IFIH1/IRF1/STAT1 promotes sepsis associated inflammatory lung injury via activating macrophage M1 polarization.

A growing body of research has shown that the phenotypic change in macrophages from M0 to M1 is essential for the start of the inflammatory process in septic acute respiratory distress syndrome (ARDS). Potential treatment targets might be identified with more knowledge of the molecular regulation of M1 macrophages in septic ARDS. A multi-microarray interrelated analysis of high-throughput experiments from ARDS patients and macrophage polarization was conducted to identify the hub genes associated with macrophage M1 polarization and septic ARDS. Lipopolysaccharide (LPS) and Poly (I:C) were utilized to stimulate bone marrow-derived macrophages (BMDMs) for M1-polarized macrophage model construction. Knock down of the hub genes on BMDMs via shRNAs was used to screen the genes regulating macrophage M1 polarization in vitro. The cecal ligation and puncture (CLP) mouse model was constructed in knockout (KO) mice and wild-type (WT) mice to explore whether the screened genes regulate macrophage M1 polarization in septic ARDS in vivo. ChIP-seq and further experiments on BMDMs were performed to investigate the molecular mechanism. The bioinformatics analysis of gene expression profiles from a clinical cohort of 26 ARDS patients and macrophage polarization found that the 5 hub genes (IFIH1, IRF1, STAT1, IFIT3, GBP1) may have a synergistic effect on macrophage M1 polarization in septic ARDS. Further in vivo investigations indicated that IFIH1, STAT1 and IRF1 contribute to macrophage M1 polarization. The histological evaluation and immunohistochemistry of the lungs from the IRF1-/- and WT mice indicated that knockout of IRF1 markedly alleviated CLP-induced lung injury and M1-polarized infiltration. Moreover, the molecular mechanism investigations indicated that knockdown of IFIH1 markedly promoted IRF1 translocation into the nucleus. Knockout of IRF1 significantly decreases the expression of STAT1. ChIP-seq and PCR further confirmed that IRF1, as a transcription factor of STAT1, binds to the promoter region of STAT1. IRF1 was identified as the key molecule that regulates macrophage M1polarization and septic ARDS development in vivo and in vitro. Moreover, as the adaptor in response to infection mimics irritants, IFIH1 promotes IRF1 (transcription factor) translocation into the nucleus to initiate STAT1 transcription.

Wang A ，Kang X ，Wang J ，Zhang S ... -  《-》

IFIH1 Contributes to M1 Macrophage Polarization in ARDS.

Accumulated evidence has demonstrated that the macrophage phenotypic switch from M0 to M1 is crucial in the initiation of the inflammatory process of acute respiratory distress syndrome (ARDS). Better insight into the molecular control of M1 macrophages in ARDS may identify potential therapeutic targets. In the current study, 36 candidate genes associated with the severity of ARDS and simultaneously involved in M1-polarized macrophages were first screened through a weighted network algorithm on all gene expression profiles from the 26 ARDS patients and empirical Bayes analysis on the gene expression profiles of macrophages. STAT1, IFIH1, GBP1, IFIT3, and IRF1 were subsequently identified as hub genes according to connectivity degree analysis and multiple external validations. Among these candidate genes, IFIH1 had the strongest connection with ARDS through the RobustRankAggreg algorithm. It was selected as a crucial gene for further investigation. For in vitro validation, the RAW264.7 cell line and BMDMs were transfected with shIFIH1 lentivirus and plasmid expression vectors of IFIH1. Cellular experimental studies further confirmed that IFIH1 was a novel regulator for promoting M1 macrophage polarization. Moreover, gene set enrichment analysis (GSEA) and in vitro validations indicated that IFIH1 regulated M1 polarization by activating IRF3. In addition, previous studies demonstrated that activation of IFIH1-IRF3 was stimulated by viral RNAs or RNA mimics. Surprisingly, the current study found that LPS could also induce IFIH1-IRF3 activation via a MyD88-dependent mechanism. We also found that only IFIH1 expression without LPS or RNA mimic stimulation could not affect IRF3 activation and M1 macrophage polarization. These findings were validated on two types of macrophages, RAW264.7 cells and BMDMs, which expanded the knowledge on the inflammatory roles of IFIH1 and IRF3, suggesting IFIH1 as a potential target for ARDS treatment.

Zhang S ，Chu C ，Wu Z ，Liu F ，Xie J ，Yang Y ，Qiu H ... -  《Frontiers in Immunology》

Irf1- and Egr1-activated transcription plays a key role in macrophage polarization: A multiomics sequencing study with partial validation.

