Fig 1: B cells regulate disease tolerance during sepsis independent of early inflammatory responses.(A) Aspartate aminotransferase (ASAT) plasma levels at 18 hr p.i. with E. coli, of lipopolysaccharide (LPS) or NaCl pretreated wildtype or lymphocyte deficient mice (Rag2-/-), which have received either phosphate-buffered saline (PBS) or splenocytes i.v. 3 weeks prior infection. (B) Schematic depiction of the treatment procedure for T cell depletion experiments. (C) ASAT plasma levels 18 hr p.i. with E. coli of mice, which were depleted from CD4+ or CD8+ T cells prior to LPS or NaCl pretreatment. (D) ASAT plasma levels of LPS or NaCl pretreated wildtype or B cell deficient (JHT) mice at 18 hr p.i. with E. coli. (E) Schematic depiction of the treatment procedure for splenocyte and B cell transfer experiments. (F) ASAT plasma levels of LPS or NaCl pretreated Rag2-/- mice at 18 hr p.i. with E. coli, which have been reconstituted with bone marrow derived B cells 3 weeks before infection. (G) ASAT plasma levels at 18 hr p.i. with E. coli of LPS or NaCl pretreated mice, which were splenectomized or sham operated 1 week before LPS or NaCl pre-exposure (i.e. 3 weeks before infection). (H–I) IL-6 levels in plasma and liver of NaCl or LPS pretreated wildtype or Rag2-/- mice at 6 hr p.i. with E. coli. (J) ASAT plasma levels of NaCl or LPS pretreated Ifnar1-/- mice at 18 hr p.i. with E. coli. (K) ASAT plasma levels of NaCl or LPS pretreated Nfkb1-/- mice at 18 hr p.i. with E. coli. Data in (A) and (G–J) are representative out of 2–3 experiments (n=3–8/experimental group). Data in (D) and (K) are pooled from 2 independent experiments (n=2–7/experimental group). Data in (C) and (F) are from a single experiment (n=6–8/group). Data are and presented as mean +/-SEM. * p≤0.05 and ** p≤0.01.
Fig 2: FXR Binds to the Promoter Site of IL-6. (A) Four potential FXR binding sites in the promoter site of IL-6 were predicted based on the databases of PROMO and NUBIscan within about 2000bp upstream of the transcription start site of IL-6 (upper). Plasmids containing different numbers of the binding site based on the basic PGL-3 plasmid were constructed and co-transfected respectively with FXR-overexpression plasmids as well as Renilla plasmids together into 293T cells. 24h after the transfection, cells were then activated with or without 5μM GW4064 for extra 24h. Luciferase activity was then evaluated with the dual luciferase assay (bottom). Data are shown as mean ± SEM. + means compared to unstimulated control and * means comparing to the luciferase activity of PGL-3 basic vector. ++ p < 0.01; +++ p < 0.001. *p < 0.05; **p < 0.01; NS, no significant difference, two-way ANOVA, followed by Sidak’s multiple comparisons test. (B) RAW 264.7 macrophages were incubated with or without 5μM FXR agonist GW4064 for 24h, and then stimulated with or without 100ng/ml LPS for 24h. Cells were then fixed with 1% w/v formaldehyde solution and chromatin from the cells was analyzed for recruitment of FXR to the last predictive binding site of the IL-6 promoter by ChIP assay. The quantification of DNA in the precipitation with FXR antibody was normalized to input chromatin and plotted relative to the Rabbit IgG. Data are shown as mean ± SEM. **p < 0.01, two-way ANOVA, followed by Sidak’s multiple comparisons test.
