Fig 1: Increased IL-22 and its signature genes in del(5q) MDS subjects.(a) Representative flow cytometry plots showing frequency of CD4+IL-22+ cells among total PBMCs in the peripheral blood of MDS patients and healthy subjects. Pre-gated on viable CD3e+CD4+ cells. Cumulative data shown in Figure 4e. (b) Expression of indicated IL-22 signature genes in CD34+ cells from healthy controls and del(5q) and non-del(5q) MDS patients. n = 17, 47, and 136 for healthy, del(5q) MDS, and non-del(5q) MDS, respectively. Kruskal-Wallis test with Dunn’s correction for multiple comparisons (b) used to calculate statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Solid lines represent median and dashed lines represent quartiles (b).
Fig 2: Butyrate promotes IL-22 production through GPR41 and HDAC inhibition.a, b CBir1 Tg CD4+ T cells were cultured with APCs and Cbir1 peptide with or without butyrate (0.5 mM) ± AR420626 (5 µM) or/and TSA (10 mM) under Th1 conditions (n = 3/group). IL-22 mRNA (a) and protein (b) were measured by qRT-PCR and ELISA at 60 h. IL-22 production was measured by flow cytometry on day 5 (c). d CD4+ T cells were cultured with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) or TSA (10 mM) (n = 3/group). Cells were collected at 24 h for analysis of HDAC activity at fluorescence intensity at excitation/emission (490/525 nm) by using the HDAC Activity Assay Kit. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA. a ****p < 0.0001, **p = 0.0016, *p = 0.0282; b ****p < 0.0001; c ****p < 0.0001, ***p = 0.0002, **p = 0.0054; d *p = 0.0144, ***p = 0.0004.
Fig 3: Butyrate promotes intestinal CD4+ T cell and ILC production of IL-22.WT mice were treated with or without 200 mM butyrate in drinking water for 3 weeks (n = 4 mice/group). a Mice were weighed daily. b Fecal pellets were collected prior and after 3-week treatment of butyrate, and butyrate levels were measured by LC–MS. Mice were killed on day 21, and IL-22 production in serum (c) and colonic organ cultures (d) were measured by ELISA. IL-22 production in CD4+ T cells (e) and ILCs (g) were analyzed in the spleen, MLN, and intestinal LP by flow cytometry. IL-22 levels in Th1, Th17, Treg cells (f), and ILCs (h) were measured in intestinal LP by flow cytometry. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t-test. b ***p = 0.0009; c *p = 0.0145; d *p = 0.0393; e middle panel: *p = 0.0158 (SP), 0.0151 (MLN), and 0.0022 (LP); right panel: *p = 0.0359 (SP), 0.0377 (MLN), and 0.0481 (LP); f middle panel: *p = 0.0356 (Th1) and 0.0375 (Th17); right panel: *p = 0.0432 (Th1) and 0.0158 (Th17); g middle panel: *p = 0.0149 (SP), 0.0227 (MLN), and 0.0232 (LP); right panel: *p = 0.0126 (SP), 0.0448 (MLN), and 0.0462 (LP); h middle panel: *p = 0.0458; right panel: *p = 0.0436.
Fig 4: SCFAs induction of IL-22 in CD4+ T cells and ILCs.Gut microbiota-derived SCFAs promote IL-22 production in CD4+ T cells and ILCs. Mechanically, butyrate promotes IL-22 production through GPR41 and HDAC inhibition. Furthermore, butyrate upregulates HIF1a and AhR, which is differentially regulated by mTOR and Stat3. HIF1a directly binds to the Il22 promoter, and butyrate increases HIF1a binding to the Il22 promoter through histone modifications.
Fig 5: Riok2 haploinsufficiency recapitulates del(5q) MDS transcriptional changes.(a to b) GSEA enrichment plots comparing proteins up-regulated (a) and down-regulated (b) upon Riok2 haploinsufficiency to the transcriptional changes seen in del(5q) MDS. (c) Schematic of mechanism underlying Riok2 haploinsufficiency-induced, IL-22 –induced anemia.
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