82130063 and 82241061), Guang Dong Basic and Applied Basic Research Foundation (2022B1515120024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGang Yuan: conceptualization, validation, writing-original draft. Qudi Qiao, Aolin Jiang, Zeihui Jiang: validation. Haihua Luo: conceptualization, methodology. Lin Huang: clinical sample collection. Jieyan Wang: conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the service provided by School of Basic Medical Sciences, Southern Medical University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBullock B, Benham MD. Bacterial sepsis. \u003cstrong\u003e2024\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003evan der Poll T, Shankar-Hari M, Wiersinga WJ. The immunology of sepsis. \u003cstrong\u003eImmunity\u003c/strong\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 54(11): 2450-2464.\u003c/li\u003e\n\u003cli\u003eKany S, Vollrath JT, Relja B. 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In response to external stimulus, its extracellular domain can be release to extracellular matrix in a paracrine or autocrine manner. However, what it plays in septic macrophages pyroptosis remain poorly understood. The role of extracellular AREG was investigated in septic macrophages, mice as well as patients. Here, we found that AREG highly expressed in sepsis increased the expression of IL-6 protein and the expression of Caspase 1, IL-1β, Nlrp3 mRNA, resulting in macrophages pyroptosis. Mechanistically, macrophages pyroptosis was aggravated by extracellular AREG pretreatment and triggered by extracellular AREG and ATP (Adenosine 5'-triphosphate). The neutralizing antibody to AREG reduced LPS-induced EGFR activation, TLR4 expression and pyroptosis. Extracellular AREG-induced macrophages pyroptosis was decreased after applying inhibitions of EGFR and NF-κB as well as knockouts of TLR4 and Myd88. Besides, oxidative extracellular AREG promotes macrophages pyroptosis. In vivo studies reveal that extracellular AREG attenuates systemic inflammation infiltration and delays animal death in septic mouse model. Furthermore, serum AREG was associated with the immune inflammatory mediator, severity and mortality rate of septic patients, and genes of AREG-mediated pyroptosis signaling pathway were highly expressed in severe patients compared normal and general septic patients. Overall, extracellular AREG aggravated or triggered macrophages pyroptosis through EGFR/TLR4/Myd88/NF-κB signaling pathway, which provided promising treatment strategies for sepsis.\u003c/p\u003e","manuscriptTitle":"LPS-induced extracellular AREG triggers macrophages pyroptosis through EGFR/TLR4 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-06 13:43:47","doi":"10.21203/rs.3.rs-5743694/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f2dddedf-8f7b-4f27-ae5f-700f42086bf5","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-09T16:38:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-06 13:43:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5743694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5743694","identity":"rs-5743694","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}