Fig 3: Proposed Model for hnRNP M-Dependent Repression for IL6 Expression in Resting, Early-, and Late-Activated Macrophages(Left) In resting macrophages, hnRNP M associates with chromatin, at the IL6 genomic locus, through interactions with RNA. These interactions may be direct with target transcripts expressed at lowlevels or spuriously or indirect via protein interactions with other RNA binding proteins or through interactions with other chromatin-associated RNAs, e.g., linc-RNAs. (Middle panel) When macrophages receive an innate immune stimulus, they transcriptionally activate genes like IL6. A population of “poised” hnRNP M can associate with chromatin-bound pre-mRNAs in these cells, slowing IL6 intron removal and preventing full maturation of IL6 nascent transcripts. (Right) As early macrophage activation proceeds, hnRNP M is phosphorylated at S574 in a p38-MAPK-dependent fashion. Phosphorylation of hnRNP M releases it from the IL6 genomic locus, relieves inhibition of IL6 splicing, and allows for full induction of IL6 gene expression. Figure generated using BioRender software.
Fig 4: hnRNP M Regulates Expression of Innate Immune Genes during Salmonella Typhimurium Infection(A) Western blot analysis and qRT-PCR of hnRNP M in RAW 264.7 macrophages with β-actin as a loading control. Valúes are mean (SD) representative of 3 biological replicates.(B) Volcano plot (t test) showing gene expression analysis of hnRNP M KD RNA-seq data from uninfected cells. x axis shows fold change of gene expression, and y axis shows statistical significance. Downregulated genes are plotted on the left, and upregulated genes are on the right.(C) Gene expression analysis of hnRNP M KD cells compared to SCR control for uninfected cells. Each column represents a biological replicate. Red, genes downregulated in hnRNP M KD; blue, genes upregulated in hnRNP M KD.(D) Gene expression analysis of hnRNP M KD cells compared to SCR control for Salmonella-infected cells. Each column represents a biological replicate.(E) Ingenuity pathway analysis of gene expression changes in uninfected and Salmonella-infected cells.(F) Manually annotated hnRNP M-dependent innate immune genes. Each column represents a biological replicate.(G) qRT-PCR of Rnf26, Rnf128, and Slc6a4 in uninfected hnRNP M KD cells.(H) qRT-PCR of mature IL6, Mx1, and Gbp5 transcripts in Salmonella-infected hnRNP M KD cells at 2 and 4 h post-infection.(I) qRT-PCR of IL1b and Tnfa transcripts in Salmonella-infected hnRNP M KD cells at 4 h post-infection.(G)-(I) represent 3 biological replicates ± SEM, n = 3. For all experiments in this study, statistical significance was determined using two-tailed Students’ t test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.
Fig 5: FXR Agonist GW4064 Inhibits IL-6 Expression in Macrophages. (A, B) RAW 264.7 macrophages were incubated with or without different concentrations of FXR agonist GW4064 for 24h, and then stimulated with or without 100ng/ml LPS. (A) The mRNA levels of TNFα, IL-1β and IL-6 from harvested cells at 6h after the stimulation are shown. (B) IL-6 levels in the supernatant from 24-hr cultures are shown. (C, D) Bone marrow-derived macrophages were incubated in the presence of DMSO, 5μM GW4064 or 1μM FXR antagonist Z-Guggulsterone for 24h. Cells were then stimulated with or without 100ng/ml LPS. (C) The mRNA levels of TNFα, IL-1β and IL-6 from harvested cells at 6h after the stimulation are shown. (D) IL-6 levels in the supernatant from 24-hr cultures are shown. (E, F) Bone marrow-derived macrophages from FXR knockout (FXR−/−) mice and the littermates were incubated with or without 5μM GW4064 for 24h, and then stimulated with or without 100ng/ml LPS. (E) The mRNA levels of TNFα, IL-1β and IL-6 from harvested cells at 6h after the stimulation are shown. (F) IL-6 levels in the supernatant from 24-hr cultures are shown. Data are shown as mean ± SEM. Experiments were repeated 3 times and representative data are shown. *p < 0.05; **p < 0.01; ***p < 0.001; NS, no significant difference; # means compared to the relative unstimulated control group, two-way ANOVA, followed by Sidak’s multiple comparisons test.
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