Fig 2: Nox4 protects against DSS-induced experimental colitis. (A) Experimental scheme for the DSS-induced murine colitis model. (B) Body weight loss for WT and Nox4-/- mice with DSS-induced colitis was measured daily (n = 6). (C) Survival probability of WT or Nox4-/- mice with DSS-induced colitis was assessed until day 14 (n = 6). (D) Macroscopic observation of the colons from WT and Nox4-/- mice with DSS-induced colitis killed on day 14. Colon lengths were assessed after the mice were killed. (E) Representative H&E staining images of colon tissues from DSS-induced colitis WT and Nox4-/- mice. (F) Colitis scoring for pathologic assessment in DSS-induced colitis WT and Nox4-/- mice: severity, inflammation severity (0–3); extent, inflammation extent (0, none; 1, mucosa; 2, submucosa; and 3, transmural), and epithelial change. (G) Disease activity index comprising assessment of body weight loss, stool consistency, and rectal bleeding measured daily. Data are expressed as means ± SD. ***P < .005, and ****P < .001 compared with WT.

Fig 3: Loss of Nox4 leads to collagen synthesis and Treg lineage commitment. (A) Scheme for bulk RNA-seq analysis of freshly isolated distal colon tissues comparing transcriptomes from the vehicle group in WT (n = 3) and Nox4-/- (n = 3) mice or the 2.5% DSS-treated colitis group in WT (n = 3) and Nox4-/- (n = 3) mice. (B) Venn diagram analysis of the 5124 DEGs comparing the numbers of up-regulated, contraregulated, and down-regulated within each control and experimental group. (C) Volcano plot identifying genes significantly down-regulated (blue) and up-regulated (red) with a more than 2-fold express level difference in Nox4-/- compared with WT samples. (D) GO biological process analysis of up-regulated DEGs in Nox4-/- mice. (E) KEGG pathway enrichment analysis of Nox4-/- DEGs. The top 14 pathways are identified, and the colors indicate statistical significance. (F) T-cell subpopulation evaluated by CIBERSORT based on WT and Nox4-/- DEGs. (G) CIBERSORT analysis of WT and Nox4-/- colon tissue. The relative proportions of 11 different immune cell types are deconvoluted from the RNA-seq data using CIBERSORT. (H) Flow cytometry analysis of freshly isolated WT or Nox4-/- distal colon tissue showing the relative number of live cells gated on CD4+Foxp3+ T cells (top, histogram) and the number of CD4+Foxp3+ T cells (bar graph, bottom). (I) Gene set enrichment analysis based on WT or Nox4-/- DEGs. Positive regulation of the TGF-ß gene set is enriched with genes up-regulated in the Nox4-/- mouse colon. BC, B cell; DC, Dendritic cell; FDR, False discovery rate; FSC-A, Forward scatter-area; FSC-H, Forward scatter-height; ES, Enrichement score; LPS, lipopolysaccharide; NES, Normalized enrichment socre; NK, Natural killer; PC, Plasma cell; PMN, Polymorphonuclear leukocyte; SSC-A, Side scatter-area; SSC-H, Side scatter-height.

Fig 4: Nox4 suppresses activation of canonical TGF-ß signaling in vivo. (A) Scheme representing the canonical TGF-ß signaling pathway. (B) Reverse-transcription quantitative PCR analysis of the expression of indicated TGF-ß regulatory genes in the WT or Nox4-/- mouse colon tissue; these mice differ from those used for RNA-seq (n = 3). Black dots represent untreated WT mice, and red dots represent untreated Nox4-/- mice. (C) Active TGF-ß level in WT (black) or Nox4-/- (red) colon tissue lysates measured by enzyme-linked immunosorbent assay. (D) Immunoblotting of TGF-ß and TGFßR1 protein levels in WT or Nox4-/- colon tissue extracts; ß-actin was used as an internal control. The band size was quantified by ImageJ. (E) Immunoblotting of pSmad2/3, Smad2/3, and Smad4 protein levels in cytosolic and nucleic fraction extracts from WT and Nox4-/- colon tissues. Smad2/3 was used as the pSmad2/3 control proteins, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HDAC were used as the cytosol and nucleus control proteins, respectively. The band size was quantified by ImageJ. Representative immunofluorescence images of WT or Nox4-/- colon sections stained with (F) TGF-ß (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue), or (G) TGF-ß (green), CD11c (violet), and DAPI. Data are expressed as means ± SD. **P < .01, and ****P < .001 compared with WT. Cy, Cytosel; KO, Knock-out; LP, Lamina propria; Nu, Nucleus; P, phosporelated.

Fig 5: Immune cell infiltration in the WT and Nox4-/-mouse colon. (A) Immunohistochemistry with antibodies against F4/80 indicating M1 macrophages, CD164 indicating M2 macrophages, CD127 indicating lymphoid cells, and Ki-67 (bottom) indicating proliferating cells. (B) Sirius-red staining showing the fibrotic area in red (top). Immunolabeling with antibodies against Tenascin-C indicating fibrosis in the colon tissue (bottom). Each positive staining area and cell was measured by ImageJ and shown as a bar graph. The WT group (N = 3) is colored gray and the Nox4-/- group (N = 3) is colored red. Data are expressed as means ± SD. *P < .5, **P < .01, and ***P < .005 compared with WT. Ki-67, marker of proliferation Ki(Kiel)-67.