Figure 10.

The effects of IL33 in intestinal microenvironment on PDL1⁺Breg cells in DSS-treated Bmal1^-/-mice. (A) Transcription levels of IL4, IL6, IL10, IL21, IL33, TGFβ, and MCP-1 in the IELs of DSS-treated and untreated Bmal1^-/- and WT mice among 24 hours. The P value was estimated by JTK cycle analysis. (B) Transcription levels of inflammatory cytokines IL4, IL6, IL10, IL21, IL33, TGFβ, and MCP-1 in the IELs of DSS-treated Bmal1^-/- and WT mice at the same time. (C) Protein expressions of Bmal1 and IL33, (D) proportion of B220⁺IL33⁺ cells in IELs of DSS-treated Bmal1^-/- and WT mice. (E) Gross appearance of colorectum, (F) body weight during DSS induction, (G) colorectal length, (H) histologic score of colon, (I) H&E of colon from DSS-treated WT mice, WT mice with IL33 antagonist antibody, Bmal1^-/- mice, and Bmal1^-/- mice with IL33 antagonist antibody. (J) FACS graphs and (K) proportion of CD5⁺CD1d⁺ cells from B220⁺ cells in IELs. (L) Fluorescence-activated cell sorter (FACS) graphs and (M) proportion of B220⁺PDL1⁺, (N) CD1d⁺PDL1⁺, and (O) CD5⁺PDL1⁺ cells in IELs. (P) PDL1 expressions in B cells isolated from WT and Bmal1^-/- spleen treated with IL21, TGFβ, or IL33 neutralizing antibodies by Western blot. (Q) PDL1 expression in B cells isolated from WT and Bmal1^-/- spleen treated with IL33 by Western blot. (R) The effects of Bmal1 and clock complexes on luciferase activities of IL33 promoter reporter with or without E-box site mutation. (S) ChIP assay to detect the binding of IL33 gene promoter with Bmal1 in SPLs and B cells. ∗P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.