IFIT3 stabilizes STING via USP18 to drive M1 macrophage polarization and early inflammation in acute lung injury

IFIT3 stabilizes STING via USP18 to drive M1 macrophage polarization and early inflammation in acute lung injury | 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 IFIT3 stabilizes STING via USP18 to drive M1 macrophage polarization and early inflammation in acute lung injury Nana Tang, Yang Yang, Yuanyuan Zeng, Jianjie Zhu, Jianjun Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6695431/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract The imbalance in macrophage M1/M2 polarization is a critical factor driving excessive inflammation during the early stages of Acute Lung Injury (ALI) /Acute Respiratory Distress Syndrome (ARDS). However, the underlying regulatory mechanisms remain poorly understood. In this study, we investigated the role of interferon-inducible protein with tetrapeptide repeats 3 (IFIT3) in the context of early-stage ALI. Our findings demonstrate that IFIT3 expression is significantly elevated in macrophages of ALI mice. We further show that IFIT3 positively regulates the cGAS-STING pathway, which promotes M1 polarization and exacerbates lung inflammation in ALI. Additionally, IFIT3 interacts with STING to inhibit its ubiquitination-mediated degradation, potentially acting as a bridging molecule facilitating the interaction between STING and the deubiquitinase USP18. These results highlight IFIT3 as a crucial player in the pathogenesis of ALI/ARDS through the modulation of macrophage polarization, suggesting that targeting IFIT3 may offer a novel therapeutic strategy for managing ALI /ARDS. IFIT3 STING Acute lung injury deubiquitinase M1 macrophage polarization USP18 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) is an acute diffuse inflammatory lung injury caused by various factors [ 1 ]. First described in 1967[ 2 ], this clinical syndrome has persisted for nearly 60 years, with an in-hospital mortality rate in ARDS patients still exceeding 40% [ 3 ], posing a significant threat to human health. Macrophages, highly plastic cells, play a pivotal role throughout the pathogenesis of ARDS[ 4 , 5 ]. During the early stages of ALI/ARDS, there is an imbalance in the proportion of two primary phenotypes of pulmonary tissue macrophages, namely the classically activated (M1) and alternatively activated (M2) phenotypes. Increased M1 macrophages lead to excessive production of proinflammatory mediators and recruitment of inflammatory cells, exacerbating pulmonary inflammation and injury[ 6 , 7 ]. However, the precise mechanisms governing the dysregulation of macrophage polarization remain incompletely understood. Investigating critical molecules involved in the early regulation of lung macrophage polarization in ALI/ARDS may elucidate the pathogenesis of the disease and provide a basis for exploring new diagnostic and therapeutic targets. Interferon-inducible protein with tetrapeptide repeats (IFIT), a member of the interferon-stimulated gene (ISG) family, with IFIT3 being a crucial member [ 8 ]. Under physiological conditions, IFIT3 is expressed at low levels in the majority of cell types, but its expression is significantly upregulated upon stimulation by viruses, bacteria, chlamydia, and interferons [ 9 – 11 ]. Recent studies have identified IFIT3 as a crucial immunoregulatory molecule involved not only in antiviral innate immunity but also in cellular functions such as proliferation, differentiation, apoptosis, and critical roles in the development of diseases like COVID-19, autoimmune disorders, and cancer[ 12 – 14 ]. However, the role of IFIT3 in the pathogenesis of ALI/ARDS remains unclear, with limited literature predominantly comprising bioinformatics analyses[ 7 , 15 ]. Currently, there is a lack of reports elucidating the function of IFIT3 in the early inflammatory response and the polarization mechanisms of pulmonary macrophages in ALI/ARDS. In this study, we found that IFIT3 is significantly upregulated in mouse lung tissue during the early phase of ALI. Silencing IFIT3 expression alleviates the M1 polarization of pulmonary macrophages and mitigates inflammatory lung injury. Under stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ), IFIT3 promotes M1 polarization of macrophages by positively regulating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Conversely, silencing IFIT3 expression inhibits the activation of the cGAS-STING pathway in the lung tissue of ALI mice, which results in a reduction of the inflammatory response. Furthermore, IFIT3 functions as a bridging protein that enhances the interaction between the deubiquitinase USP18 and STING by inhibiting STING ubiquitination and improving its stability. Overall, our study suggests that IFIT3 is a crucial regulatory molecule contributing to the imbalanced macrophage polarization and excessive inflammation during the early stages of ALI/ARDS, which may provide novel therapeutic targets for ALI/ARDS. Materials and methods Mice Our study exclusively examined male mice. It is unknown whether the findings are relevant for female mice. Wild-type C57BL/6 mice were procured from the Experimental Animal Center of Soochow University and housed under specific pathogen-free (SPF) conditions at room temperature. Male mice weighing 20-22 g, aged 6-8 weeks, were used in this study. All animal procedures were approved by and conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee of Soochow University. Cell lines The RAW264.7 murine macrophage cell line was obtained from Procell (Wuhan, China), while immortalized mouse bone marrow-derived macrophages (iBMDM) were procured from iCell Bioscience (Shanghai, China). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO 2 at 37°C. Primary cells Isolation and culture of primary bone marrow-derived macrophages (BMDM) were conducted using C57BL/6 mice (6-8 weeks old)[16]. Mouse peritoneal macrophages (PM) were isolated and cultured via peritoneal lavage[17]. Identifying differentially expressed genes (DEGs) Gene expression profiles of human macrophage polarization (GSE57614 and GSE61298) were obtained from the Gene Expression Omnibus (GEO) database and analyzed to identify differentially expressed genes (DEGs) in M1 macrophages compared to the control group (M0+M2). GEO2R was employed for DEG identification and analysis, using a significance threshold of |log2(fold change)| ≥ 3 and an adjusted p -value < 0.05 for statistical significance. Volcano plots depicting DEGs and Venn diagrams illustrating co-upregulated DEGs were generated using the 'ggplot' package (version 3.3.6) within the R software environment. Functional enrichment analysis of co-upregulated DEGs The co-upregulated DEGs were subjected to analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for comprehensive functional annotation. GO analysis encompassed three domains: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Functional enrichment analysis was conducted utilizing the Database for Annotation, Visualization, and Integrated Discovery web tool. The results of the analysis were visualized using the 'clusterProfiler' package within the R programming environment. Construction of PPI networks and identification of hub genes The protein-protein interaction (PPI) network of the co-upregulated DEGs was constructed utilizing the STRING website. The resulting PPI network, with a minimum interaction score of 0.4, was visualized using Cytoscape version 3.9.1. The identification of hub genes was facilitated by using CytoHubba plugins. Detection of hub gene expression in ALI/ARDS-related datasets Mouse ALI-associated datasets (GSE2411 and GSE18341) and ARDS patient-related datasets (GSE40885 and GSE68610) were selected from the GEO database as validation cohorts to assess the expression of hub genes, aiding in the identification of hub genes highly expressed in ALI/ARDS and associated with M1 polarization. Heatmaps illustrating the expression differences of hub genes were generated using the "ComplexHeatmap" package in the R programming environment. Animal models of LPS-induced ALI and lung tissue IFIT3 knockdown WT C57BL/6 mice were induced with the ALI model by intraperitoneal injection of LPS derived from E. coli 055:B5 (Sigma-Aldrich, USA) at a dosage of 10 mg/kg body weight for 12h. Control group mice received intraperitoneal injection of an equivalent volume of saline. Ad-shNC and Ad-shIFIT3 (1×10 9 pfu/mouse) were administered to the adenovirus gene delivery groups five days before LPS administration using intratracheal injection. Mice were anesthetized, and the adenovirus was directly injected into the trachea following surgical visualization. Mice were sacrificed after 12 hours, and the lung tissues were collected under aseptic conditions. Inducing macrophage polarization to M1 phenotypes The mouse cell lines RAW264.7 and iBMDM, as well as primary mouse cells, including BMDM and PM, were stimulated with LPS at a concentration of 500 ng/mL, combined with IFN-γ at 20 ng/mL, for 12 hours to induce M1 polarization. Histology and immunohistochemistry Mouse lung tissues were fixed in 4% paraformaldehyde, followed by dehydration through a series of graded alcohol solutions. The tissues were then embedded in paraffin and cut into 5 µm sections. For histological analysis, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, the sections were deparaffinized, rehydrated, and washed before undergoing antigen retrieval. To minimize non-specific binding, the sections were blocked with 5% bovine serum albumin (BSA) and subsequently incubated overnight at 4°C with an IFIT3 antibody. Immunoreactivity was assessed using a horseradish peroxidase (HRP)-linked secondary antibody, and visualization was achieved through the application of 3,3'-diaminobenzidine tetrahydrochloride (DAB) (ZSGB-BIO, China). Immunofluorescence Staining Cultured cells were seeded on sterile glass coverslips in 24-well plates and allowed to adhere overnight. After treatment, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h. Primary antibodies diluted in blocking buffer were incubated overnight at 4℃. After washing with PBS, cells were incubated with fluorophore-conjugated secondary antibodies (1:500) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI (1μg/mL) for 10 min. Finally, coverslips were mounted onto slides using antifade mounting medium and imaged using a confocal microscope. Plasmid transfection and viral infection Mouse Flag-IFIT3 was purchased from GeneChem (China), and mouse HA-STING, His-STING, and Flag-USP18 were purchased from YouBio (China). All constructs were confirmed using DNA sequencing, and the plasmids were then transfected into 293T cells procured from Procell (China) using the Lipofectamine 2000 (Invitrogen, USA). The recombinant lentivirus containing the specific mouse shRNAs for IFIT3 was purchased from GeneChem (China). The recombinant lentivirus containing the mouse IFIT3 gene and recombinant adenovirus containing the mouse shRNA for IFIT3 were purchased from GenePharma (China). The shRNA target sequences are listed in Table S2. Detection of Reactive Oxygen Species (ROS) ROS levels were measured using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). Cells were cultured in six-well plates and treated according to the experimental design. DCFH-DA was diluted 1:1000 in serum-free DMEM. Following the removal of the culture medium, cells were washed three times with PBS and incubated with 1 mL of the diluted DCFH-DA solution for 20 minutes at 37°C. After incubation, cells were washed three times with serum-free DMEM to remove uninternalized DCFH-DA. Cells were then harvested using trypsinization, collected in centrifuge tubes, and centrifuged at 2000 rpm for 3 minutes. The supernatant was discarded, and the cell pellets were resuspended in 200 µL of PBS. For the blank control group, identical procedures were followed without DCFH-DA exposure. Flow cytometry was used to analyze the samples, and data were processed using FlowJo software. Generation of USP18 Knockout Cell Lines The lenti CRISPR V2 plasmid was used to construct USP18 KO cells, and the following sgRNA sequences were used: USP18 sg1: 5′- TCGGCAGGATAACAGTGCCT-3′; USP18 sg2: 5′-TTCCTCTCTTCTGCACTCCG’. The USP18 lenti CRISPR V2 plasmid and packaging plasmids pVSVg and psPAX2 were transfected into HEK293T cells. The lentiviruses were collected, and USP18 KO iBMDM were selected with puromycin (2.5 μg/mL) for 7 days. Statistical analysis Statistical analyses were conducted using GraphPad Prism 8.0. Data are expressed as the mean ± standard deviation (SD). For comparisons between the two groups, a two-tailed unpaired Student's t-test was employed. When comparing three or more groups, a one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was utilized. Statistical significance was defined as p < 0.05. Results IFIT3 identified as a key regulator of macrophage M1 polarization in ALI/ARDS Two GEO datasets (GSE57614 and GSE61298) were analyzed to identify potential critical genes associated with macrophage polarization. In the GSE57614 dataset, 310 differentially expressed genes (DEGs) were identified, including 274 upregulated and 36 downregulated genes (Figure 1A). The GSE61298 dataset revealed 664 DEGs, with 485 upregulated and 179 downregulated genes (Figure 1B). A total of 102 co-upregulated DEGs were found in both datasets (Figure 1C). Functional enrichment analyses of these DEGs were conducted using GO and KEGG pathway analyses (Figures 1D, 1E, and S1A). The protein-protein interaction (PPI) network of the co-upregulated DEGs was constructed using the STRING database (Figure S1B). The CytoHubba plugin was used to identify essential nodes in the network, resulting in the top 10 hub genes (Figure 1F). RAW264.7 cells were polarized into the M1 phenotype using LPS, and qRT-PCR assessed the expression of hub genes. Compared to the M0 control group, the mRNA expression levels of several highly conserved human-mouse hub genes, including IL-6, IL-1β, CCL5, TNFα, IFIT3, CD80, CD274, RSAD2, and IRF1, were significantly upregulated in the M1 group (Figure 1G), consistent with the bioinformatics analysis results. To further explore genes associated with M1 macrophage polarization in ALI/ARDS, human ARDS datasets (GSE40885 and GSE68610) and mouse ALI datasets (GSE2411 and GSE18341) were queried from the GEO database. Differential expression analysis of the top ten critical genes related to M1 macrophages between the ALI/ARDS groups and normal controls in these datasets revealed that IFIT3 exhibited significantly higher expression levels in the ALI/ARDS groups compared to the normal controls (Figures 1H-1K). Increased expression of IFIT3 in M1 macrophages and early ALI mice lungs After 12 hours of stimulation with LPS and IFN-γ, qRT-PCR revealed elevated expression levels of the M1 polarization markers CD86 and iNOS (NOS2), along with increased levels of the inflammatory cytokine IL-6 in RAW264.7 and iBMDM cells compared to the control group. Conversely, no significant changes were observed in the expression of the M2 polarization marker CD206 (Figures 2A and 2B). Western blot analysis further corroborated the enhanced expression of iNOS protein relative to the control group, confirming the successful M1 polarization of RAW264.7 and iBMDM cells induced by LPS and IFN-γ (Figure 2C). Collectively, qRT-PCR, Western blot, and immunofluorescence assays demonstrated a significant upregulation of IFIT3 expression in M1 macrophages (Figures 2A-2D). Following intraperitoneal injection of LPS to establish the ALI model in C57BL/6 mice, the wet/dry weight ratios of lung tissues from LPS-injected mice at 6, 12, 18, and 24 hours were significantly increased compared to the control group (Figure 2F). H&E staining of lung tissue sections revealed thickening of alveolar walls, infiltration of inflammatory cells, increased exudation in alveolar spaces, and marked congestion (Figure 2E). These results suggest that intraperitoneal injection of LPS (10 mg/mL) for 6 to 24 hours successfully establishes an early ALI model in mice. qRT-PCR, Western blot, and immunohistochemistry results indicated that the mRNA and protein expression levels of IFIT3 in the lung tissues of ALI mice were elevated following LPS treatment at 6, 12, 18, and 24 hours compared to the control group (Figures 2G-2H). IFIT3 promotes M1 macrophage polarization and inflammation To investigate the role of IFIT3 in M1 macrophage polarization, stable knockdown of IFIT3 expression was achieved in RAW264.7 cells via lentiviral infection (Figures 3A, 3D). qRT-PCR results demonstrated a significant increase in the mRNA levels of M1 markers (CD86, NOS2), proinflammatory cytokines (IL-6, TNFα, and IL-1β), and chemokines (Cxcl2, Cxcl3, and Ccl2) in cells stimulated with LPS and IFN-γ compared to the shNC group. Upon IFIT3 knockdown and subsequent stimulation with LPS and IFN-γ, the expression of these genes was significantly reduced in the shIFIT3 group compared to the shNC group, indicating that silencing IFIT3 expression inhibits LPS and IFN-γ-induced M1 polarization of macrophages and reduces the production of multiple proinflammatory cytokines and chemokines (Figures 3B-3D, 3G-3I, and S4A-4C). ROS levels in RAW264.7 cells were measured by flow