, CCR-7, did not significantly differ between B6 and CD69−/− CD4 T cells (Fig. 4D). The expression of CCR-9, an intestine homing marker, was increased only in the spleen of CD69-deficient mice (Fig. 4E), while no differences were observed in the expression of α4β7 (Fig. 4F). These data indicated that the expression of main intestine homing markers is generally not affected by CD69. However, CD69 regulates naïve/memory cell ratio with CD69−/− mice having a reduced fraction of memory CD44+ and CD103+ CD4 T cells and increased fraction of naïve CD62L+ CD4 T cells.
Figure 4. B6 and CD69−/− CD4 T cells differ in the expression of naïve and memory surface cell markers.
Cells were isolated from the spleen (spl), mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP), colonic lamina propria (cLP) and blood (bld) of non-treated B6 and CD69−/− mice and analyzed by flow cytometry. The surface expression of CD44 (A), CD103 (B), CD62L (C), CCR-7 (D), CCR-9 (E) and α4β7 integrin (F) by CD4 T cells were analysed. Graphs represent mean (± SEM) of CD4 T cell fraction expressing the indicated molecule for four mice per each strain per tissue. Black bars represent data for B6 and white bars for CD69−/− cells. *p≤0.05.
Administration of 2% DSS in the drinking water induced severe colitis in CD69-deficient mice as observed by the accelerated body weight loss compared to the DSS treated B6 animals and non-treated control group (Fig. 5A). Severity of intestinal inflammation is evaluated by histopathological examination of the colon. Average colitis score was significantly increased in the group of DSS-treated CD69−/− mice when compared to the DSS-treated B6 and non-treated control groups (Fig. 5B). Colonic tissue of the control mice showed normal architecture and cellularity on the histological slides, while after the DSS treatment in B6 animals the signs of mild inflammation were observed (Fig. 5C). In CD69−/− mice a massive infiltration of inflammatory cells and the los of tissue architecture confirmed the severity of the disease (Fig. 5C). Relative quantification of gene expression by RT-PCR was conducted using the RNA isolated from the frozen tissue sections of proximal colon taken from DSS administrated B6 and CD69−/− mice and non-treated control animals. These analyzes revealed significant increase in the expression of CCL-1, CXCL-10 and CCL-19 in the inflamed colon tissue of DSS-treated CD69−/− mice when compared to the other experimental groups (Fig. 5D–F). The treatment of DSS-induced colitis in CD69−/− mice with the mixture of Abs that neutralize CCL-1, CXCL-10 and CCL-19 did not affect the accelerated body weight loss (Fig. 5A), but it reduced significantly the histopathological severity of the disease (Fig. 5B). CD69-deficient animals treated with neutralizing Abs showed the signs of mild colitis, with normal number of goblet cells, slightly elongated colonic crypts and few infiltrating inflammatory cells (Fig. 5C). Severe colonic inflammation with infiltration of high number of inflammatory cells into the colonic wall observed in CD69−/− mice after DSS administration could be caused by the increased expression of pro-inflammatory chemokine ligands.
Figure 5. Increased susceptibility of CD69−/− mice to DSS induced colitis is partially due to increased chemokine-dependent CD4 T cell migration to cLP.
B6 or CD69−/− mice are administrated 2% dextrane sodium sulphate (DSS) in the drinking water for 5 days and then provided with normal sterile water. One group of DSS-administrated CD69−/− mice were treated with the mixture of CCL-1, CXCL-10 and CCL-19 blocking Abs by i.p. injection at days 0, 2, 4 and 6 (CD69−/−+Ch-block group). The control group represents non-treated B6 animals. A. Mean (± SEM) of body weight loss (%) of at least 5 mice per group is shown. B. Colitis scores of the histological colon sections of DSS treated B6 and CD69−/− mice, control and CD69−/−+Ch-block groups. Mean (± SEM) for at least five mice per group are presented. C. Histopathological colon tissue samples taken from control B6 mice, DSS treated B6 or CD69−/− animals and CD69−/−+Ch-block group were embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with H&E. Representative images of one individual mouse per group (from at least five mice per group analyzed) are shown (original magnification×20). RNA was isolated from the frozen colon tissue samples of control B6 animals and DSS treated B6 or CD69−/− mice and reverse transcribed to complementary DNA. Relative expression of CCL-1 (D), CXCL-10 (E) and CCL-19 (F) genes compared to β-actin gene is measured by qRT-PCR. Mean (± SEM) for at least five mice per group are presented. N.S. – not statistically significant; *p≤0.05; **p≤0.005.
