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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Structure of the complex monolayer of gemini surfactant and DNA at the air/water interface.

The properties of the complex monolayers composed of cationic gemini surfactants, [C(18)H(37)(CH(3))(2)N(+)-(CH(2))(s)-N(+)(CH(3))(2)C(18)H(37)],2Br(-) (18-s-18 with s = 3, 4, 6, 8, 10 and 12), and ds-DNA or ss-DNA at the air/water interface were in situ studied by the surface pressure-area per molecule (π-A) isotherm measurement and the infrared reflection absorption spectroscopy (IRRAS). The corresponding Langmuir-Blodgett (LB) films were also investigated by the atomic force microscopy (AFM), the Fourier transform infrared spectroscopy (FT-IR), and the circular dichroism spectroscopy (CD). The π-A isotherms and AFM images reveal that the spacer of gemini surfactant has a significant effect on the surface properties of the complex monolayers. As s ≤ 6, the gemini/ds-DNA complex monolayers can both laterally and normally aggregate to form fibril structures with heights of 2.0-7.0 nm and widths of from several tens to ~300 nm. As s > 6, they can laterally condense to form the platform structure with about 1.4 nm height. Nevertheless, FT-IR, IRRAS, and CD spectra, as well as AFM images, suggest that DNA retains its double-stranded character when complexed. This is very important and meaningful for gene therapy because it is crucial to maintain the extracellular genes undamaged to obtain a high transfection efficiency. In addition, when s ≤ 6, the gemini/ds-DNA complex monolayers can experience a transition of DNA molecule from the double-stranded helical structure to a typical ψ-phase with a supramolecular chiral order.