cytometry. Results showed that intracellular DCF fluorescence intensity in the (shNC+LPS+IFN-γ) group was significantly higher compared to the shNC group. In contrast, the intensity in the (shIFIT3+LPS+IFN-γ) group was markedly lower compared to the (shNC+LPS+IFN-γ) group (Figures 3E, 3F), indicating that IFIT3 knockdown significantly inhibits ROS generation in macrophages induced by LPS and IFN-γ stimulation. ELISA was used to assess the levels of inflammatory cytokines IL-6, TNFα, and IL-1β in the cell culture supernatant. The results revealed that IFIT3 knockdown had no significant impact on the secretion of IL-6, TNFα, and IL-1β under basal conditions. However, it significantly suppressed the secretion of these proinflammatory cytokines induced by LPS and IFN-γ stimulation (Figures 3J-3L). Knockdown of IFIT3 in mouse lung tissue alleviates early lung injury induced by LPS The animal experimental procedures are illustrated in Figure 4A. qRT-PCR and Western blot analyses revealed that the expression of IFIT3 in lung tissues from the (shIFIT3+LPS) group was significantly reduced compared to the (shNC+LPS) group, indicating effective silencing of IFIT3 gene expression in mouse lung tissue following intratracheal administration of ADV-shIFIT3 (Figures 4D, 4K). The lung wet-to-dry weight ratio results showed a significant increase in water content in lung tissues of both the LPS group and the (shNC+LPS) group compared to the control group. However, the lung water content in the (shIFIT3+LPS) group was significantly lower than that in the (shNC+LPS) group and the LPS group (Figure 4C), suggesting that knockdown of IFIT3 effectively alleviated LPS-induced pulmonary edema. H&E staining results further demonstrated that compared to the LPS group and the (shNC+LPS) group, inflammatory cell infiltration was significantly reduced in the lung tissues of the (shIFIT3+LPS) group, with marked thinning of alveolar walls, reduced exudation in alveolar cavities, and decreased alveolar collapse (Figure 4B). qRT-PCR results indicated that intraperitoneal injection of LPS for 12 hours induced increased expression of NOS2, CXCL2, CXCL3, TNFα, IL-6, and IL-1β in mouse lung tissue, promoting the early onset of pulmonary inflammation in acute lung injury (ALI) (Figures 4F, 4G, and S5A-S5G). The expression levels of these genes in the lung tissue of mice in the (shIFIT3+LPS) group were lower compared to the (shNC+LPS) group and the LPS group. ELISA results also indicated that the levels of inflammatory cytokines in the lung tissue of mice in the (shIFIT3+LPS) group were lower than those in the (shNC+LPS) group and the LPS group (Figures 4H-4J). Western blot and immunohistochemistry (IHC) analyses revealed that the protein expression of the M1 macrophage marker CD86 and the M2 macrophage marker CD206 were elevated in the lung tissue of mice in the LPS group and the (shNC+LPS) group compared to the control group. In the (shIFIT3+LPS) group, the protein levels of CD86 and the proinflammatory mediator iNOS in the lung tissue, as well as the phosphorylation levels of the inflammatory signaling molecule p65, were lower than those in the LPS group and the (shNC+LPS) group. In contrast, the expression of CD206 protein showed no significant difference among the groups (Figures 4K-4N). These results suggest that silencing the gene expression of IFIT3 in lung tissue can inhibit M1 macrophage polarization and proinflammatory activity, thereby improving LPS-induced inflammatory lung injury. JAK-STAT3 pathway upregulates IFIT3 expression in LPS-induced M1 macrophages Stimulation of RAW264.7 cells with LPS and IFN-γ for 12 hours resulted in increased phosphorylation levels of JAK1, JAK2, and STAT3 proteins compared to the control group (Figure 5A). Treatment with the STAT3-specific phosphorylation inhibitor Stattic (2 μM) for 2 hours effectively inhibited STAT3 protein phosphorylation. Subsequent stimulation with LPS and IFN-γ for 12 hours revealed that in the (Stattic+LPS+IFN-γ) group, the protein expression levels of IFIT3 and iNOS were lower compared to the (LPS+IFN-γ) group (Figure 5B). This suggests that activated STAT3 promotes M1 macrophage polarization and IFIT3 protein expression in response to LPS and IFN-γ stimulation. Knockdown of IFIT3 expression in RAW264.7 cells was achieved using lentiviral vectors, and its effect on STAT3 protein expression was assessed. Western blot analysis showed no significant differences in the levels of total STAT3 protein and phosphorylated STAT3 between the (shNC+LPS+IFN-γ) and (shIFIT3+LPS+IFN-γ) groups (Figure 5C). This indicates that IFIT3 knockdown does not affect STAT3 protein expression or phosphorylation, suggesting that IFIT3 functions downstream of the JAK-STAT3 pathway. Knocking down IFIT3 inhibits the activation of the LPS and IFN-γ induced cGAS-STING signaling pathway in macrophages The Western blot analysis demonstrated that co-stimulation of RAW264.7 cells (Figure 5D) and iBMDM cells (Figure S4D) with LPS and IFN-γ resulted in a significant upregulation of cGAS, STING, p-TBK1, and p-IRF3 protein levels in the (shNC+LPS+IFN-γ) group compared to the shNC group. This indicates that LPS and IFN-γ induce activation of the cGAS-STING signaling pathway. In contrast, the (shIFIT3+LPS+IFN-γ) group showed no significant difference in cGAS protein expression compared to the (shNC+LPS+IFN-γ) group. However, the levels of STING, p-TBK1, and p-IRF3 proteins were significantly reduced in the (shIFIT3+LPS+IFN-γ) group, suggesting that IFIT3 knockdown inhibits the activation of the cGAS-STING pathway induced by LPS and IFN-γ. IFIT3's regulatory effect on this pathway is likely targeted at the STING molecule. Additionally, phosphorylation levels of NF-κB p65 and MAPK p38 proteins were significantly increased in the (shNC+LPS+IFN-γ) group relative to the shNC group, indicating that LPS and IFN-γ stimulate the activation of NF-κB p65 and MAPK p38 proteins in RAW264.7 cells, thus triggering related inflammatory pathways. In the (shIFIT3+LPS+IFN-γ) group, the phosphorylation levels of NF-κB p65 and MAPK p38 were markedly lower compared to the (shNC+LPS+IFN-γ) group (Figure 5E). This suggests that IFIT3 knockdown suppresses the activation of NF-κB and MAPK inflammatory signaling pathways induced by LPS and IFN-γ. Furthermore, after IFIT3 knockdown in RAW264.7 cells, both cytoplasmic and nuclear levels of phosphorylated p65 and IRF3 proteins were lower in the (shIFIT3+LPS+IFN-γ) group compared to the (shNC+LPS+IFN-γ) group (Figure 5F). This finding implies that IFIT3 promotes the phosphorylation of nuclear transcription factors NF-κB p65 and IRF3, facilitating their nuclear translocation in response to LPS and IFN-γ. Immunofluorescence staining also revealed that LPS and IFN-γ enhance the phosphorylation of TBK1 protein in RAW264.7 cells. In the (shIFIT3+LPS+IFN-γ) group, TBK1 phosphorylation levels in the cytoplasm were significantly lower than those in the (shNC+LPS+IFN-γ) group (Figure 5G), suggesting that IFIT3 knockdown inhibits TBK1 phosphorylation induced by LPS and IFN-γ. In vivo experiments showed that in the lung tissues of mice from the LPS group and (shNC+LPS) group, cGAS and STING protein levels, along with the phosphorylation of TBK1 and IRF3 proteins, were significantly upregulated compared to the control group. This indicates the activation of the cGAS-STING signaling pathway in ALI mouse lung tissues. Conversely, in the lung tissues of mice from the (shIFIT3+LPS) group, cGAS, and STING protein levels, as well as the phosphorylation of TBK1 and IRF3, were lower than those in the LPS and (shNC+LPS) groups (Figure 4K). These results suggest that early-stage upregulation of IFIT3 in ALI mouse lung tissues activates the cGAS-STING signaling pathway, promoting macrophage polarization to the M1 phenotype and enhancing the inflammatory response. IFIT3 increases the stability of STING protein by inhibiting the proteasomal degradation pathway Knockdown of IFIT3 expression did not significantly affect the mRNA levels of STING. Still, it led to a substantial reduction in STING protein levels (Figures 6A, 6B), suggesting that IFIT3 may regulate STING protein expression through post-translational modifications. Endogenous co-immunoprecipitation (Co-IP) performed in iBMDM cells (Figures 6C, 6D) and exogenous Co-IP conducted in 293T cells (Figures 6E, 6F) confirmed the interaction between IFIT3 and STING proteins. Immunofluorescence experiments demonstrated the co-localization of IFIT3 and STING in the cytoplasm (Figure 6G), further supporting the interaction between these two proteins. Since alterations in IFIT3 expression affect only STING protein levels without impacting mRNA expression, it was hypothesized that IFIT3 might influence STING protein degradation and stability. To test this hypothesis, RAW264.7 cells were treated with LPS and IFN-γ for 12 hours to induce IFIT3 expression, followed by the addition of cycloheximide (CHX) to inhibit STING protein synthesis and assess the effect of IFIT3 on STING protein half-life. Results showed that the half-life of STING protein was significantly reduced in the shIFIT3 group compared to the shNC group, indicating that IFIT3 inhibits STING protein degradation (Figures 6H, 6I). Furthermore, inhibition of the proteasomal degradation pathway using MG132 led to a partial reversal of the reduction in STING protein levels observed in the shIFIT3 group 24 hours after CHX treatment (Figure 6J). These findings suggest that IFIT3 enhances STING protein stability by inhibiting the proteasomal degradation pathway. IFIT3 enhances STING protein stability by inhibiting ubiquitination Ubiquitination plays a critical role in regulating STING homeostasis[18-20]. Endogenous ubiquitination Co-IP assays showed that the level of ubiquitination in the shIFIT3 group RAW264.7 cells was higher than in the shNC group (Figure 7A). This indicates that knocking down IFIT3 expression increases STING protein ubiquitination, suggesting that high expression of IFIT3 induced by LPS and IFN-γ can inhibit STING protein ubiquitination. Previous studies have shown that IFIT3 lacks enzymatic activity, but its structural characteristics facilitate interactions with other proteins, thereby affecting their functions[8]. Therefore, we hypothesize that IFIT3 may inhibit STING ubiquitination by promoting the binding of other proteins to STING. To identify proteins that might bind to IFIT3 and potentially influence protein ubiquitination, we performed Co-IP combined with mass spectrometry (Figure S6A outlines the experimental workflow). KEGG analysis of the mass spectrometry results showed enrichment in the "Ubiquitin-mediated proteolysis pathway" (Figure 7B). Proteins detected in the IFIT3 overexpression (OE) group with expression levels at least 10 times higher than the Vector group (FC ≥ 10) were selected as candidate proteins. A total of 150 proteins potentially interacting specifically with IFIT3 were identified (Figure 6C), including five from the top ten proteins predicted to bind with IFIT3 according to the STRING database (Figure S6C): USP18, IFIT1, IFIH1, RSAD2, and ISG15. Among these, USP18 belongs to the deubiquitinase family, and studies have shown that it can promote the inhibition of STING protein ubiquitination and degradation[21]. In addition to USP18, the mass spectrometry results also revealed interactions between IFIT3 and the deubiquitinases OTUD5 and OTUD6B, with literature reporting that OTUD5 can also deubiquitinate STING[22]. These findings support our hypothesis that IFIT3 acts as a bridging protein, enhancing the binding of deubiquitinases to STING, thereby inhibiting STING ubiquitination and increasing STING protein stability. Protein interaction simulation analysis showed that upon the addition of IFIT3, the binding potential energy between STING and USP18 decreased, indicating that IFIT3 significantly enhances the stability of the binding between STING and USP18 proteins (Figures 7D, 7E). Co-IP experiments in 293T cells confirmed the interaction between STING and USP18 (Figures 7F, 7G). Further,successful knockout of USP18 in iBMDM followed by induction of M1 phenotype polarization showed no significant effects on IFIT3 and cGAS protein expression. However, it markedly reduced STING protein expression along with its downstream phosphorylation levels of TBK1 and IRF3, as well as iNOS protein expression. suggesting that IFIT3 may act as a bridging protein to promote the binding of deubiquitinase USP18 to STING. These results demonstrate that IFIT3 acts as a bridging protein to promote the interaction between USP18 and STING, thereby enhancing STING protein stability. Discussion The pathogenesis of ALI/ARDS remains incompletely elucidated, involving inflammatory responses in lung tissue and multiple systemic systems, coagulation abnormalities, and dysregulation of signaling pathways, which are initially the body's normal defense responses to infection and injury. However, once excessively and extensively activated, these functions may lead to tissue damage, ultimately triggering ALI/ARDS. ARDS can be classified pathologically into early (acute exudative phase), proliferative phase, and fibrotic phase[ 23 ]. During the early stages of ARDS, excessive inflammatory response is the most prominent feature, with typical pathological changes characterized by neutrophilic alveolitis and the formation of hyaline membranes representing diffuse alveolar damage [ 24 ]. The primary instigator of induced neutrophil recruitment is pulmonary tissue macrophages[ 25 , 26 ]. Pulmonary macrophages, pivotal effector cells in lung tissue's response to external stimuli, play a critical role in the pathogenesis of pulmonary inflammation [ 27 ]. Macrophages exhibit high plasticity and heterogeneity; under different pathological and physiological conditions and surrounding microenvironments, they undergo phenotypic and functional changes induced by various signals and cytokines from an inactive M0 state, a process known as macrophage polarization [ 28 ]. Macrophage polarization primarily divides into two subtypes: classically activated M1 and alternatively activated M2 [ 28 , 29 ]. An imbalance in M1/M2 polarization can lead to the occurrence and development of various diseases [ 30 – 32 ]. In the early stages (acute exudative phase) of ALI/ARDS, lung macrophages primarily polarize towards M1. Sustained M1 polarization can release multiple inflammatory mediators and genes, such as IL-6, TNFα, IL-1β, NOS, and reactive oxygen species (ROS), recruiting neutrophils from the bloodstream into lung tissues and alveolar spaces, thereby triggering severe inflammatory reactions and progressing lung injury[ 33 ]. Regulation of the imbalance in M1/M2 polarization of macrophages can mitigate lung tissue damage and improve the prognosis of ALI/ARDS. IFIT3, an essential member of the interferon-stimulated genes and the IFIT family has been increasingly recognized for its critical role not only in various pathogen infections, especially in antiviral defense and immune responses, but also in influencing diverse cellular functions [ 8 , 9 , 34 , 35 ]. However, the specific role of IFIT3 in the pathogenesis of ALI/ARDS remains unclear. In this study, we utilized bioinformatics to identify hub genes related to M1 macrophage polarization that are highly expressed in lung samples of ARDS/ALI, using data from the GEO database. Validation was performed through the construction of an M1 cell model and subsequent qRT-PCR experiments. The results identified four well-established cytokines (IL-6, IL-1β, CCL5, and TNFα) as the highest-expressed hub genes. Notably, IFIT3 ranked fifth in expression, but its role in ALI/ARDS is unclear, with no existing mechanistic studies. Consequently, we selected IFIT3 for further investigation to assess its influence on macrophage polarization and its potential roles and mechanisms in ALI/ARDS. Referencing previous literature[ 36 , 37 ] and our experimental verification, we found that stimulation of mouse macrophages with LPS combined with IFN-γ for 12 hours significantly increased the expression of CD86 and iNOS compared to the control group, while CD206 showed no difference, indicating that this induction condition could polarize macrophages toward an M1 phenotype. We also observed a significant increase in the expression of three proinflammatory factors (IL-6, IL-1β, and TNFα) after induction in RAW264.7 cells. However, iBMDM cells exhibited a less pronounced inflammatory phenotype compared to RAW264.7 cells. Thus, RAW264.7 was primarily used as the cell model for our study, with iBMDM cells serving as experimental supplements. These two macrophage cell lines showed some differences in their inflammatory phenotypes post-induction, likely due to factors such as cellular origin and differential responses to inducers. Under normal physiological conditions, the expression of IFIT3 is low in most cells, but it significantly increases under pathological conditions induced by stimuli [ 12 ]. Our experimental results also demonstrated that the basal expression levels of IFIT3 in primary macrophages (PM and BMDM) and macrophage cell lines (RAW264.7 and iBMDM) were low. Still, its expression significantly increased upon induction of M1 polarization, indicating