To investigate the influence of the factors other than chemokines in DSS colitis model, we analyzed expression/production of pro-inflammatory cytokines as well. We saw that DSS-administrated CD69-deficient mice have significantly increased serum level of IL-17 as compared to the DSS-treated B6 and non-treated control group (Fig. 6A). Also, expression of the gene encoding pro-inflammatory cytokine IFN-γ was increased in the colonic tissue of the CD69−/− animals treated with DSS (Fig. 6B). Furthermore, three days after the DSS administration CD69−/− mice had reduced number of CD4 T cells in MLN and increased number of these cells in cLP when compared to B6 animals (Fig. 6C). Further analyses of these CD4 T cells showed that 3 days after the DSS administration CD4 T cell population in cLP of CD69-deficient mice contained significantly reduced fraction of Foxp3-expressing Treg cells compared to B6 mice, while in blood and MLN there was no difference (Fig. 6D,E). This early influx of CD4 T cells devoid of Treg cells together with the increased levels of pro-inflammatory cytokines contribute to the severe pathology observed in the colon of CD69−/− mice treated with DSS.
Figure 6. Severe colitis in DSS treated CD69−/− mice is associated with increased pro-inflammatory response and early influx of CD4 T cells devoid of Foxp3 Treg cell into the colon.
A. IL-17 concentration was determined by ELISA in the sera of control B6 mice and DSS treated B6 and CD69−/− mice 7 days after the DSS administration. Mean (± SEM) of at least five mice per group is presented. B. RNA was isolated from frozen colon tissue samples of B6 and CD69−/− mice treated with DSS, seven days after the DSS administration, or the control B6 mice and reverse transcribed to cDNA. Relative expression of IFN-γ gene was measured by qRT-PCR and presented as mean ± SEM of at least five mice per group. C. Mean (± SEM) total number of CD4+ T cells in blood, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) of 3 days DSS treated B6 and CD69−/− mice are shown. At least five mice per each strain were analysed. D. The expression of Foxp3 by CD4 T cells from blood, MLN and cLP of B6 and CD69−/− mice treated with DSS for 3 days was analysed by flow cytometry. Data from an individual, representative mouse (out of at least 5 mice per group analysed) are shown. Numbers indicate the percentage of CD4 T cells that express the transcriptional factor Foxp3. E. Mean (± SEM) total number of CD4+ Foxp3+ T cells in blood, MLN and cLP of 3 days DSS treated B6 and CD69−/− mice are shown. At least five mice per each strain were analyzed. N.S. – not statistically significant; *p≤0.05; **p≤0.005.
Antigen-specific transfer colitis model was established to study the inflammatory response of CD4 T cells in the presence or absence of specific antigen in intestinal lumen. CD45RBhigh CD4 T cells were sorted from the splenocytes of OT-II or OT-II×CD69−/− mice and adoptively transferred into the RAG−/− hosts. After the transfer hosts were fed or not every second day with 1 mg OVA protein. Both groups fed with OVA developed colitis, but the disease was more severe in the hosts transferred with OT-II×CD69−/− cells and fed with OVA as showed by accelerated body weight loss compared to all the other groups (Fig. 7A) and by severe histopathology in colon (Fig. 7B,C). The hosts transferred with OT-II or OT-II×CD69−/− CD45RBhigh CD4 T cells that were not fed with OVA, normally gain body weight as the control group (Fig. 7A), although OT-II×CD69−/− cell-transferred mice showed more severe histopathology in colonic tissue (Fig. 7B,C). Total number of OT-II CD4 T cells in cLP of the hosts was significantly increased compared to the control in the presence of antigen (Fig. 7D). The number of cLP OT-II×CD69−/− CD4 T cells was increased significantly in both groups fed or not fed with OVA when compared to the control group (Fig.