Macrophage polarization has a causal role in the pathogenesis and resolution of various clinical diseases. DNA-binding transcription factors (TFs) have been identified as essential factors during gene transcription. Better insight into the TFs that regulate macrophage polarization could provide novel therapeutic targets. IFN-γ (50 ng/mL) or IL4 (20 ng/mL) was utilized to stimulate bone marrow-derived macrophages from mice for 24 h for M1- and M2-polarized macrophage model construction, respectively. First, ATAC-seq (Assay for Targeting Accessible-Chromatin with high throughout sequencing) and motif analysis were conducted to identify potential transcription factors (TFs) involved in M1 and M2 macrophage polarization. Second, essential TFs were identified through RNA-seq, after which, their expression was compared between M0-polarized and M1/M2-polarized macrophages. Furthermore, a multiomic analysis of RNA-seq (siRNA knock down of the identified TFs), ChIP-seq and ATAC-seq was utilized to explore the TF-regulated molecular network. GO and KEGG analyses were used to expound the main functions of the TF-regulated molecular network. Finally, the top 5 TF-regulated genes were validated through flow cytometry, ELISA and qPCR. The cut-off values for high-throughput sequencing and qPCR were FDR < 0.05 and P < 0.05, respectively. Compared with M0 macrophages, 10,771 and 4,848 peaks were identified by ATAC-seq during M1 and M2 macrophage polarization, respectively (FDR < 0.05). Fifty and 62 TF binding motifs were identified for the TFs that participate in M1 and M2 macrophage polarization, respectively. The most significantly highly expressed TFs in M1 and M2 macrophages were identified by RNA-seq as Irf1 and Egr1, with LogFC values of 3.2 and 2.8, respectively. Multiomic analyses further found that Irf1 regulated the transcription of 90 genes and that Egr1 regulated the transcription of 116 genes. The Irf1-regulated molecular network played a key role in the inflammatory response and viral defence of M1 macrophages, and 116 Egr1-regulated genes included anti-inflammatory and cell proliferation genes. Validation experiments indicated that IFN-γ-induced Gbp5, Nos2, CD86, Cxcl10 and Cxcl5 expression was significantly downregulated in siIrf1-BMDMs, and IL4-induced Itgax, Nipal1, Bhlhe40, CD206 and Ffar4 expression was significantly downregulated in siEgr1-BMDMs (P < 0.05). Through multiomic analyses of epigenetic sequencing and RNA-seq with partial validation, the current study found that Irf1- and Egr1-induced transcription plays key roles in M1 and M2 macrophage polarization, respectively.

Chu YB ，Li J ，Jia P ，Cui J ，Zhang R ，Kang X ，Lv M ，Zhang S ... -  《-》

IBR1, a novel endogenous IFIH1-binding dsRNA, governs IFIH1 activation and M1 macrophage polarisation in ARDS.

Zhang S ，Huang W ，Wu X ，Chen H ，Wang L ，Chao J ，Xie J ，Qiu H ... -  《Clinical and Translational Medicine》

CD274 (PD-L1) negatively regulates M1 macrophage polarization in ALI/ARDS.

Acute lung injury (ALI)/severe acute respiratory distress syndrome (ARDS) is a serious clinical syndrome characterized by a high mortality rate. The pathophysiological mechanisms underlying ALI/ARDS remain incompletely understood. Considering the crucial role of immune infiltration and macrophage polarization in the pathogenesis of ALI/ARDS, this study aims to identify key genes associated with both ALI/ARDS and M1 macrophage polarization, employing a combination of bioinformatics and experimental approaches. The findings could potentially reveal novel biomarkers for the diagnosis and management of ALI/ARDS. Gene expression profiles relevant to ALI were retrieved from the GEO database to identify co-upregulated differentially expressed genes (DEGs). GO and KEGG analyses facilitated functional annotation and pathway elucidation. PPI networks were constructed to identify hub genes, and differences in immune cell infiltration were subsequently examined. The expression of hub genes in M1 versus M2 macrophages was evaluated using macrophage polarization datasets. The diagnostic utility of CD274 (PD-L1) for ARDS was assessed by receiver operating characteristic (ROC) analysis in a validation dataset. Experimental confirmation was conducted using two LPS-induced M1 macrophage models and an ALI mouse model. The role of CD274 (PD-L1) in M1 macrophage polarization and associated proinflammatory cytokine production was further investigated by siRNA-mediated silencing. A total of 99 co-upregulated DEGs were identified in two ALI-linked datasets. Enrichment analysis revealed that these DEGs were mainly involved in immune-inflammatory pathways. The following top 10 hub genes were identified from the PPI network: IL-6, IL-1β, CXCL10, CD274, CCL2, TLR2, CXCL1, CCL3, IFIT1, and IFIT3. Immune infiltration analysis revealed a significantly increased abundance of M1 and M2 macrophages in lung tissue from the ALI group compared to the control group. Subsequent analysis confirmed that CD274 (PD-L1), a key immunological checkpoint molecule, was highly expressed within M1 macrophages. ROC analysis validated CD274 (PD-L1) as a promising biomarker for the diagnosis of ARDS. Both in vitro and in vivo experiments supported the bioinformatics analysis and confirmed that the JAK-STAT3 pathway promotes CD274 (PD-L1) expression on M1 macrophages. Importantly, knockdown of CD274 (PD-L1) expression potentiated M1 macrophage polarization and enhanced proinflammatory cytokines production. This study demonstrates a significant correlation between CD274 (PD-L1) and M1 macrophages in ALI/ARDS. CD274 (PD-L1) functions as a negative regulator of M1 polarization and the secretion of proinflammatory cytokines in macrophages. These findings suggest potential new targets for the diagnosis and treatment of ALI/ARDS.

Tang N ，Yang Y ，Xie Y ，Yang G ，Wang Q ，Li C ，Liu Z ，Huang JA ... -  《Frontiers in Immunology》