high expression of IFIT3 in M1 macrophages. IFIT3 exhibited low-level expression in normal control mouse lung tissues, and its mRNA and protein expression significantly increased in early ALI mouse lung tissues. These in vitro and in vivo experiments further confirmed the accuracy and reliability of the bioinformatics analysis results. Limited studies have investigated whether IFIT3 can regulate macrophage phenotype polarization and function. Subsequent to stable knockdown of IFIT3 expression using lentiviral vectors in vitro, we found a significant attenuation of M1 phenotype polarization of macrophages induced by LPS and IFN-γ: downregulation of M1 markers (CD86 and iNOS), proinflammatory factors (IL-6, IL-1β, and TNFα), and chemokines (CCL2, CXCL2, and CXCL3), along with reduced ROS generation. Conversely, stable overexpression of IFIT3 further enhanced the expression of iNOS, IL-6, IL-1β, and TNFα induced by LPS and IFN-γ. These results suggest that high expression of IFIT3 promotes M1 polarization and related inflammatory responses in macrophages. Additionally, we observed that without induction stimulation by LPS and IFN-γ, overexpression of IFIT3 alone did not promote macrophage polarization or expression of inflammatory factors, indicating that IFIT3 is not an inducer of M1 polarization but rather a regulatory molecule influencing the process of M1 polarization. IFIT3 positively regulates macrophage M1 phenotype polarization and inflammatory responses. Furthermore, the experiment in vivo showed that silencing IFIT3 expression in lung tissues significantly improved lung tissue damage and inflammatory responses in early ALI mice. In summary, we have confirmed that IFIT3 promotes excessive inflammation and lung injury in early ALI/ARDS by regulating M1 polarization of pulmonary macrophages. The JAK-STAT pathway is widely implicated in the pathogenesis of ARDS [ 38 – 40 ]. Under pathological conditions, phosphorylated STAT1, STAT2, and IRF9 form the ISGF3 complex, which translocates to the nucleus and binds to ISRE elements in the promoter regions of IFIT family genes, thereby stimulating the expression of IFIT family genes[ 41 , 42 ]. Whether other members of the STAT family can also regulate IFIT3 expression remains unclear. Whether STAT3 also regulates the expression of IFIT3 in macrophages remains to be explored. Our study found that in the LPS and IFN-γ induced M1 polarization model of macrophages, the phosphorylation levels of JAK1, JAK2, and STAT3 proteins were significantly increased, indicating the activation of the JAK-STAT3 pathway. In the early LPS-induced ALI mouse model, the phosphorylation of STAT3 protein in lung tissues was also significantly increased, suggesting the activation of STAT3 protein in M1 macrophages and ALI mouse lung tissues. After inhibiting STAT3 phosphorylation with Stattic and then inducing macrophages with LPS and IFN-γ, the protein levels of IFIT3 and iNOS were significantly reduced, indicating that activated STAT3 promotes IFIT3 expression and M1 polarization of macrophages. Conversely, knocking down IFIT3 expression did not affect the expression and phosphorylation levels of STAT3 protein, confirming that the JAK-STAT3 pathway is upstream of IFIT3 and that activated STAT3 positively regulates the polarization of macrophage M1 phenotype and IFIT3 expression under inducer stimulation. The cGAS-STING signaling pathway is a crucial innate immune pathway that is linked to inflammation, infection, autoimmunity, degenerative diseases, and cancer[ 43 , 44 ]. The essence of ARDS lies in its inflammatory lung injury, where the aberrant presence of endogenous and exogenous DNA can serve as PAMP/DAMP to induce and promote ARDS progression, hence garnering attention to the role of the cGAS-STING pathway in ARDS in recent years. Existing research indicates that the cGAS-STING signaling pathway promotes the occurrence and development of ALI/ARDS [ 45 – 49 ], but the specific regulatory mechanisms await further clarification. Currently, there is limited research on the association between IFIT3 and the cGAS-STING pathway, with relevant literature primarily focused on viral infections and autoimmune diseases [ 35 , 50 ]. Increased activity of the cGAS-STING signaling pathway and expression levels of IFIT3 were observed in PBMCs of systemic lupus erythematosus (SLE) patients compared to healthy controls. Co-IP detection suggests that IFIT3 interacts with STING and TBK1, activating the cGAS-STING signaling pathway, thereby promoting disease activity in SLE patients[ 51 ], although the molecular mechanisms remain to be elucidated. High-throughput sequencing and in vivo experiments in mice have shown that IFIT3 is one of the core genes regulating Sjögren's Syndrome (SS), leading to aberrant activation of autophagy through the cGAS-STING pathway, which is a crucial factor in SS pathogenesis[ 52 ]. However, the related regulatory mechanisms are not yet precise. In summary, the activation of the cGAS-STING signaling pathway has been implicated in ALI/ARDS, although the regulatory mechanisms remain poorly understood. Our study investigates the role of IFIT3 in macrophage polarization and its influence on the cGAS-STING pathway in ALI/ARDS. In vitro experiments, it was demonstrated that LPS and IFN-γ-induced M1 macrophage polarization led to increased expression of cGAS, STING, and TBK1 phosphorylation, highlighting the pathway's activation in M1 macrophages. Notably, stable knockdown of IFIT3 in macrophages resulted in decreased STING levels and reduced phosphorylation of TBK1, IRF3, p65, and p38, suggesting that IFIT3 regulates the cGAS-STING pathway primarily through STING. In vivo studies in ALI mice confirmed that IFIT3 knockdown inhibited cGAS-STING pathway activity and M1 polarization, alleviating inflammatory responses. Interestingly, a decrease in cGAS levels in lung tissues was observed in vivo, which contrasts with in vitro findings. This discrepancy may arise because in vitro experiments focused on macrophages. At the same time, the in vivo approach affected multiple cell types, necessitating further investigations to elucidate the underlying mechanisms of IFIT3's role in ALI/ARDS. We further investigated the potential mechanisms by which IFIT3 regulates the cGAS-STING pathway. The IFIT3 protein is predominantly located in the cytoplasm, and currently, no known enzymatic activity has been found for it. IFIT3 protein contains multiple structural motifs of tetratricopeptide repeats (TPR), which enable IFIT3 to form complexes with other proteins to carry out various cellular functions[ 12 ]. Through protein docking simulations, immunofluorescence, and Co-IP experiments, we confirmed that IFIT3 can interact with STING protein. We also observed that knocking down IFIT3 had no significant effect on STING mRNA expression both in vivo and in vitro, which was inconsistent with the changes in STING protein expression, suggesting that IFIT3 may regulate STING protein levels by affecting post-translational modifications and protein stability. Our study showed that upregulated IFIT3 inhibits STING protein ubiquitination and degradation through the ubiquitination-proteasomal system in the M1 macrophage model, thereby increasing the stability and abundance of STING protein. Co-IP combined with protein mass spectrometry (MS) showed that the most robust proteins interacting with IFIT3 included the deubiquitinating enzyme USP18. Prediction from the STRING database suggested that the top ten proteins interacting with IFIT3 also included USP18, and protein interaction simulation analysis revealed that after the addition of IFIT3, the stability of the interaction between STING and USP18 proteins was enhanced. Co-IP experiments also confirmed the interaction between STING and USP18 proteins. USP18 is a unique member of the ubiquitin-specific protease (USP) family, previously believed to be unresponsive to ubiquitin but capable of removing ubiquitin-like (UbI) protein ISG15 (interferon-stimulated gene 15) from substrate proteins[ 53 , 54 ]. Additionally, USP18 can interact with type I interferon receptors, inhibiting interferon signaling [ 54 – 56 ]. Contrary to initial findings, recent research has demonstrated that USP18 also possesses deubiquitinase activity[ 57 – 60 ]. USP18 deubiquitinates the cGAS protein and inhibits cGAS protein degradation [ 61 , 62 ]. USP18 recruits USP20 to deconjugate K48-linked ubiquitin chains from STING, enhancing STING protein stability as well as the expression of type I IFN and proinflammatory cytokines. Knockout of USP18 leads to increased K48-linked ubiquitination of STING and accelerated degradation of STING, impairing downstream activation of IRF3 and NF-κB[ 21 ]. The MS results in our study revealed that in addition to USP18, the deubiquitinating enzymes that interact with IFIT3 and exhibit high expression levels include OTUD5 and OTUD6B. Previous studies have shown that the deubiquitinase OTUD5 can interact with STING, cleaving its K48-linked polyubiquitin chains and promoting STING protein stability [ 22 ]. However, the role of the deubiquitinase OTUD6B in regulating STING ubiquitination levels remains unexplored and warrants further investigation. In summary, our study found that in the early stages of ALI/ARDS, the upregulated expression of IFIT3 in lung tissue acts as a bridging protein, promoting the binding of STING to deubiquitinases such as USP18. This interaction inhibits the ubiquitin-mediated degradation pathway of STING, thereby increasing STING protein stability and activating the cGAS-STING pathway. Consequently, this process enhances M1 polarization of pulmonary macrophages and inflammatory responses, facilitating the progression of ALI/ARDS. These findings contribute to a deeper understanding of the pathogenesis of ALI/ARDS and provide new targets for drug research and therapeutic interventions. Limitations of the study Our study has several limitations. First, the in vitro experiments were conducted using only two mouse macrophage cell lines. To improve the generalizability of the findings, future research should incorporate primary macrophages and human-derived macrophages. The role of IFIT3 as a bridging protein between deubiquitinases and STING requires further validation, including more precise identification of the interaction sites. Additionally, while we utilized adenoviral vector-mediated RNAi for transient IFIT3 knockdown, future studies should employ gene knockout mouse models to provide a more comprehensive understanding of IFIT3 function. It is also necessary to use flow cytometry of alveolar lavage fluid to assess changes in IFIT3 expression in alveolar macrophages during early-stage ALI. Finally, clinical observational studies are needed to evaluate the diagnostic and prognostic value of IFIT3 in ARDS. Declarations Author contributions Nana Tang and Yang Yang contributed equally to this work and are considered co-first authors. Nana Tang and Yang Yang designed and performed the experiments, analyzed the data, and wrote the manuscript. Yuanyuan Zeng, Zhu Jianjie, Li Jianjun, Wang Jiajia, and Ding Ling assisted with experimental procedures and data collection. Jianan Huang and Zeyi Liu supervised the study, provided critical revisions, and are the corresponding authors responsible for overall project direction and manuscript finalization. Funding This study was funded by the Guizhou Science and Technology Department Plan Supporting Project of China (No. 2021 General 086); the Jiangsu Provincial Medical Key Discipline (No. ZDXK202201); NSFC Cultivation Program Project of the Affiliated Hospital of Guizhou Medical University (No. GYFYNSFC2023-55); National Postdoctoral Fellowship Program (No. GZC20231895). Data availability All data supporting the findings of this study are available in the article and Supplementary Material files. All original data for this study can be obtained from the corresponding author. Declarations Ethics approval and consent to participate Ethical approval Mouse studies were performed under protocols approved by Soochow University's IACUC (Approval ID: 2023-551), following institutional animal care standards. Consent to participate Not applicable. Consent for publication All the authors consent to the publication of this study. Competing interests The authors declare that they have no competing interests. Acknowledgements Not applicable. References Gorman EA, O'Kane CM, McAuley DF(2022) Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management . 400 : 1157-1170. Villar J, Szakmany T, Grasselli G, Camporota L(2023) Redefining ARDS: a paradigm shift . 27 : 416. Bellani G, et al(2016) Epidemiology, Patterns of Care, and Mortality for Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries . 315 : 788-800. Liu C, Xi L, Liu Y, Mak JCW, Mao S, Wang Z, Zheng Y(2023) An Inhalable Hybrid Biomimetic Nanoplatform for Sequential Drug Release and Remodeling Lung Immune Homeostasis in Acute Lung Injury Treatment . 17 : 11626-11644. Wang L, Wang D, Zhang T, Ma Y, Tong X, Fan H(2023) The role of immunometabolism in macrophage polarization and its impact on acute lung injury/acute respiratory distress syndrome . 14 : 1117548. Liu C, Xiao K, Xie L(2022) Advances in the Regulation of Macrophage Polarization by Mesenchymal Stem Cells and Implications for ALI/ARDS Treatment . 13 : 928134. Zhang S, Chu C, Wu Z, Liu F, Xie J, Yang Y, Qiu H(2020) IFIH1 Contributes to M1 Macrophage Polarization in ARDS . 11 : 580838. Zhang W, Li Y, Xin S, Yang L, Jiang M, Xin Y, Wang Y, Cao P, Zhang S, Yang Y, Lu J(2023) The emerging roles of IFIT3 in antiviral innate immunity and cellular biology . 95 : e28259. Schindewolf C, et al(2023) SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3 . 97 : e0153222. Xu S, Huang J, Xun Z, Li S, Fu Y, Lin N, Wu W, Chen T, Liu C, Ou Q(2022) IFIT3 Is Increased in Serum from Patients with Chronic Hepatitis B Virus (HBV) Infection and Promotes the Anti-HBV Effect of Interferon Alpha via JAK-STAT2 In Vitro . 10 : e0155722. Zhang W, Jiang M, Liao X, Li Y, Xin S, Yang L, Xin Y, Umar A, Lu J(2023) IFIT3 inhibits Epstein-Barr virus reactivation via upregulating innate immunity . 95 : e29237. Wu YY, Xing J, Li XF, Yang YL, Shao H, Li J(2023) Roles of interferon induced protein with tetratricopeptide repeats (IFIT) family in autoimmune disease . 22 : 103453. Wang Z, et al(2020) Inflammatory IFIT3 renders chemotherapy resistance by regulating post-translational modification of VDAC2 in pancreatic cancer . 10 : 7178-7192. Margaroli C, et al(2023) Interferon-dependent signaling is critical for viral clearance in airway neutrophils . 8. Yang X, Wang J, Liu W(2023) Molecular markers of type II alveolar epithelial cells in acute lung injury by bioinformatics analysis . 13 : 17797. Tang N, Yang Y, Xie Y, Yang G, Wang Q, Li C, Liu Z, Huang JA(2024) CD274 (PD-L1) negatively regulates M1 macrophage polarization in ALI/ARDS . 15 : 1344805. Yang Y, Wu J, Li Q, Wang J, Mu L, Hui L, Li M, Xu W, Yang H, Wei L(2022) A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx . 11. Cho W, et al(2023) Targeted Protein Upregulation of STING for Boosting the Efficacy of Immunotherapy . 62 : e202300978. Mutlu M, et al(2024) Small molecule induced STING degradation facilitated by the HECT ligase HERC4 . 15 : 4584. Tao Y, Yin S, Liu Y, Li C, Chen Y, Han D, Huang J, Xu S, Zou Z, Yu Y(2023) UFL1 promotes antiviral immune response by maintaining STING stability independent of UFMylation . 30 : 16-26. Zhang M, Zhang MX, Zhang Q, Zhu GF, Yuan L, Zhang DE, Zhu Q, Yao J, Shu HB, Zhong B(2016) USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA . 26 : 1302-1319. Guo Y, et al(2021) OTUD5 promotes innate antiviral and antitumor immunity through deubiquitinating and stabilizing STING . 18 : 1945-1955. Matthay MA, Zemans RL, Zimmerman GA, Arabi YM, Beitler JR, Mercat A, Herridge M, Randolph AG, Calfee CS(2019) Acute respiratory distress syndrome . 5 : 18. Thille AW, Peñuelas O, Lorente JA, Fernández-Segoviano P, Rodriguez JM, Aramburu JA, Panizo J, Esteban A, Frutos-Vivar F(2017) Predictors of diffuse alveolar damage in patients with acute respiratory distress syndrome: a retrospective analysis of clinical autopsies . 21 : 254. Bos LDJ, Ware LB(2022) Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes . 400 : 1145-1156. Kumar V(2020) Pulmonary Innate Immune Response Determines the Outcome of Inflammation During Pneumonia and Sepsis-Associated Acute Lung Injury . 11 : 1722. Chen X, Tang J, Shuai W, Meng J, Feng J, Han Z(2020) Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome . 69 : 883-895. Locati M, Curtale G, Mantovani A(2020) Diversity, Mechanisms, and Significance of Macrophage Plasticity . 15 : 123-147. Xue S, Su Z, Liu D(2023) Immunometabolism and immune response regulate macrophage function in atherosclerosis . 90 : 101993. Wang C, Ma C, Gong L, Guo Y, Fu K, Zhang Y, Zhou H, Li Y(2021) Macrophage Polarization and Its Role in Liver Disease . 12 : 803037. Funes SC, Rios M, Escobar-Vera J, Kalergis AM(2018) Implications of macrophage polarization in autoimmunity . 154 : 186-195. Kadomoto S, Izumi K, Mizokami A(2021) Macrophage Polarity and Disease Control . 23. Wang Z, Wang Z(2023) The role of macrophages polarization in sepsis-induced acute lung injury . 