Failure of T cell homing, reduced CD4/CD8alphaalpha intraepithelial lymphocytes, and inflammation in the gut of vitamin D receptor KO mice

Abstract

Specific pathogen-free IL-10 KO mice failed to develop inflammatory bowel disease (IBD), whereas IL-10/vitamin D receptor (VDR) double KO mice developed fulminating IBD. WT CD4 T cells inhibited experimental IBD, while VDR KO CD4 T cells failed to suppress IBD. VDR KO mice had normal numbers and functions of regulatory T cells. The percentages of IL-17- and IFN-gamma-secreting T cells in the gut of mice reconstituted with WT and VDR KO CD4 T cells were also not different. Instead, there were twice as many CD8alphaalpha intraepithelial lymphocytes (IEL) in mice that were reconstituted with WT CD4 T cells than in mice reconstituted with VDR KO CD4 T cells. Furthermore, VDR KO mice had reduced numbers of CD8alphaalpha IEL, absent CD4/CD8alphaalpha populations, and as a result low IL-10 production in the IEL. The lack of CD8alphaalpha IEL was due in part to decreased CCR9 expression on T cells that resulted in the failure of the VDR KO T cells to home to the small intestine. We conclude that the VDR mediates T cell homing to the gut and as a result the VDR KO mouse has reduced numbers of CD8alphaalpha IEL with low levels of IL-10 leading to increased inflammatory response to the normally harmless commensal flora.

Fig. 1.

CD4 T cells from SPF DKO mice transfer IBD to SPF Rag KO mice. All donors and recipients were SPF. (A) The percentage change in BW over time is plotted ± SEM for each group of recipients. DKO values were significantly different from VDR KO and IL-10 KO values, P < 0.05. (B) Representative colonic sections from the Rag KO recipients of CD4 T cells (scoring system in Materials and Methods). Colon sections shown were rated VDR KO normal, IL-10 KO normal, and DKO mild. (C) The percentage of mice that showed normal, mild, or severe symptoms of IBD is recorded for each group of Rag KO mice receiving CD4 T cells.

Fig. 2.

Failure of total CD4 T cells from VDR KO mice to suppress IBD. (A) CD4 T cells were purified from WT or VDR KO mice and injected into DKO recipients that showed symptoms of IBD (squares, n = 5–6) or that were symptom free (circles, n = 3–5). The percentage change in BW over time as a function of age is shown. Symptomatic DKO mice were injected at 4 weeks of age and asymptomatic mice were injected at 2 weeks of age. (B) Changes in BW of recipient Rag KO mice following transfer of no cells, DKO splenocytes, DKO splenocytes plus WT CD4 T cells (WT CD4), or DKO splenocytes plus VDR KO CD4 T cells (VDR KO CD4). Values represent n = 3–4 mice per group and one representative of two independent experiments. At the end of the experiment the VDR KO CD4 group lost significantly more BW than the WT CD4 group and significantly less than the DKO group, P < 0.05.

Fig. 3.

T regs from VDR KO mice are functionally normal. CD4⁺/CD25⁻ and CD4⁺/CD25⁺ T cells were isolated from VDR KO and WT mice. (A) WT CD25⁻ proliferation was inhibited equally by WT and VDR KO CD4/CD25⁺ cells at all ratios tested. (B) VDR KO CD25⁻ proliferation was inhibited equally by WT and VDR KO CD4/CD25⁺ cells at all ratios tested. (C) VDR KO T regs suppress IBD in the Rag KO mouse. The percentage change in BW of Rag KO mice that did not receive any cells (control, CTRL), received only WT CD4/CD25⁻ T cells (CD25⁻), received WT CD4/CD25⁻ and WT CD4/CD25⁺ (WT T reg), or received WT CD4/CD25⁻ and VDR KO CD4/CD25⁺ (VDR KO T reg) is shown. Only Rag KO recipients of CD25⁻ cells lost significantly more BW by the end of the experiment than the CTRL, WT T reg, or VDR KO T reg Rag KO recipients, P < 0.05.

Fig. 4.

CD8αα T cell populations in the IEL. (A) The IEL from Rag KO recipients of DKO splenocytes, DKO splenocytes plus WT CD4 T cells (WT CD4), and DKO splenocytes plus VDR KO CD4 T cells (VDR KO CD4) cells (same mice as in Fig. 2B) were isolated and stained for CD4, CD8α, and CD8β. *, values in the WT CD4 group were significantly different from those in the VDR KO CD4 and DKO group, P < 0.05. **, values in the DKO only group were significantly different from those in the WT or VDR KO group, P < 0.05. (B) IEL were isolated from 6–8 VDR KO and WT mice and stained for CD4, CD8α, CD8β, and TCRβ. *, values in the WT group were significantly different from those in the VDR KO group, P < 0.01. Fig. S2 shows the isotype controls for staining and representative histograms.

Fig. 5.

Cytokine production and homing to the IEL. (A) Intracellular staining for IL-17 and IFN-γ in IEL from Rag KO recipients of DKO splenocytes, DKO splenocytes plus WT CD4 T cells, and DKO splenocytes plus VDR KO CD4 T cells (same mice as in Fig. 2B). Values from five to six mice per group ± SEM are shown. (B) Intracellular staining for IL-10 in IEL from VDR KO and WT recipients. The histogram shows isotype control staining (shaded) and VDR KO (dotted line) and WT (solid line) IEL staining for IL-10. The values in the table represent percentage of IL-10 secreting in the total IEL and CD8αα IEL of three individual mice per group ± SEM. *, values in the WT group were significantly higher than those in the VDR KO group, P < 0.05. (C) T cell reconstitution of the spleen and IEL of Rag KO mice following injection of a 1:1 mixture of WT (CD45.1) and VDR KO (CD45.2) cells. VDR KO cells are the TCRβ⁺ and CD45.1⁻ staining cells and WT cells are the TCRβ⁺ and CD45.1⁺ cells. *, the percentages of T cells from WT and VDR KO mice in the spleen and IEL of Rag KO mice were compared in seven Rag KO recipients ± SEM and the percentages of T cells from the WT mice were significantly higher than those from the VDR KO mice in the IEL, P < 0.001.