14 : 1209438. Xu F, Song H, An B, Xiao Q, Cheng G, Tan G(2019) NF-κB-Dependent IFIT3 Induction by HBx Promotes Hepatitis B Virus Replication . 10 : 2382. Chikhalya A, Dittmann M, Zheng Y, Sohn SY, Rice CM, Hearing P(2021) Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression . 12 : e0282921. Huang Y, Tian C, Li Q, Xu Q(2019) TET1 Knockdown Inhibits Porphyromonas gingivalis LPS/IFN-γ-Induced M1 Macrophage Polarization through the NF-κB Pathway in THP-1 Cells . 20. Zhou F, Mei J, Han X, Li H, Yang S, Wang M, Chu L, Qiao H, Tang T(2019) Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF-κB/MAPK signaling and protecting chondrocytes . 9 : 973-985. Gajjela BK, Zhou MM(2022) Calming the cytokine storm of COVID-19 through inhibition of JAK2/STAT3 signaling . 27 : 390-400. Zhang Y, Gao Z, Jiang F, Yan H, Yang B, He Q, Luo P, Xu Z, Yang X(2023) JAK-STAT signaling as an ARDS therapeutic target: Status and future trends . 208 : 115382. Purohit M, Gupta G, Afzal O, Altamimi ASA, Alzarea SI, Kazmi I, Almalki WH, Gulati M, Kaur IP, Singh SK, Dua K(2023) Janus kinase/signal transducers and activator of transcription (JAK/STAT) and its role in Lung inflammatory disease . 371 : 110334. Song J, Guo Y, Wang D, Quan R, Wang J, Liu J(2023) Seneca Valley virus 3C(pro) antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals . 97 : e0072723. Nowicka H, Sekrecka A, Blaszczyk K, Kluzek K, Chang CY, Wesoly J, Lee CK, Bluyssen HAR(2023) ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs . 24. Sun Z, Hornung V(2022) cGAS-STING signaling . 32 : R730-r734. Chen C, Xu P(2023) Cellular functions of cGAS-STING signaling . 33 : 630-648. Zhao J, Zhen N, Zhou Q, Lou J, Cui W, Zhang G, Tian B(2023) NETs Promote Inflammatory Injury by Activating cGAS-STING Pathway in Acute Lung Injury . 24. Messaoud-Nacer Y, Culerier E, Rose S, Maillet I, Rouxel N, Briault S, Ryffel B, Quesniaux VFJ, Togbe D(2022) STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS) . 13 : 269. Joshi JC, et al(2020) SPHK2-Generated S1P in CD11b(+) Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages . 30 : 4096-4109.e4095. Ning L, Wei W, Wenyang J, Rui X, Qing G(2020) Cytosolic DNA-STING-NLRP3 axis is involved in murine acute lung injury induced by lipopolysaccharide . 10 : e228. Wu YT, Xu WT, Zheng L, Wang S, Wei J, Liu MY, Zhou HP, Li QF, Shi X, Lv X(2023) 4-octyl itaconate ameliorates alveolar macrophage pyroptosis against ARDS via rescuing mitochondrial dysfunction and suppressing the cGAS/STING pathway . 118 : 110104. Liu XY, Chen W, Wei B, Shan YF, Wang C(2011) IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1 . 187 : 2559-2568. Wang J, et al(2018) Association of Abnormal Elevations in IFIT3 With Overactive Cyclic GMP-AMP Synthase/Stimulator of Interferon Genes Signaling in Human Systemic Lupus Erythematosus Monocytes . 70 : 2036-2045. Zhu W, Wang Y, Guan Y, Lu Y, Li Y, Sun L, Wang Y(2024) Rapamycin can alleviate the submandibular gland pathology of Sjögren's syndrome by limiting the activation of cGAS-STING signaling pathway . 32 : 1113-1131. Basters A, Geurink PP, Röcker A, Witting KF, Tadayon R, Hess S, Semrau MS, Storici P, Ovaa H, Knobeloch KP, Fritz G(2017) Structural basis of the specificity of USP18 toward ISG15 . 24 : 270-278. Martin-Fernandez M, et al(2022) A partial form of inherited human USP18 deficiency underlies infection and inflammation . 219. Gothe F, et al(2022) Aberrant inflammatory responses to type I interferon in STAT2 or IRF9 deficiency . 150 : 955-964.e916. Arimoto KI, et al(2023) Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis . 14 : 251. Kang JA, Jeon YJ(2020) Emerging Roles of USP18: From Biology to Pathophysiology . 21. Song C, Peng J, Wei Y, Shao J, Chen X, Zhang X, Xu J(2021) USP18 promotes tumor metastasis in esophageal squamous cell carcinomas via deubiquitinating ZEB1 . 409 : 112884. Liu W, Ge X, Zhou Z, Jiang D, Rong Y, Wang J, Ji C, Fan J, Yin G, Cai W(2021) Deubiquitinase USP18 regulates reactive astrogliosis by stabilizing SOX9 . 69 : 1782-1798. An S, et al(2017) USP18 protects against hepatic steatosis and insulin resistance through its deubiquitinating activity . 66 : 1866-1884. Ma ZR, et al(2023) USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy . 122 : 110617. Tang L, Liu X, Wang C, Shu C(2023) USP18 promotes innate immune responses and apoptosis in influenza A virus-infected A549 cells via cGAS-STING pathway . 585 : 240-247. Supplementary Files SupplementalMaterial.docx TheoriginalimagesofWesternblot2025.05.09.pdf Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 02 Sep, 2025 Reviewers agreed at journal 16 Aug, 2025 Reviewers invited by journal 11 Jun, 2025 Editor assigned by journal 21 May, 2025 First submitted to journal 20 May, 2025 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-6695431","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":469939239,"identity":"3569626a-01af-40d0-b36d-3ddbde5fe7ca","order_by":0,"name":"Nana Tang","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Tang","suffix":""},{"id":469939240,"identity":"374b9e6a-8e34-41f7-b017-18ac6500a4d6","order_by":1,"name":"Yang Yang","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":469939241,"identity":"4aae5438-e889-4c56-94c5-1d7392c5cce5","order_by":2,"name":"Yuanyuan Zeng","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Zeng","suffix":""},{"id":469939242,"identity":"14cc6038-8d54-4c23-8708-16a9812455f0","order_by":3,"name":"Jianjie Zhu","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jianjie","middleName":"","lastName":"Zhu","suffix":""},{"id":469939243,"identity":"415404b1-061a-4e23-a790-d8c80a14828d","order_by":4,"name":"Jianjun Li","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Li","suffix":""},{"id":469939244,"identity":"b6617141-90a8-4c0e-945c-b1eb0a27ca4a","order_by":5,"name":"Jiajia Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"Wang","suffix":""},{"id":469939245,"identity":"840ca56b-6304-4f86-8daf-e184b5de8738","order_by":6,"name":"Ling Ding","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Ding","suffix":""},{"id":469939246,"identity":"3388a125-d60a-445a-be91-d689c3171ef3","order_by":7,"name":"Jian-an Huang","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jian-an","middleName":"","lastName":"Huang","suffix":""},{"id":469939247,"identity":"7458a82e-7a00-48fa-8157-c35c66dcdb1d","order_by":8,"name":"Zeyi Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACPmYwJQHECYwPEipqCGthQ9LCbPDgzDEitCCYCWySD1uYidDCzmP84WObhb3B8eRnFYkNbAz87d0JBBzGY2A4s00iccOZZ2Y3EnfIMEicObuBoJZk3jaJBIMbCUAtZ9gYDCRyCWs5/LdNwt7gRvq3gsQ2ZqK0GDYztkkwbriRY8ZApBa2YsaecxKJM8+8KZZIOHOMh6Bf+PkPb/7wo6zOnu94+saPPypq5Pjbe/FrAQNGpNjhIawcDP4QqW4UjIJRMApGJgAAvTxD48j2UgIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2528-6909","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":true,"prefix":"","firstName":"Zeyi","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-05-19 06:03:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6695431/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6695431/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-06016-w","type":"published","date":"2026-02-13T15:58:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84558059,"identity":"47ce7b7e-3691-4544-b643-45d7ed6ef21a","added_by":"auto","created_at":"2025-06-13 12:08:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":361425,"visible":true,"origin":"","legend":"\u003cp\u003eScreening upregulated hub genes associated with macrophage M1 polarization in ALI/ARDS.\u003c/p\u003e\n\u003cp\u003e(A-B) Volcano plots showing differentially expressed genes (DEGs) in the datasets GSE57614 and GSE61298.\u003c/p\u003e\n\u003cp\u003e(C) Venn diagrams showing the co-upregulated DEGs in the datasets GSE57614 and GSE61298.\u003c/p\u003e\n\u003cp\u003e(D-E) KEGG enrichment analysis of upregulated DEGs in the datasets GSE57614 and GSE61298.\u003c/p\u003e\n\u003cp\u003e(F) Top 10 hub genes in the PPI network of co-upregulated DEGs.\u003c/p\u003e\n\u003cp\u003e(G) Validation of hub genes expression in M1 macrophages (RAW264.7) by qRT-PCR.\u003c/p\u003e\n\u003cp\u003e(H-I) Validation of hub genes expression in ARDS-related datasets GSE40885 and GSE68610.\u003c/p\u003e\n\u003cp\u003e(J-K) Validation of hub genes expression in mouse ALI-related datasets GSE2411 and GSE18341.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/83d35ec409a286a3fdab83fb.png"},{"id":84558178,"identity":"fed30c8f-1702-40ea-b878-44a1db13b91b","added_by":"auto","created_at":"2025-06-13 12:16:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":515757,"visible":true,"origin":"","legend":"\u003cp\u003eElevated IFIT3 expression in M1-polarized macrophages and the lungs of early ALI mice.\u003c/p\u003e\n\u003cp\u003e(A-B) RAW264.7 cells and murine immortalized bone marrow-derived macrophages (iBMDM) were stimulated with LPS (500 ng/mL) combined with IFN-γ (20 ng/mL) for 12 hours. qRT-PCR was performed to evaluate changes in gene expression.\u003c/p\u003e\n\u003cp\u003e(C) Western blot analysis showing the levels of IFIT3 and iNOS in RAW264.7 cells and iBMDM.\u003c/p\u003e\n\u003cp\u003e(D) Immunofluorescence staining for IFIT3 in RAW264.7 cells and iBMDM. Scale bars: 20 μm.\u003c/p\u003e\n\u003cp\u003e(E) Histological examination of lung tissue from C57BL/6 mice following intraperitoneal LPS injection (10 mg/kg) using H\u0026amp;E staining. Scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e(F) Measurement of the Wet/Dry weight ratio in mouse lung tissue at various time points following intraperitoneal LPS injection.\u003c/p\u003e\n\u003cp\u003e(G-H) qRT-PCR analysis and Western blot analysis of IFIT3 expression in mouse lung tissue at different time points following intraperitoneal LPS injection. ns: not significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/e7558dbcf4ea7fd612ac6bf1.png"},{"id":84558031,"identity":"a5e0b1db-788c-4eed-991e-84fce1cdad61","added_by":"auto","created_at":"2025-06-13 12:08:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":313041,"visible":true,"origin":"","legend":"\u003cp\u003eIFIT3 promotes macrophage M1 polarization and inflammation.\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR showing the efficiency of IFIT3 gene knockdown in RAW264.7 cells.\u003c/p\u003e\n\u003cp\u003e(B-D) qRT-PCR and Western blot analysis of IFIT3 knockdown and its effects on M1 macrophage markers (NOS2 and CD86).\u003c/p\u003e\n\u003cp\u003e(E-F) Flow cytometry analysis of ROS generation following IFIT3 gene knockdown.\u003c/p\u003e\n\u003cp\u003e(G-I) qRT-PCR analysis of IFIT3 knockdown and its effects on chemokine expression (CXCL2, CXCL3, and CCL2).\u003c/p\u003e\n\u003cp\u003e(J-L) ELISA measurement of cytokine secretion levels (IL-6, TNFα, and IL-1β) following IFIT3 gene knockdown. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/da820be7203ba0778c473e3d.png"},{"id":84558067,"identity":"1900aa74-3404-46a2-9468-b2fa657e0b95","added_by":"auto","created_at":"2025-06-13 12:08:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1090644,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of IFIT3 in mouse lung tissue alleviates early lung injury induced by LPS.\u003c/p\u003e\n\u003cp\u003e(A) Animal experiment flowchart.\u003c/p\u003e\n\u003cp\u003e(B) H\u0026amp;E staining to evaluate the pathological changes in lung tissue from different groups. Scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e(C) Lung Wet/Dry weight ratio to assess pulmonary edema in each group.\u003c/p\u003e\n\u003cp\u003e(D-G) qRT-PCR detection of IFIT3, NOS2, CXCL2, and CXLC3 expression in lung tissues from different groups.\u003c/p\u003e\n\u003cp\u003e(H-J) ELISA detected the levels of cytokines IL-6, TNFα, and IL-1β in lung tissues from different groups.\u003c/p\u003e\n\u003cp\u003e(K) Western blot showing that knockdown of IFIT3 expression inhibits M1 polarization of macrophages and activation of the cGAS-STING signaling pathway in early ALI mouse lung tissue.\u003c/p\u003e\n\u003cp\u003e(L-N) The expression levels of CD86 and CD206 in lung tissues from different groups were analyzed by IHC. Scale bar = 100 μm. ns: not significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/491f718a811d5e7d19647b13.png"},{"id":84557910,"identity":"9e18c75d-d77d-42bb-90a8-99fa0fc9f00d","added_by":"auto","created_at":"2025-06-13 12:08:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":508468,"visible":true,"origin":"","legend":"\u003cp\u003eThe JAK-STAT3 pathway upregulates IFIT3 expression, which in turn positively regulates the cGAS-STING signaling pathway in LPS and IFN-γ induced M1 macrophages.\u003c/p\u003e\n\u003cp\u003e(A) Western blot shows that stimulation of RAW264.7 cells with LPS and IFN-γ for 12 hours induces the activation of the JAK-STAT3 pathway.\u003c/p\u003e\n\u003cp\u003e(B) The treatment with the STAT3 phosphorylation inhibitor Stattic (2 μM) effectively inhibited the phosphorylation of STAT3 protein, resulting in significant suppression of IFIT3 and iNOS protein expression induced by LPS and IFN-γin RAW264.7 cells.\u003c/p\u003e\n\u003cp\u003e(C) Knockdown of IFIT3 does not affect the protein expression or phosphorylation of STAT3 in RAW264.7 cells.\u003c/p\u003e\n\u003cp\u003e(D) Knockdown of IFIT3 suppresses the activation of the cGAS-STING pathway induced by LPS and IFN-γ in RAW264.7 cells.\u003c/p\u003e\n\u003cp\u003e(E) Knockdown of IFIT3 inhibits the activation of NF-κB (p65) and MAPK (p38) inflammatory pathways induced by LPS and IFN-γ in RAW264.7 cells.\u003c/p\u003e\n\u003cp\u003e(F) Knockdown of IFIT3 inhibits the phosphorylation and nuclear translocation of IRF3 and p65 induced by LPS and IFN-γ in RAW264.7 cells.\u003c/p\u003e\n\u003cp\u003e(G) Immunofluorescence analysis shows that the knockdown of IFIT3 inhibits the phosphorylation of TBK1 induced by LPS and IFN-γ in RAW264.7 cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/3b70ea3c667d7b12eb3a82e3.png"},{"id":84557915,"identity":"5505671a-0e08-49a6-91ce-bfbe78f452cc","added_by":"auto","created_at":"2025-06-13 12:08:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":462739,"visible":true,"origin":"","legend":"\u003cp\u003eIFIT3 enhances STING protein stability by inhibiting the proteasomal degradation pathway.\u003c/p\u003e\n\u003cp\u003e(A) Knockdown of IFIT3 expression in RAW264.7 cells does not significantly affect the mRNA expression levels of STING. NS = not significant (p \u0026gt; 0.05), **p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e(B) Knockdown of IFIT3 expression in RAW264.7 cells decreases the protein expression levels of STING.\u003c/p\u003e\n\u003cp\u003e(C, D) Endogenous co-immunoprecipitation (Co-IP) was performed in iBMDM using IFIT3 / STING antibody to facilitate immunoprecipitation.\u003c/p\u003e\n\u003cp\u003e(E-F) Exogenous Co-IP was performed in 293T cells using Flag / HA antibody to facilitate immunoprecipitation.\u003c/p\u003e\n\u003cp\u003e(G) Immunofluorescence staining was performed to detect the localization of IFIT3 and STING in RAW264.7 cells. Scale bars = 20 μm.\u003c/p\u003e\n\u003cp\u003e(H, I) Knockdown of IFIT3 resulted in a shortened half-life of the STING protein and decreased protein stability, as indicated by the addition of cycloheximide (CHX, 100 μg/mL) to inhibit protein synthesis.\u003c/p\u003e\n\u003cp\u003e(J) RAW264.7 cells were treated with CHX for 24 hours, and MG132 (10 μM) was added 6 hours prior to harvesting protein samples to inhibit the proteasomal degradation pathway, partially reversing the decrease in STING protein levels caused by the knockdown of IFIT3 expression. ns: not significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/23ec38b317ec06701715e9d4.png"},{"id":84558032,"identity":"27ace6da-4cb4-43f8-b95e-ec007a198ab6","added_by":"auto","created_at":"2025-06-13 12:08:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":599217,"visible":true,"origin":"","legend":"\u003cp\u003eIFIT3 promotes the binding of deubiquitinase USP18 to STING and inhibits the ubiquitination of STING protein.\u003c/p\u003e\n\u003cp\u003e(A) RAW264.7 cells were stimulated with LPS and IFN-γ for 12 hours, with MG132 added to the culture medium 6 hours post-stimulation. Following the 12-hour stimulation period, cells were collected, and Co-IP experiments were performed using an anti-STING antibody. Western blot analysis demonstrated that knockdown of IFIT3 resulted in increased levels of STING protein ubiquitination (Weak exp: weak exposure; Strong exp: strong exposure).\u003c/p\u003e\n\u003cp\u003e(B) Docking simulation of the protein interaction between STING and USP18.\u003c/p\u003e\n\u003cp\u003e(C) Simulation analysis of protein interactions indicates that the binding stability between STING and USP18 is augmented in the presence of IFIT3.\u003c/p\u003e\n\u003cp\u003e(D) Immunofluorescence images showing comparable colocalization of IFIT3 (green) and STING (red) in both wild-type and USP18 knockout iBMDM, suggesting that USP18 deficiency does not impair their interaction. Nuclei were counterstained with DAPI (blue).\u003c/p\u003e\n\u003cp\u003e(E, F) Exogenous Co-IP was performed in 293T cells using Flag / His antibody to facilitate immunoprecipitation.\u003c/p\u003e\n\u003cp\u003e(G) USP18 knockout in iBMDM significantly attenuated LPS and IFN-γ induced STING protein expression, phosphorylation of downstream TBK1 and IRF3 in the cGAS-STING pathway, and M1 polarization marker iNOS expression, while showing no significant effects on IFIT3 or cGAS protein levels.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/53a4e7920398466198de8c3d.png"},{"id":84558066,"identity":"09a3954e-550a-4095-99a2-364186150cc9","added_by":"auto","created_at":"2025-06-13 12:08:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":215128,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of IFIT3 mediated macrophage polarization and early inflammation in acute lung injury\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/0e1e889528300b859f0e03b9.png"},{"id":102786154,"identity":"8c638259-8bd5-4eec-849e-f46505e0e912","added_by":"auto","created_at":"2026-02-16 16:12:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4774631,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/dfe65890-68f2-496a-a790-5d4059eb525b.pdf"},{"id":84557971,"identity":"cc123326-85f3-4a62-9b68-fc6f4cc34e2b","added_by":"auto","created_at":"2025-06-13 12:08:48","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5872389,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/2180834656554121947629c4.docx"},{"id":84558000,"identity":"bf478c81-6fff-44a6-992b-7301e5986e25","added_by":"auto","created_at":"2025-06-13 12:08:49","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":997023,"visible":true,"origin":"","legend":"","description":"","filename":"TheoriginalimagesofWesternblot2025.05.09.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6695431/v1/820d9cbcf8d44f9fab17cbb6.pdf"}],"financialInterests":"","formattedTitle":"IFIT3 stabilizes STING via USP18 to drive M1 macrophage polarization and early inflammation in acute lung injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute lung injury (ALI) / acute respiratory distress syndrome (ARDS) is an acute diffuse inflammatory lung injury caused by various factors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. First described in 1967[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], this clinical syndrome has persisted for nearly 60 years, with an in-hospital mortality rate in ARDS patients still exceeding 40% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], posing a significant threat to human health. Macrophages, highly plastic cells, play a pivotal role throughout the pathogenesis of ARDS[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. During the early stages of ALI/ARDS, there is an imbalance in the proportion of two primary phenotypes of pulmonary tissue macrophages, namely the classically activated (M1) and alternatively activated (M2) phenotypes. Increased M1 macrophages lead to excessive production of proinflammatory mediators and recruitment of inflammatory cells, exacerbating pulmonary inflammation and injury[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the precise mechanisms governing the dysregulation of macrophage polarization remain incompletely understood. Investigating critical molecules involved in the early regulation of lung macrophage polarization in ALI/ARDS may elucidate the pathogenesis of the disease and provide a basis for exploring new diagnostic and therapeutic targets.\u003c/p\u003e \u003cp\u003eInterferon-inducible protein with tetrapeptide repeats (IFIT), a member of the interferon-stimulated gene (ISG) family, with IFIT3 being a crucial member [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Under physiological conditions, IFIT3 is expressed at low levels in the majority of cell types, but its expression is significantly upregulated upon stimulation by viruses, bacteria, chlamydia, and interferons [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent studies have identified IFIT3 as a crucial immunoregulatory molecule involved not only in antiviral innate immunity but also in cellular functions such as proliferation, differentiation, apoptosis, and critical roles in the development of diseases like COVID-19, autoimmune disorders, and cancer[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the role of IFIT3 in the pathogenesis of ALI/ARDS remains unclear, with limited literature predominantly comprising bioinformatics analyses[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Currently, there is a lack of reports elucidating the function of IFIT3 in the early inflammatory response and the polarization mechanisms of pulmonary macrophages in ALI/ARDS.\u003c/p\u003e \u003cp\u003eIn this study, we found that IFIT3 is significantly upregulated in mouse lung tissue during the early phase of ALI. Silencing IFIT3 expression alleviates the M1 polarization of pulmonary macrophages and mitigates inflammatory lung injury. Under stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ), IFIT3 promotes M1 polarization of macrophages by positively regulating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Conversely, silencing IFIT3 expression inhibits the activation of the cGAS-STING pathway in the lung tissue of ALI mice, which results in a reduction of the inflammatory response. Furthermore, IFIT3 functions as a bridging protein that enhances the interaction between the deubiquitinase USP18 and STING by inhibiting STING ubiquitination and improving its stability. Overall, our study suggests that IFIT3 is a crucial regulatory molecule contributing to the imbalanced macrophage polarization and excessive inflammation during the early stages of ALI/ARDS, which may provide novel therapeutic targets for ALI/ARDS.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study exclusively examined male mice. It is unknown whether the findings are relevant for female mice. Wild-type C57BL/6 mice were procured from the Experimental Animal Center of Soochow University and housed under specific pathogen-free (SPF) conditions at room temperature. Male mice weighing 20-22 g, aged 6-8 weeks, were used in this study. All animal procedures were approved by and conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee of Soochow University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RAW264.7 murine macrophage cell line was obtained from Procell (Wuhan, China), while immortalized mouse bone marrow-derived macrophages (iBMDM) were procured from iCell Bioscience (Shanghai, China). Cells were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation and culture of primary bone marrow-derived macrophages (BMDM) were conducted using C57BL/6 mice (6-8 weeks old)[16]. Mouse peritoneal macrophages (PM) were isolated and cultured via peritoneal lavage[17].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentifying differentially expressed genes (DEGs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression profiles of human macrophage polarization (GSE57614 and GSE61298) were obtained from the Gene Expression Omnibus (GEO) database and analyzed to identify differentially expressed genes (DEGs) in M1 macrophages compared to the control group (M0+M2). GEO2R was employed for DEG identification and analysis, using a significance threshold of |log2(fold change)| \u0026ge; 3 and an adjusted \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 for statistical significance. Volcano plots depicting DEGs and Venn diagrams illustrating co-upregulated DEGs were generated using the \u0026apos;ggplot\u0026apos; package (version 3.3.6) within the R software environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of co-upregulated DEGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe co-upregulated DEGs were subjected to analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for comprehensive functional annotation. GO analysis encompassed three domains: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Functional enrichment analysis was conducted utilizing the Database for Annotation, Visualization, and Integrated Discovery web tool. The results of the analysis were visualized using the \u0026apos;clusterProfiler\u0026apos; package within the R programming environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of PPI networks and identification of hub genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein-protein interaction (PPI) network of the co-upregulated DEGs was constructed utilizing the STRING website. The resulting PPI network, with a minimum interaction score of 0.4, was visualized using Cytoscape version 3.9.1. The identification of hub genes was facilitated by using CytoHubba plugins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of hub gene expression in ALI/ARDS-related datasets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse ALI-associated datasets (GSE2411 and GSE18341) and ARDS patient-related datasets (GSE40885 and GSE68610) were selected from the GEO database as validation cohorts to assess the expression of hub genes, aiding in the identification of hub genes highly expressed in ALI/ARDS and associated with M1 polarization. Heatmaps illustrating the expression differences of hub genes were generated using the \u0026quot;ComplexHeatmap\u0026quot; package in the R programming environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal models of LPS-induced ALI and lung tissue IFIT3 knockdown\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWT C57BL/6 mice were induced with the ALI model by intraperitoneal injection of LPS derived from E. coli 055:B5 (Sigma-Aldrich, USA) at a dosage of 10 mg/kg body weight for 12h. Control group mice received intraperitoneal injection of an equivalent volume of saline. Ad-shNC and Ad-shIFIT3 (1\u0026times;10\u003csup\u003e9\u003c/sup\u003e pfu/mouse) were administered to the adenovirus gene delivery groups five days before LPS administration using intratracheal injection. Mice were anesthetized, and the adenovirus was directly injected into the trachea following surgical visualization. Mice were sacrificed after 12 hours, and the lung tissues were collected under aseptic conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInducing macrophage polarization to M1 phenotypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse cell lines RAW264.7 and iBMDM, as well as primary mouse cells, including BMDM and PM, were stimulated with LPS at a concentration of 500 ng/mL, combined with IFN-\u0026gamma; at 20 ng/mL, for 12 hours to induce M1 polarization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse lung tissues were fixed in 4% paraformaldehyde, followed by dehydration through a series of graded alcohol solutions. The tissues were then embedded in paraffin and cut into 5 \u0026micro;m sections. For histological analysis, the sections were stained with hematoxylin and eosin (H\u0026amp;E). For immunohistochemical analysis, the sections were deparaffinized, rehydrated, and washed before undergoing antigen retrieval. To minimize non-specific binding, the sections were blocked with 5% bovine serum albumin (BSA) and subsequently incubated overnight at 4\u0026deg;C with an IFIT3 antibody. Immunoreactivity was assessed using a horseradish peroxidase (HRP)-linked secondary antibody, and visualization was achieved through the application of 3,3\u0026apos;-diaminobenzidine tetrahydrochloride (DAB) (ZSGB-BIO, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured cells were seeded on sterile glass coverslips in 24-well plates and allowed to adhere overnight. After treatment, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h. Primary antibodies diluted in blocking buffer were incubated overnight at 4℃. After washing with PBS, cells were incubated with fluorophore-conjugated secondary antibodies (1:500) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI (1\u0026mu;g/mL) for 10 min. Finally, coverslips were mounted onto slides using antifade mounting medium and imaged using a confocal microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid transfection and viral infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse Flag-IFIT3 was purchased from GeneChem (China), and mouse HA-STING, His-STING, and Flag-USP18 were purchased from YouBio (China). All constructs were confirmed using DNA sequencing, and the plasmids were then transfected into 293T cells procured from Procell (China) using the Lipofectamine 2000 (Invitrogen, USA). The recombinant lentivirus containing the specific mouse shRNAs for IFIT3 was purchased from GeneChem (China). The recombinant lentivirus containing the mouse IFIT3 gene and recombinant adenovirus containing the mouse shRNA for IFIT3 were purchased from GenePharma (China). The shRNA target sequences are listed in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Reactive Oxygen Species (ROS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eROS levels were measured using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). Cells were cultured in six-well plates and treated according to the experimental design. DCFH-DA was diluted 1:1000 in serum-free DMEM. Following the removal of the culture medium, cells were washed three times with PBS and incubated with 1 mL of the diluted DCFH-DA solution for 20 minutes at 37\u0026deg;C. After incubation, cells were washed three times with serum-free DMEM to remove uninternalized DCFH-DA. Cells were then harvested using trypsinization, collected in centrifuge tubes, and centrifuged at 2000 rpm for 3 minutes. The supernatant was discarded, and the cell pellets were resuspended in 200 \u0026micro;L of PBS. For the blank control group, identical procedures were followed without DCFH-DA exposure. Flow cytometry was used to analyze the samples, and data were processed using FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of USP18 Knockout Cell Lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lenti CRISPR V2 plasmid was used to construct USP18 KO cells, and the following sgRNA sequences were used: USP18 sg1: 5\u0026prime;- TCGGCAGGATAACAGTGCCT-3\u0026prime;; USP18 sg2: 5\u0026prime;-TTCCTCTCTTCTGCACTCCG\u0026rsquo;. The USP18 lenti CRISPR V2 plasmid and packaging plasmids pVSVg and psPAX2 were transfected into HEK293T cells. The lentiviruses were collected, and USP18 KO iBMDM were selected with puromycin (2.5 \u0026mu;g/mL) for 7 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using GraphPad Prism 8.0. Data are expressed as the mean \u0026plusmn; standard deviation (SD). For comparisons between the two groups, a two-tailed unpaired Student\u0026apos;s t-test was employed. When comparing three or more groups, a one-way analysis of variance (ANOVA) followed by Tukey\u0026apos;s post-hoc test was utilized. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIFIT3 identified as a key regulator of macrophage M1 polarization in ALI/ARDS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo GEO datasets (GSE57614 and GSE61298) were analyzed to identify potential critical genes associated with macrophage polarization. In the GSE57614 dataset, 310 differentially expressed genes (DEGs) were identified, including 274 upregulated and 36 downregulated genes (Figure 1A). The GSE61298 dataset revealed 664 DEGs, with 485 upregulated and 179 downregulated genes (Figure 1B). A total of 102 co-upregulated DEGs were found in both datasets (Figure 1C). Functional enrichment analyses of these DEGs were conducted using GO and KEGG pathway analyses (Figures 1D, 1E, and S1A). The protein-protein interaction (PPI) network of the co-upregulated DEGs was constructed using the STRING database (Figure S1B). The CytoHubba plugin was used to identify essential nodes in the network, resulting in the top 10 hub genes (Figure 1F). RAW264.7 cells were polarized into the M1 phenotype using LPS, and qRT-PCR assessed the expression of hub genes. Compared to the M0 control group, the mRNA expression levels of several highly conserved human-mouse hub genes, including IL-6, IL-1\u0026beta;, CCL5, TNF\u0026alpha;, IFIT3, CD80, CD274, RSAD2, and IRF1, were significantly upregulated in the M1 group (Figure 1G), consistent with the bioinformatics analysis results. To further explore genes associated with M1 macrophage polarization in ALI/ARDS, human ARDS datasets (GSE40885 and GSE68610) and mouse ALI datasets (GSE2411 and GSE18341) were queried from the GEO database. Differential expression analysis of the top ten critical genes related to M1 macrophages between the ALI/ARDS groups and normal controls in these datasets revealed that IFIT3 exhibited significantly higher expression levels in the ALI/ARDS groups compared to the normal controls (Figures 1H-1K).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncreased expression of IFIT3 in M1 macrophages and early ALI mice lungs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 12 hours of stimulation with LPS and IFN-\u0026gamma;, qRT-PCR revealed elevated expression levels of the M1 polarization markers CD86 and iNOS (NOS2), along with increased levels of the inflammatory cytokine IL-6 in RAW264.7 and iBMDM cells compared to the control group. Conversely, no significant changes were observed in the expression of the M2 polarization marker CD206 (Figures 2A and 2B). Western blot analysis further corroborated the enhanced expression of iNOS protein relative to the control group, confirming the successful M1 polarization of RAW264.7 and iBMDM cells induced by LPS and IFN-\u0026gamma; (Figure 2C). Collectively, qRT-PCR, Western blot, and immunofluorescence assays demonstrated a significant upregulation of IFIT3 expression in M1 macrophages (Figures 2A-2D). Following intraperitoneal injection of LPS to establish the ALI model in C57BL/6 mice, the wet/dry weight ratios of lung tissues from LPS-injected mice at 6, 12, 18, and 24 hours were significantly increased compared to the control group (Figure 2F). H\u0026amp;E staining of lung tissue sections revealed thickening of alveolar walls, infiltration of inflammatory cells, increased exudation in alveolar spaces, and marked congestion (Figure 2E). These results suggest that intraperitoneal injection of LPS (10 mg/mL) for 6 to 24 hours successfully establishes an early ALI model in mice. qRT-PCR, Western blot, and immunohistochemistry results indicated that the mRNA and protein expression levels of IFIT3 in the lung tissues of ALI mice were elevated following LPS treatment at 6, 12, 18, and 24 hours compared to the control group (Figures 2G-2H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFIT3 promotes M1 macrophage polarization and inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of IFIT3 in M1 macrophage polarization, stable knockdown of IFIT3 expression was achieved in RAW264.7 cells via lentiviral infection (Figures 3A, 3D). qRT-PCR results demonstrated a significant increase in the mRNA levels of M1 markers (CD86, NOS2), proinflammatory cytokines (IL-6, TNF\u0026alpha;, and IL-1\u0026beta;), and chemokines (Cxcl2, Cxcl3, and Ccl2) in cells stimulated with LPS and IFN-\u0026gamma; compared to the shNC group. Upon IFIT3 knockdown and subsequent stimulation with LPS and IFN-\u0026gamma;, the expression of these genes was significantly reduced in the shIFIT3 group compared to the shNC group, indicating that silencing IFIT3 expression inhibits LPS and IFN-\u0026gamma;-induced M1 polarization of macrophages and reduces the production of multiple proinflammatory cytokines and chemokines (Figures 3B-3D, 3G-3I, and S4A-4C). ROS levels in RAW264.7 cells were measured by flow cytometry. Results showed that intracellular DCF fluorescence intensity in the (shNC+LPS+IFN-\u0026gamma;) group was significantly higher compared to the shNC group. In contrast, the intensity in the (shIFIT3+LPS+IFN-\u0026gamma;) group was markedly lower compared to the (shNC+LPS+IFN-\u0026gamma;) group (Figures 3E, 3F), indicating that IFIT3 knockdown significantly inhibits ROS generation in macrophages induced by LPS and IFN-\u0026gamma; stimulation. ELISA was used to assess the levels of inflammatory cytokines IL-6, TNF\u0026alpha;, and IL-1\u0026beta; in the cell culture supernatant. The results revealed that IFIT3 knockdown had no significant impact on the secretion of IL-6, TNF\u0026alpha;, and IL-1\u0026beta; under basal conditions. However, it significantly suppressed the secretion of these proinflammatory cytokines induced by LPS and IFN-\u0026gamma; stimulation (Figures 3J-3L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of IFIT3 in mouse lung tissue alleviates early lung injury induced by LPS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experimental procedures are illustrated in Figure 4A. qRT-PCR and Western blot analyses revealed that the expression of IFIT3 in lung tissues from the (shIFIT3+LPS) group was significantly reduced compared to the (shNC+LPS) group, indicating effective silencing of IFIT3 gene expression in mouse lung tissue following intratracheal administration of ADV-shIFIT3 (Figures 4D, 4K). The lung wet-to-dry weight ratio results showed a significant increase in water content in lung tissues of both the LPS group and the (shNC+LPS) group compared to the control group. However, the lung water content in the (shIFIT3+LPS) group was significantly lower than that in the (shNC+LPS) group and the LPS group (Figure 4C), suggesting that knockdown of IFIT3 effectively alleviated LPS-induced pulmonary edema. H\u0026amp;E staining results further demonstrated that compared to the LPS group and the (shNC+LPS) group, inflammatory cell infiltration was significantly reduced in the lung tissues of the (shIFIT3+LPS) group, with marked thinning of alveolar walls, reduced exudation in alveolar cavities, and decreased alveolar collapse (Figure 4B). qRT-PCR results indicated that intraperitoneal injection of LPS for 12 hours induced increased expression of NOS2, CXCL2, CXCL3, TNF\u0026alpha;, IL-6, and IL-1\u0026beta; in mouse lung tissue, promoting the early onset of pulmonary inflammation in acute lung injury (ALI) (Figures 4F, 4G, and S5A-S5G). The expression levels of these genes in the lung tissue of mice in the (shIFIT3+LPS) group were lower compared to the (shNC+LPS) group and the LPS group. ELISA results also indicated that the levels of inflammatory cytokines in the lung tissue of mice in the (shIFIT3+LPS) group were lower than those in the (shNC+LPS) group and the LPS group (Figures 4H-4J). Western blot and immunohistochemistry (IHC) analyses revealed that the protein expression of the M1 macrophage marker CD86 and the M2 macrophage marker CD206 were elevated in the lung tissue of mice in the LPS group and the (shNC+LPS) group compared to the control group. In the (shIFIT3+LPS) group, the protein levels of CD86 and the proinflammatory mediator iNOS in the lung tissue, as well as the phosphorylation levels of the inflammatory signaling molecule p65, were lower than those in the LPS group and the (shNC+LPS) group. In contrast, the expression of CD206 protein showed no significant difference among the groups (Figures 4K-4N). These results suggest that silencing the gene expression of IFIT3 in lung tissue can inhibit M1 macrophage polarization and proinflammatory activity, thereby improving LPS-induced inflammatory lung injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJAK-STAT3 pathway upregulates IFIT3 expression in LPS-induced M1 macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStimulation of RAW264.7 cells with LPS and IFN-\u0026gamma; for 12 hours resulted in increased phosphorylation levels of JAK1, JAK2, and STAT3 proteins compared to the control group (Figure 5A). Treatment with the STAT3-specific phosphorylation inhibitor Stattic (2 \u0026mu;M) for 2 hours effectively inhibited STAT3 protein phosphorylation. Subsequent stimulation with LPS and IFN-\u0026gamma; for 12 hours revealed that in the (Stattic+LPS+IFN-\u0026gamma;) group, the protein expression levels of IFIT3 and iNOS were lower compared to the (LPS+IFN-\u0026gamma;) group (Figure 5B). This suggests that activated STAT3 promotes M1 macrophage polarization and IFIT3 protein expression in response to LPS and IFN-\u0026gamma; stimulation. Knockdown of IFIT3 expression in RAW264.7 cells was achieved using lentiviral vectors, and its effect on STAT3 protein expression was assessed. Western blot analysis showed no significant differences in the levels of total STAT3 protein and phosphorylated STAT3 between the (shNC+LPS+IFN-\u0026gamma;) and (shIFIT3+LPS+IFN-\u0026gamma;) groups (Figure 5C). This indicates that IFIT3 knockdown does not affect STAT3 protein expression or phosphorylation, suggesting that IFIT3 functions downstream of the JAK-STAT3 pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnocking down IFIT3 inhibits the activation of the LPS and IFN-\u0026gamma; induced cGAS-STING signaling pathway in macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Western blot analysis demonstrated that co-stimulation of RAW264.7 cells (Figure 5D) and iBMDM cells (Figure S4D) with LPS and IFN-\u0026gamma; resulted in a significant upregulation of cGAS, STING, p-TBK1, and p-IRF3 protein levels in the (shNC+LPS+IFN-\u0026gamma;) group compared to the shNC group. This indicates that LPS and IFN-\u0026gamma; induce activation of the cGAS-STING signaling pathway. In contrast, the (shIFIT3+LPS+IFN-\u0026gamma;) group showed no significant difference in cGAS protein expression compared to the (shNC+LPS+IFN-\u0026gamma;) group. However, the levels of STING, p-TBK1, and p-IRF3 proteins were significantly reduced in the (shIFIT3+LPS+IFN-\u0026gamma;) group, suggesting that IFIT3 knockdown inhibits the activation of the cGAS-STING pathway induced by LPS and IFN-\u0026gamma;. IFIT3\u0026apos;s regulatory effect on this pathway is likely targeted at the STING molecule. Additionally, phosphorylation levels of NF-\u0026kappa;B p65 and MAPK p38 proteins were significantly increased in the (shNC+LPS+IFN-\u0026gamma;) group relative to the shNC group, indicating that LPS and IFN-\u0026gamma; stimulate the activation of NF-\u0026kappa;B p65 and MAPK p38 proteins in RAW264.7 cells, thus triggering related inflammatory pathways. In the (shIFIT3+LPS+IFN-\u0026gamma;) group, the phosphorylation levels of NF-\u0026kappa;B p65 and MAPK p38 were markedly lower compared to the (shNC+LPS+IFN-\u0026gamma;) group (Figure 5E). This suggests that IFIT3 knockdown suppresses the activation of NF-\u0026kappa;B and MAPK inflammatory signaling pathways induced by LPS and IFN-\u0026gamma;. Furthermore, after IFIT3 knockdown in RAW264.7 cells, both cytoplasmic and nuclear levels of phosphorylated p65 and IRF3 proteins were lower in the (shIFIT3+LPS+IFN-\u0026gamma;) group compared to the (shNC+LPS+IFN-\u0026gamma;) group (Figure 5F). This finding implies that IFIT3 promotes the phosphorylation of nuclear transcription factors NF-\u0026kappa;B p65 and IRF3, facilitating their nuclear translocation in response to LPS and IFN-\u0026gamma;. Immunofluorescence staining also revealed that LPS and IFN-\u0026gamma; enhance the phosphorylation of TBK1 protein in RAW264.7 cells. In the (shIFIT3+LPS+IFN-\u0026gamma;) group, TBK1 phosphorylation levels in the cytoplasm were significantly lower than those in the (shNC+LPS+IFN-\u0026gamma;) group (Figure 5G), suggesting that IFIT3 knockdown inhibits TBK1 phosphorylation induced by LPS and IFN-\u0026gamma;. In vivo experiments showed that in the lung tissues of mice from the LPS group and (shNC+LPS) group, cGAS and STING protein levels, along with the phosphorylation of TBK1 and IRF3 proteins, were significantly upregulated compared to the control group. This indicates the activation of the cGAS-STING signaling pathway in ALI mouse lung tissues. Conversely, in the lung tissues of mice from the (shIFIT3+LPS) group, cGAS, and STING protein levels, as well as the phosphorylation of TBK1 and IRF3, were lower than those in the LPS and (shNC+LPS) groups (Figure 4K). These results suggest that early-stage upregulation of IFIT3 in ALI mouse lung tissues activates the cGAS-STING signaling pathway, promoting macrophage polarization to the M1 phenotype and enhancing the inflammatory response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFIT3 increases the stability of STING protein by inhibiting the proteasomal degradation pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKnockdown of IFIT3 expression did not significantly affect the mRNA levels of STING. Still, it led to a substantial reduction in STING protein levels (Figures 6A, 6B), suggesting that IFIT3 may regulate STING protein expression through post-translational modifications. Endogenous co-immunoprecipitation (Co-IP) performed in iBMDM cells (Figures 6C, 6D) and exogenous Co-IP conducted in 293T cells (Figures 6E, 6F) confirmed the interaction between IFIT3 and STING proteins. Immunofluorescence experiments demonstrated the co-localization of IFIT3 and STING in the cytoplasm (Figure 6G), further supporting the interaction between these two proteins. Since alterations in IFIT3 expression affect only STING protein levels without impacting mRNA expression, it was hypothesized that IFIT3 might influence STING protein degradation and stability. To test this hypothesis, RAW264.7 cells were treated with LPS and IFN-\u0026gamma; for 12 hours to induce IFIT3 expression, followed by the addition of cycloheximide (CHX) to inhibit STING protein synthesis and assess the effect of IFIT3 on STING protein half-life. Results showed that the half-life of STING protein was significantly reduced in the shIFIT3 group compared to the shNC group, indicating that IFIT3 inhibits STING protein degradation (Figures 6H, 6I). Furthermore, inhibition of the proteasomal degradation pathway using MG132 led to a partial reversal of the reduction in STING protein levels observed in the shIFIT3 group 24 hours after CHX treatment (Figure 6J). These findings suggest that IFIT3 enhances STING protein stability by inhibiting the proteasomal degradation pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFIT3 enhances STING protein stability by inhibiting ubiquitination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUbiquitination plays a critical role in regulating STING homeostasis[18-20]. Endogenous ubiquitination Co-IP assays showed that the level of ubiquitination in the shIFIT3 group RAW264.7 cells was higher than in the shNC group (Figure 7A). This indicates that knocking down IFIT3 expression increases STING protein ubiquitination, suggesting that high expression of IFIT3 induced by LPS and IFN-\u0026gamma; can inhibit STING protein ubiquitination. Previous studies have shown that IFIT3 lacks enzymatic activity, but its structural characteristics facilitate interactions with other proteins, thereby affecting their functions[8]. Therefore, we hypothesize that IFIT3 may inhibit STING ubiquitination by promoting the binding of other proteins to STING. To identify proteins that might bind to IFIT3 and potentially influence protein ubiquitination, we performed Co-IP combined with mass spectrometry (Figure S6A outlines the experimental workflow). KEGG analysis of the mass spectrometry results showed enrichment in the \u0026quot;Ubiquitin-mediated proteolysis pathway\u0026quot; (Figure 7B). Proteins detected in the IFIT3 overexpression (OE) group with expression levels at least 10 times higher than the Vector group (FC \u0026ge; 10) were selected as candidate proteins. A total of 150 proteins potentially interacting specifically with IFIT3 were identified (Figure 6C), including five from the top ten proteins predicted to bind with IFIT3 according to the STRING database (Figure S6C): USP18, IFIT1, IFIH1, RSAD2, and ISG15. Among these, USP18 belongs to the deubiquitinase family, and studies have shown that it can promote the inhibition of STING protein ubiquitination and degradation[21]. In addition to USP18, the mass spectrometry results also revealed interactions between IFIT3 and the deubiquitinases OTUD5 and OTUD6B, with literature reporting that OTUD5 can also deubiquitinate STING[22]. These findings support our hypothesis that IFIT3 acts as a bridging protein, enhancing the binding of deubiquitinases to STING, thereby inhibiting STING ubiquitination and increasing STING protein stability. Protein interaction simulation analysis showed that upon the addition of IFIT3, the binding potential energy between STING and USP18 decreased, indicating that IFIT3 significantly enhances the stability of the binding between STING and USP18 proteins (Figures 7D, 7E). Co-IP experiments in 293T cells confirmed the interaction between STING and USP18 (Figures 7F, 7G). \u0026nbsp;Further,successful knockout of USP18 in iBMDM followed by induction of M1 phenotype polarization showed no significant effects on IFIT3 and cGAS protein expression. However, it markedly reduced STING protein expression along with its downstream phosphorylation levels of TBK1 and IRF3, as well as iNOS protein expression. suggesting that IFIT3 may act as a bridging protein to promote the binding of deubiquitinase USP18 to STING. These results demonstrate that IFIT3 acts as a bridging protein to promote the interaction between USP18 and STING, thereby enhancing STING protein stability.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pathogenesis of ALI/ARDS remains incompletely elucidated, involving inflammatory responses in lung tissue and multiple systemic systems, coagulation abnormalities, and dysregulation of signaling pathways, which are initially the body's normal defense responses to infection and injury. However, once excessively and extensively activated, these functions may lead to tissue damage, ultimately triggering ALI/ARDS. ARDS can be classified pathologically into early (acute exudative phase), proliferative phase, and fibrotic phase[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. During the early stages of ARDS, excessive inflammatory response is the most prominent feature, with typical pathological changes characterized by neutrophilic alveolitis and the formation of hyaline membranes representing diffuse alveolar damage [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The primary instigator of induced neutrophil recruitment is pulmonary tissue macrophages[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Pulmonary macrophages, pivotal effector cells in lung tissue's response to external stimuli, play a critical role in the pathogenesis of pulmonary inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Macrophages exhibit high plasticity and heterogeneity; under different pathological and physiological conditions and surrounding microenvironments, they undergo phenotypic and functional changes induced by various signals and cytokines from an inactive M0 state, a process known as macrophage polarization [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Macrophage polarization primarily divides into two subtypes: classically activated M1 and alternatively activated M2 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. An imbalance in M1/M2 polarization can lead to the occurrence and development of various diseases [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the early stages (acute exudative phase) of ALI/ARDS, lung macrophages primarily polarize towards M1. Sustained M1 polarization can release multiple inflammatory mediators and genes, such as IL-6, TNFα, IL-1β, NOS, and reactive oxygen species (ROS), recruiting neutrophils from the bloodstream into lung tissues and alveolar spaces, thereby triggering severe inflammatory reactions and progressing lung injury[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Regulation of the imbalance in M1/M2 polarization of macrophages can mitigate lung tissue damage and improve the prognosis of ALI/ARDS. IFIT3, an essential member of the interferon-stimulated genes and the IFIT family has been increasingly recognized for its critical role not only in various pathogen infections, especially in antiviral defense and immune responses, but also in influencing diverse cellular functions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the specific role of IFIT3 in the pathogenesis of ALI/ARDS remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we utilized bioinformatics to identify hub genes related to M1 macrophage polarization that are highly expressed in lung samples of ARDS/ALI, using data from the GEO database. Validation was performed through the construction of an M1 cell model and subsequent qRT-PCR experiments. The results identified four well-established cytokines (IL-6, IL-1β, CCL5, and TNFα) as the highest-expressed hub genes. Notably, IFIT3 ranked fifth in expression, but its role in ALI/ARDS is unclear, with no existing mechanistic studies. Consequently, we selected IFIT3 for further investigation to assess its influence on macrophage polarization and its potential roles and mechanisms in ALI/ARDS. Referencing previous literature[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and our experimental verification, we found that stimulation of mouse macrophages with LPS combined with IFN-γ for 12 hours significantly increased the expression of CD86 and iNOS compared to the control group, while CD206 showed no difference, indicating that this induction condition could polarize macrophages toward an M1 phenotype. We also observed a significant increase in the expression of three proinflammatory factors (IL-6, IL-1β, and TNFα) after induction in RAW264.7 cells.\u003c/p\u003e \u003cp\u003eHowever, iBMDM cells exhibited a less pronounced inflammatory phenotype compared to RAW264.7 cells. Thus, RAW264.7 was primarily used as the cell model for our study, with iBMDM cells serving as experimental supplements. These two macrophage cell lines showed some differences in their inflammatory phenotypes post-induction, likely due to factors such as cellular origin and differential responses to inducers. Under normal physiological conditions, the expression of IFIT3 is low in most cells, but it significantly increases under pathological conditions induced by stimuli [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our experimental results also demonstrated that the basal expression levels of IFIT3 in primary macrophages (PM and BMDM) and macrophage cell lines (RAW264.7 and iBMDM) were low. Still, its expression significantly increased upon induction of M1 polarization, indicating high expression of IFIT3 in M1 macrophages. IFIT3 exhibited low-level expression in normal control mouse lung tissues, and its mRNA and protein expression significantly increased in early ALI mouse lung tissues. These in vitro and in vivo experiments further confirmed the accuracy and reliability of the bioinformatics analysis results. Limited studies have investigated whether IFIT3 can regulate macrophage phenotype polarization and function. Subsequent to stable knockdown of IFIT3 expression using lentiviral vectors in vitro, we found a significant attenuation of M1 phenotype polarization of macrophages induced by LPS and IFN-γ: downregulation of M1 markers (CD86 and iNOS), proinflammatory factors (IL-6, IL-1β, and TNFα), and chemokines (CCL2, CXCL2, and CXCL3), along with reduced ROS generation.\u003c/p\u003e \u003cp\u003eConversely, stable overexpression of IFIT3 further enhanced the expression of iNOS, IL-6, IL-1β, and TNFα induced by LPS and IFN-γ. These results suggest that high expression of IFIT3 promotes M1 polarization and related inflammatory responses in macrophages. Additionally, we observed that without induction stimulation by LPS and IFN-γ, overexpression of IFIT3 alone did not promote macrophage polarization or expression of inflammatory factors, indicating that IFIT3 is not an inducer of M1 polarization but rather a regulatory molecule influencing the process of M1 polarization. IFIT3 positively regulates macrophage M1 phenotype polarization and inflammatory responses. Furthermore, the experiment in vivo showed that silencing IFIT3 expression in lung tissues significantly improved lung tissue damage and inflammatory responses in early ALI mice. In summary, we have confirmed that IFIT3 promotes excessive inflammation and lung injury in early ALI/ARDS by regulating M1 polarization of pulmonary macrophages.\u003c/p\u003e \u003cp\u003eThe JAK-STAT pathway is widely implicated in the pathogenesis of ARDS [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Under pathological conditions, phosphorylated STAT1, STAT2, and IRF9 form the ISGF3 complex, which translocates to the nucleus and binds to ISRE elements in the promoter regions of IFIT family genes, thereby stimulating the expression of IFIT family genes[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Whether other members of the STAT family can also regulate IFIT3 expression remains unclear. Whether STAT3 also regulates the expression of IFIT3 in macrophages remains to be explored. Our study found that in the LPS and IFN-γ induced M1 polarization model of macrophages, the phosphorylation levels of JAK1, JAK2, and STAT3 proteins were significantly increased, indicating the activation of the JAK-STAT3 pathway. In the early LPS-induced ALI mouse model, the phosphorylation of STAT3 protein in lung tissues was also significantly increased, suggesting the activation of STAT3 protein in M1 macrophages and ALI mouse lung tissues. After inhibiting STAT3 phosphorylation with Stattic and then inducing macrophages with LPS and IFN-γ, the protein levels of IFIT3 and iNOS were significantly reduced, indicating that activated STAT3 promotes IFIT3 expression and M1 polarization of macrophages. Conversely, knocking down IFIT3 expression did not affect the expression and phosphorylation levels of STAT3 protein, confirming that the JAK-STAT3 pathway is upstream of IFIT3 and that activated STAT3 positively regulates the polarization of macrophage M1 phenotype and IFIT3 expression under inducer stimulation.\u003c/p\u003e \u003cp\u003eThe cGAS-STING signaling pathway is a crucial innate immune pathway that is linked to inflammation, infection, autoimmunity, degenerative diseases, and cancer[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The essence of ARDS lies in its inflammatory lung injury, where the aberrant presence of endogenous and exogenous DNA can serve as PAMP/DAMP to induce and promote ARDS progression, hence garnering attention to the role of the cGAS-STING pathway in ARDS in recent years. Existing research indicates that the cGAS-STING signaling pathway promotes the occurrence and development of ALI/ARDS [\u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], but the specific regulatory mechanisms await further clarification. Currently, there is limited research on the association between IFIT3 and the cGAS-STING pathway, with relevant literature primarily focused on viral infections and autoimmune diseases [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Increased activity of the cGAS-STING signaling pathway and expression levels of IFIT3 were observed in PBMCs of systemic lupus erythematosus (SLE) patients compared to healthy controls. Co-IP detection suggests that IFIT3 interacts with STING and TBK1, activating the cGAS-STING signaling pathway, thereby promoting disease activity in SLE patients[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], although the molecular mechanisms remain to be elucidated. High-throughput sequencing and in vivo experiments in mice have shown that IFIT3 is one of the core genes regulating Sj\u0026ouml;gren's Syndrome (SS), leading to aberrant activation of autophagy through the cGAS-STING pathway, which is a crucial factor in SS pathogenesis[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, the related regulatory mechanisms are not yet precise.\u003c/p\u003e \u003cp\u003eIn summary, the activation of the cGAS-STING signaling pathway has been implicated in ALI/ARDS, although the regulatory mechanisms remain poorly understood. Our study investigates the role of IFIT3 in macrophage polarization and its influence on the cGAS-STING pathway in ALI/ARDS. In vitro experiments, it was demonstrated that LPS and IFN-γ-induced M1 macrophage polarization led to increased expression of cGAS, STING, and TBK1 phosphorylation, highlighting the pathway's activation in M1 macrophages. Notably, stable knockdown of IFIT3 in macrophages resulted in decreased STING levels and reduced phosphorylation of TBK1, IRF3, p65, and p38, suggesting that IFIT3 regulates the cGAS-STING pathway primarily through STING. In vivo studies in ALI mice confirmed that IFIT3 knockdown inhibited cGAS-STING pathway activity and M1 polarization, alleviating inflammatory responses. Interestingly, a decrease in cGAS levels in lung tissues was observed in vivo, which contrasts with in vitro findings. This discrepancy may arise because in vitro experiments focused on macrophages. At the same time, the in vivo approach affected multiple cell types, necessitating further investigations to elucidate the underlying mechanisms of IFIT3's role in ALI/ARDS.\u003c/p\u003e \u003cp\u003eWe further investigated the potential mechanisms by which IFIT3 regulates the cGAS-STING pathway. The IFIT3 protein is predominantly located in the cytoplasm, and currently, no known enzymatic activity has been found for it. IFIT3 protein contains multiple structural motifs of tetratricopeptide repeats (TPR), which enable IFIT3 to form complexes with other proteins to carry out various cellular functions[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Through protein docking simulations, immunofluorescence, and Co-IP experiments, we confirmed that IFIT3 can interact with STING protein. We also observed that knocking down IFIT3 had no significant effect on STING mRNA expression both in vivo and in vitro, which was inconsistent with the changes in STING protein expression, suggesting that IFIT3 may regulate STING protein levels by affecting post-translational modifications and protein stability. Our study showed that upregulated IFIT3 inhibits STING protein ubiquitination and degradation through the ubiquitination-proteasomal system in the M1 macrophage model, thereby increasing the stability and abundance of STING protein. Co-IP combined with protein mass spectrometry (MS) showed that the most robust proteins interacting with IFIT3 included the deubiquitinating enzyme USP18. Prediction from the STRING database suggested that the top ten proteins interacting with IFIT3 also included USP18, and protein interaction simulation analysis revealed that after the addition of IFIT3, the stability of the interaction between STING and USP18 proteins was enhanced. Co-IP experiments also confirmed the interaction between STING and USP18 proteins.\u003c/p\u003e \u003cp\u003eUSP18 is a unique member of the ubiquitin-specific protease (USP) family, previously believed to be unresponsive to ubiquitin but capable of removing ubiquitin-like (UbI) protein ISG15 (interferon-stimulated gene 15) from substrate proteins[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Additionally, USP18 can interact with type I interferon receptors, inhibiting interferon signaling [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Contrary to initial findings, recent research has demonstrated that USP18 also possesses deubiquitinase activity[\u003cspan additionalcitationids=\"CR58 CR59\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. USP18 deubiquitinates the cGAS protein and inhibits cGAS protein degradation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. USP18 recruits USP20 to deconjugate K48-linked ubiquitin chains from STING, enhancing STING protein stability as well as the expression of type I IFN and proinflammatory cytokines. Knockout of USP18 leads to increased K48-linked ubiquitination of STING and accelerated degradation of STING, impairing downstream activation of IRF3 and NF-κB[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The MS results in our study revealed that in addition to USP18, the deubiquitinating enzymes that interact with IFIT3 and exhibit high expression levels include OTUD5 and OTUD6B. Previous studies have shown that the deubiquitinase OTUD5 can interact with STING, cleaving its K48-linked polyubiquitin chains and promoting STING protein stability [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the role of the deubiquitinase OTUD6B in regulating STING ubiquitination levels remains unexplored and warrants further investigation.\u003c/p\u003e \u003cp\u003eIn summary, our study found that in the early stages of ALI/ARDS, the upregulated expression of IFIT3 in lung tissue acts as a bridging protein, promoting the binding of STING to deubiquitinases such as USP18. This interaction inhibits the ubiquitin-mediated degradation pathway of STING, thereby increasing STING protein stability and activating the cGAS-STING pathway. Consequently, this process enhances M1 polarization of pulmonary macrophages and inflammatory responses, facilitating the progression of ALI/ARDS. These findings contribute to a deeper understanding of the pathogenesis of ALI/ARDS and provide new targets for drug research and therapeutic interventions.\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eLimitations of the study\u003c/h2\u003e \u003cp\u003eOur study has several limitations. First, the in vitro experiments were conducted using only two mouse macrophage cell lines. To improve the generalizability of the findings, future research should incorporate primary macrophages and human-derived macrophages. The role of IFIT3 as a bridging protein between deubiquitinases and STING requires further validation, including more precise identification of the interaction sites. Additionally, while we utilized adenoviral vector-mediated RNAi for transient IFIT3 knockdown, future studies should employ gene knockout mouse models to provide a more comprehensive understanding of IFIT3 function. It is also necessary to use flow cytometry of alveolar lavage fluid to assess changes in IFIT3 expression in alveolar macrophages during early-stage ALI. Finally, clinical observational studies are needed to evaluate the diagnostic and prognostic value of IFIT3 in ARDS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNana Tang and Yang Yang contributed equally to this work and are considered co-first authors. Nana Tang and Yang Yang designed and performed the experiments, analyzed the data, and wrote the manuscript. Yuanyuan Zeng, Zhu Jianjie, Li Jianjun, Wang Jiajia, and Ding Ling assisted with experimental procedures and data collection. Jianan Huang and Zeyi Liu supervised the study, provided critical revisions, and are the corresponding authors responsible for overall project direction and manuscript finalization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Guizhou Science and Technology Department Plan Supporting Project of China (No. 2021 General 086); the Jiangsu Provincial Medical Key Discipline (No. ZDXK202201); NSFC Cultivation Program Project of the Affiliated Hospital of Guizhou Medical University (No. GYFYNSFC2023-55); National Postdoctoral Fellowship Program (No. GZC20231895).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available in the article and Supplementary Material files. All original data for this study can be obtained from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eMouse studies were performed under protocols approved by Soochow University\u0026apos;s IACUC (Approval ID: 2023-551), following institutional animal care standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All the authors consent to the publication of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGorman EA, O\u0026apos;Kane CM, McAuley DF(2022) Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management\u003cstrong\u003e.\u003c/strong\u003e 400\u003cstrong\u003e:\u003c/strong\u003e1157-1170.\u003c/li\u003e\n\u003cli\u003eVillar J, Szakmany T, Grasselli G, Camporota L(2023) Redefining ARDS: a paradigm shift\u003cstrong\u003e.\u003c/strong\u003e 27\u003cstrong\u003e:\u003c/strong\u003e416.\u003c/li\u003e\n\u003cli\u003eBellani G, et al(2016) Epidemiology, Patterns of Care, and Mortality for Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries\u003cstrong\u003e.\u003c/strong\u003e 315\u003cstrong\u003e:\u003c/strong\u003e788-800.\u003c/li\u003e\n\u003cli\u003eLiu C, Xi L, Liu Y, Mak JCW, Mao S, Wang Z, Zheng Y(2023) An Inhalable Hybrid Biomimetic Nanoplatform for Sequential Drug Release and Remodeling Lung Immune Homeostasis in Acute Lung Injury Treatment\u003cstrong\u003e.\u003c/strong\u003e 17\u003cstrong\u003e:\u003c/strong\u003e11626-11644.\u003c/li\u003e\n\u003cli\u003eWang L, Wang D, Zhang T, Ma Y, Tong X, Fan H(2023) The role of immunometabolism in macrophage polarization and its impact on acute lung injury/acute respiratory distress syndrome\u003cstrong\u003e.\u003c/strong\u003e 14\u003cstrong\u003e:\u003c/strong\u003e1117548.\u003c/li\u003e\n\u003cli\u003eLiu C, Xiao K, Xie L(2022) Advances in the Regulation of Macrophage Polarization by Mesenchymal Stem Cells and Implications for ALI/ARDS Treatment\u003cstrong\u003e.\u003c/strong\u003e 13\u003cstrong\u003e:\u003c/strong\u003e928134.\u003c/li\u003e\n\u003cli\u003eZhang S, Chu C, Wu Z, Liu F, Xie J, Yang Y, Qiu H(2020) IFIH1 Contributes to M1 Macrophage Polarization in ARDS\u003cstrong\u003e.\u003c/strong\u003e 11\u003cstrong\u003e:\u003c/strong\u003e580838.\u003c/li\u003e\n\u003cli\u003eZhang W, Li Y, Xin S, Yang L, Jiang M, Xin Y, Wang Y, Cao P, Zhang S, Yang Y, Lu J(2023) The emerging roles of IFIT3 in antiviral innate immunity and cellular biology\u003cstrong\u003e.\u003c/strong\u003e 95\u003cstrong\u003e:\u003c/strong\u003ee28259.\u003c/li\u003e\n\u003cli\u003eSchindewolf C, et al(2023) SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3\u003cstrong\u003e.\u003c/strong\u003e 97\u003cstrong\u003e:\u003c/strong\u003ee0153222.\u003c/li\u003e\n\u003cli\u003eXu S, Huang J, Xun Z, Li S, Fu Y, Lin N, Wu W, Chen T, Liu C, Ou Q(2022) IFIT3 Is Increased in Serum from Patients with Chronic Hepatitis B Virus (HBV) Infection and Promotes the Anti-HBV Effect of Interferon Alpha via JAK-STAT2 In Vitro\u003cstrong\u003e.\u003c/strong\u003e 10\u003cstrong\u003e:\u003c/strong\u003ee0155722.\u003c/li\u003e\n\u003cli\u003eZhang W, Jiang M, Liao X, Li Y, Xin S, Yang L, Xin Y, Umar A, Lu J(2023) IFIT3 inhibits Epstein-Barr virus reactivation via upregulating innate immunity\u003cstrong\u003e.\u003c/strong\u003e 95\u003cstrong\u003e:\u003c/strong\u003ee29237.\u003c/li\u003e\n\u003cli\u003eWu YY, Xing J, Li XF, Yang YL, Shao H, Li J(2023) Roles of interferon induced protein with tetratricopeptide repeats (IFIT) family in autoimmune disease\u003cstrong\u003e.\u003c/strong\u003e 22\u003cstrong\u003e:\u003c/strong\u003e103453.\u003c/li\u003e\n\u003cli\u003eWang Z, et al(2020) Inflammatory IFIT3 renders chemotherapy resistance by regulating post-translational modification of VDAC2 in pancreatic cancer\u003cstrong\u003e.\u003c/strong\u003e 10\u003cstrong\u003e:\u003c/strong\u003e7178-7192.\u003c/li\u003e\n\u003cli\u003eMargaroli C, et al(2023) Interferon-dependent signaling is critical for viral clearance in airway neutrophils\u003cstrong\u003e.\u003c/strong\u003e 8.\u003c/li\u003e\n\u003cli\u003eYang X, Wang J, Liu W(2023) Molecular markers of type II alveolar epithelial cells in acute lung injury by bioinformatics analysis\u003cstrong\u003e.\u003c/strong\u003e 13\u003cstrong\u003e:\u003c/strong\u003e17797.\u003c/li\u003e\n\u003cli\u003eTang N, Yang Y, Xie Y, Yang G, Wang Q, Li C, Liu Z, Huang JA(2024) CD274 (PD-L1) negatively regulates M1 macrophage polarization in ALI/ARDS\u003cstrong\u003e.\u003c/strong\u003e 15\u003cstrong\u003e:\u003c/strong\u003e1344805.\u003c/li\u003e\n\u003cli\u003eYang Y, Wu J, Li Q, Wang J, Mu L, Hui L, Li M, Xu W, Yang H, Wei L(2022) A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx\u003cstrong\u003e.\u003c/strong\u003e 11.\u003c/li\u003e\n\u003cli\u003eCho W, et al(2023) Targeted Protein Upregulation of STING for Boosting the Efficacy of Immunotherapy\u003cstrong\u003e.\u003c/strong\u003e 62\u003cstrong\u003e:\u003c/strong\u003ee202300978.\u003c/li\u003e\n\u003cli\u003eMutlu M, et al(2024) Small molecule induced STING degradation facilitated by the HECT ligase HERC4\u003cstrong\u003e.\u003c/strong\u003e 15\u003cstrong\u003e:\u003c/strong\u003e4584.\u003c/li\u003e\n\u003cli\u003eTao Y, Yin S, Liu Y, Li C, Chen Y, Han D, Huang J, Xu S, Zou Z, Yu Y(2023) UFL1 promotes antiviral immune response by maintaining STING stability independent of UFMylation\u003cstrong\u003e.\u003c/strong\u003e 30\u003cstrong\u003e:\u003c/strong\u003e16-26.\u003c/li\u003e\n\u003cli\u003eZhang M, Zhang MX, Zhang Q, Zhu GF, Yuan L, Zhang DE, Zhu Q, Yao J, Shu HB, Zhong B(2016) USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA\u003cstrong\u003e.\u003c/strong\u003e 26\u003cstrong\u003e:\u003c/strong\u003e1302-1319.\u003c/li\u003e\n\u003cli\u003eGuo Y, et al(2021) OTUD5 promotes innate antiviral and antitumor immunity through deubiquitinating and stabilizing STING\u003cstrong\u003e.\u003c/strong\u003e 18\u003cstrong\u003e:\u003c/strong\u003e1945-1955.\u003c/li\u003e\n\u003cli\u003eMatthay MA, Zemans RL, Zimmerman GA, Arabi YM, Beitler JR, Mercat A, Herridge M, Randolph AG, Calfee CS(2019) Acute respiratory distress syndrome\u003cstrong\u003e.\u003c/strong\u003e 5\u003cstrong\u003e:\u003c/strong\u003e18.\u003c/li\u003e\n\u003cli\u003eThille AW, Pe\u0026ntilde;uelas O, Lorente JA, Fern\u0026aacute;ndez-Segoviano P, Rodriguez JM, Aramburu JA, Panizo J, Esteban A, Frutos-Vivar F(2017) Predictors of diffuse alveolar damage in patients with acute respiratory distress syndrome: a retrospective analysis of clinical autopsies\u003cstrong\u003e.\u003c/strong\u003e 21\u003cstrong\u003e:\u003c/strong\u003e254.\u003c/li\u003e\n\u003cli\u003eBos LDJ, Ware LB(2022) Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes\u003cstrong\u003e.\u003c/strong\u003e 400\u003cstrong\u003e:\u003c/strong\u003e1145-1156.\u003c/li\u003e\n\u003cli\u003eKumar V(2020) Pulmonary Innate Immune Response Determines the Outcome of Inflammation During Pneumonia and Sepsis-Associated Acute Lung Injury\u003cstrong\u003e.\u003c/strong\u003e 11\u003cstrong\u003e:\u003c/strong\u003e1722.\u003c/li\u003e\n\u003cli\u003eChen X, Tang J, Shuai W, Meng J, Feng J, Han Z(2020) Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome\u003cstrong\u003e.\u003c/strong\u003e 69\u003cstrong\u003e:\u003c/strong\u003e883-895.\u003c/li\u003e\n\u003cli\u003eLocati M, Curtale G, Mantovani A(2020) Diversity, Mechanisms, and Significance of Macrophage Plasticity\u003cstrong\u003e.\u003c/strong\u003e 15\u003cstrong\u003e:\u003c/strong\u003e123-147.\u003c/li\u003e\n\u003cli\u003eXue S, Su Z, Liu D(2023) Immunometabolism and immune response regulate macrophage function in atherosclerosis\u003cstrong\u003e.\u003c/strong\u003e 90\u003cstrong\u003e:\u003c/strong\u003e101993.\u003c/li\u003e\n\u003cli\u003eWang C, Ma C, Gong L, Guo Y, Fu K, Zhang Y, Zhou H, Li Y(2021) Macrophage Polarization and Its Role in Liver Disease\u003cstrong\u003e.\u003c/strong\u003e 12\u003cstrong\u003e:\u003c/strong\u003e803037.\u003c/li\u003e\n\u003cli\u003eFunes SC, Rios M, Escobar-Vera J, Kalergis AM(2018) Implications of macrophage polarization in autoimmunity\u003cstrong\u003e.\u003c/strong\u003e 154\u003cstrong\u003e:\u003c/strong\u003e186-195.\u003c/li\u003e\n\u003cli\u003eKadomoto S, Izumi K, Mizokami A(2021) Macrophage Polarity and Disease Control\u003cstrong\u003e.\u003c/strong\u003e 23.\u003c/li\u003e\n\u003cli\u003eWang Z, Wang Z(2023) The role of macrophages polarization in sepsis-induced acute lung injury\u003cstrong\u003e.\u003c/strong\u003e 14\u003cstrong\u003e:\u003c/strong\u003e1209438.\u003c/li\u003e\n\u003cli\u003eXu F, Song H, An B, Xiao Q, Cheng G, Tan G(2019) NF-\u0026kappa;B-Dependent IFIT3 Induction by HBx Promotes Hepatitis B Virus Replication\u003cstrong\u003e.\u003c/strong\u003e 10\u003cstrong\u003e:\u003c/strong\u003e2382.\u003c/li\u003e\n\u003cli\u003eChikhalya A, Dittmann M, Zheng Y, Sohn SY, Rice CM, Hearing P(2021) Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression\u003cstrong\u003e.\u003c/strong\u003e 12\u003cstrong\u003e:\u003c/strong\u003ee0282921.\u003c/li\u003e\n\u003cli\u003eHuang Y, Tian C, Li Q, Xu Q(2019) TET1 Knockdown Inhibits Porphyromonas gingivalis LPS/IFN-\u0026gamma;-Induced M1 Macrophage Polarization through the NF-\u0026kappa;B Pathway in THP-1 Cells\u003cstrong\u003e.\u003c/strong\u003e 20.\u003c/li\u003e\n\u003cli\u003eZhou F, Mei J, Han X, Li H, Yang S, Wang M, Chu L, Qiao H, Tang T(2019) Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF-\u0026kappa;B/MAPK signaling and protecting chondrocytes\u003cstrong\u003e.\u003c/strong\u003e 9\u003cstrong\u003e:\u003c/strong\u003e973-985.\u003c/li\u003e\n\u003cli\u003eGajjela BK, Zhou MM(2022) Calming the cytokine storm of COVID-19 through inhibition of JAK2/STAT3 signaling\u003cstrong\u003e.\u003c/strong\u003e 27\u003cstrong\u003e:\u003c/strong\u003e390-400.\u003c/li\u003e\n\u003cli\u003eZhang Y, Gao Z, Jiang F, Yan H, Yang B, He Q, Luo P, Xu Z, Yang X(2023) JAK-STAT signaling as an ARDS therapeutic target: Status and future trends\u003cstrong\u003e.\u003c/strong\u003e 208\u003cstrong\u003e:\u003c/strong\u003e115382.\u003c/li\u003e\n\u003cli\u003ePurohit M, Gupta G, Afzal O, Altamimi ASA, Alzarea SI, Kazmi I, Almalki WH, Gulati M, Kaur IP, Singh SK, Dua K(2023) Janus kinase/signal transducers and activator of transcription (JAK/STAT) and its role in Lung inflammatory disease\u003cstrong\u003e.\u003c/strong\u003e 371\u003cstrong\u003e:\u003c/strong\u003e110334.\u003c/li\u003e\n\u003cli\u003eSong J, Guo Y, Wang D, Quan R, Wang J, Liu J(2023) Seneca Valley virus 3C(pro) antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals\u003cstrong\u003e.\u003c/strong\u003e 97\u003cstrong\u003e:\u003c/strong\u003ee0072723.\u003c/li\u003e\n\u003cli\u003eNowicka H, Sekrecka A, Blaszczyk K, Kluzek K, Chang CY, Wesoly J, Lee CK, Bluyssen HAR(2023) ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs\u003cstrong\u003e.\u003c/strong\u003e 24.\u003c/li\u003e\n\u003cli\u003eSun Z, Hornung V(2022) cGAS-STING signaling\u003cstrong\u003e.\u003c/strong\u003e 32\u003cstrong\u003e:\u003c/strong\u003eR730-r734.\u003c/li\u003e\n\u003cli\u003eChen C, Xu P(2023) Cellular functions of cGAS-STING signaling\u003cstrong\u003e.\u003c/strong\u003e 33\u003cstrong\u003e:\u003c/strong\u003e630-648.\u003c/li\u003e\n\u003cli\u003eZhao J, Zhen N, Zhou Q, Lou J, Cui W, Zhang G, Tian B(2023) NETs Promote Inflammatory Injury by Activating cGAS-STING Pathway in Acute Lung Injury\u003cstrong\u003e.\u003c/strong\u003e 24.\u003c/li\u003e\n\u003cli\u003eMessaoud-Nacer Y, Culerier E, Rose S, Maillet I, Rouxel N, Briault S, Ryffel B, Quesniaux VFJ, Togbe D(2022) STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS)\u003cstrong\u003e.\u003c/strong\u003e 13\u003cstrong\u003e:\u003c/strong\u003e269.\u003c/li\u003e\n\u003cli\u003eJoshi JC, et al(2020) SPHK2-Generated S1P in CD11b(+) Macrophages Blocks STING to Suppress the Inflammatory Function of Alveolar Macrophages\u003cstrong\u003e.\u003c/strong\u003e 30\u003cstrong\u003e:\u003c/strong\u003e4096-4109.e4095.\u003c/li\u003e\n\u003cli\u003eNing L, Wei W, Wenyang J, Rui X, Qing G(2020) Cytosolic DNA-STING-NLRP3 axis is involved in murine acute lung injury induced by lipopolysaccharide\u003cstrong\u003e.\u003c/strong\u003e 10\u003cstrong\u003e:\u003c/strong\u003ee228.\u003c/li\u003e\n\u003cli\u003eWu YT, Xu WT, Zheng L, Wang S, Wei J, Liu MY, Zhou HP, Li QF, Shi X, Lv X(2023) 4-octyl itaconate ameliorates alveolar macrophage pyroptosis against ARDS via rescuing mitochondrial dysfunction and suppressing the cGAS/STING pathway\u003cstrong\u003e.\u003c/strong\u003e 118\u003cstrong\u003e:\u003c/strong\u003e110104.\u003c/li\u003e\n\u003cli\u003eLiu XY, Chen W, Wei B, Shan YF, Wang C(2011) IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1\u003cstrong\u003e.\u003c/strong\u003e 187\u003cstrong\u003e:\u003c/strong\u003e2559-2568.\u003c/li\u003e\n\u003cli\u003eWang J, et al(2018) Association of Abnormal Elevations in IFIT3 With Overactive Cyclic GMP-AMP Synthase/Stimulator of Interferon Genes Signaling in Human Systemic Lupus Erythematosus Monocytes\u003cstrong\u003e.\u003c/strong\u003e 70\u003cstrong\u003e:\u003c/strong\u003e2036-2045.\u003c/li\u003e\n\u003cli\u003eZhu W, Wang Y, Guan Y, Lu Y, Li Y, Sun L, Wang Y(2024) Rapamycin can alleviate the submandibular gland pathology of Sj\u0026ouml;gren\u0026apos;s syndrome by limiting the activation of cGAS-STING signaling pathway\u003cstrong\u003e.\u003c/strong\u003e 32\u003cstrong\u003e:\u003c/strong\u003e1113-1131.\u003c/li\u003e\n\u003cli\u003eBasters A, Geurink PP, R\u0026ouml;cker A, Witting KF, Tadayon R, Hess S, Semrau MS, Storici P, Ovaa H, Knobeloch KP, Fritz G(2017) Structural basis of the specificity of USP18 toward ISG15\u003cstrong\u003e.\u003c/strong\u003e 24\u003cstrong\u003e:\u003c/strong\u003e270-278.\u003c/li\u003e\n\u003cli\u003eMartin-Fernandez M, et al(2022) A partial form of inherited human USP18 deficiency underlies infection and inflammation\u003cstrong\u003e.\u003c/strong\u003e 219.\u003c/li\u003e\n\u003cli\u003eGothe F, et al(2022) Aberrant inflammatory responses to type I interferon in STAT2 or IRF9 deficiency\u003cstrong\u003e.\u003c/strong\u003e 150\u003cstrong\u003e:\u003c/strong\u003e955-964.e916.\u003c/li\u003e\n\u003cli\u003eArimoto KI, et al(2023) Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis\u003cstrong\u003e.\u003c/strong\u003e 14\u003cstrong\u003e:\u003c/strong\u003e251.\u003c/li\u003e\n\u003cli\u003eKang JA, Jeon YJ(2020) Emerging Roles of USP18: From Biology to Pathophysiology\u003cstrong\u003e.\u003c/strong\u003e 21.\u003c/li\u003e\n\u003cli\u003eSong C, Peng J, Wei Y, Shao J, Chen X, Zhang X, Xu J(2021) USP18 promotes tumor metastasis in esophageal squamous cell carcinomas via deubiquitinating ZEB1\u003cstrong\u003e.\u003c/strong\u003e 409\u003cstrong\u003e:\u003c/strong\u003e112884.\u003c/li\u003e\n\u003cli\u003eLiu W, Ge X, Zhou Z, Jiang D, Rong Y, Wang J, Ji C, Fan J, Yin G, Cai W(2021) Deubiquitinase USP18 regulates reactive astrogliosis by stabilizing SOX9\u003cstrong\u003e.\u003c/strong\u003e 69\u003cstrong\u003e:\u003c/strong\u003e1782-1798.\u003c/li\u003e\n\u003cli\u003eAn S, et al(2017) USP18 protects against hepatic steatosis and insulin resistance through its deubiquitinating activity\u003cstrong\u003e.\u003c/strong\u003e 66\u003cstrong\u003e:\u003c/strong\u003e1866-1884.\u003c/li\u003e\n\u003cli\u003eMa ZR, et al(2023) USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy\u003cstrong\u003e.\u003c/strong\u003e 122\u003cstrong\u003e:\u003c/strong\u003e110617.\u003c/li\u003e\n\u003cli\u003eTang L, Liu X, Wang C, Shu C(2023) USP18 promotes innate immune responses and apoptosis in influenza A virus-infected A549 cells via cGAS-STING pathway\u003cstrong\u003e.\u003c/strong\u003e 585\u003cstrong\u003e:\u003c/strong\u003e240-247.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"IFIT3, STING, Acute lung injury, deubiquitinase, M1 macrophage polarization, USP18","lastPublishedDoi":"10.21203/rs.3.rs-6695431/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6695431/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe imbalance in macrophage M1/M2 polarization is a critical factor driving excessive inflammation during the early stages of Acute Lung Injury (ALI) /Acute Respiratory Distress Syndrome (ARDS). However, the underlying regulatory mechanisms remain poorly understood. In this study, we investigated the role of interferon-inducible protein with tetrapeptide repeats 3 (IFIT3) in the context of early-stage ALI. Our findings demonstrate that IFIT3 expression is significantly elevated in macrophages of ALI mice. We further show that IFIT3 positively regulates the cGAS-STING pathway, which promotes M1 polarization and exacerbates lung inflammation in ALI. Additionally, IFIT3 interacts with STING to inhibit its ubiquitination-mediated degradation, potentially acting as a bridging molecule facilitating the interaction between STING and the deubiquitinase USP18. These results highlight IFIT3 as a crucial player in the pathogenesis of ALI/ARDS through the modulation of macrophage polarization, suggesting that targeting IFIT3 may offer a novel therapeutic strategy for managing ALI /ARDS.\u003c/p\u003e","manuscriptTitle":"IFIT3 stabilizes STING via USP18 to drive M1 macrophage polarization and early inflammation in acute lung injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 12:08:30","doi":"10.21203/rs.3.rs-6695431/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-09-02T11:34:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-16T06:54:03+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-11T16:27:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-21T06:59:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-05-21T01:28:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"debdaab3-a171-4829-9cec-68954411cb95","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-6695431","link":"https://doi.org/10.1007/s00018-025-06016-w","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2026-02-13 15:58:09","publishedOnDateReadable":"February 13th, 2026"},"versionCreatedAt":"2025-06-13 12:08:30","video":"","vorDoi":"10.1007/s00018-025-06016-w","vorDoiUrl":"https://doi.org/10.1007/s00018-025-06016-w","workflowStages":[]},"version":"v1","identity":"rs-6695431","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6695431","identity":"rs-6695431","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}