Mechanisms of Protection of Alpha4-Deficient T Cells in a Model of Chronic Experimental Colitis

Abstract

Gastrointestinal (GI) tract represents an important interphase between the body and the outside world. Under steady-state conditions, T-cellassociated α4 integrins (α4β7, α4β1) play an important role in T-cell recruitment to the gut-associated lymphoid tissue (GALT: Peyer's Patches, lymphoid follicles), mesenteric lymph nodes (MLNs) and small intestine. Moreover, these molecules are important contributors to the pathogenesis of experimental and human Crohn's disease (CD). Although the effectiveness of anti-adhesion therapy targeting α4 integrins is thought to be primarily due to blocking effector T cell recruitment into intestinal tissues, there is little direct evidence to confirm this, since α4 integrins can be also found on natural killer cells, monocytes, and granulocytes. Therefore, the objective of the current study was to ascertain the importance of T cell-associated α4 integrins in the induction of chronic gut inflammation. We utilized cre/loxP technology to generate conditional mutant mice with T cells lacking α4 integrins (α4-/-). Using adoptive T cell transfer model of CD, we reconstituted recombination activating gene-deficient (RAG-1-/-) mice with α4-/- or control α4+/+ CD4+CD45RBhigh T cells and monitored development of disease. Blinded histopathological colon scores, infiltrating leukocyte numbers and phenotype, and cytokine production by colon lamina propria (LP) cells were determined. To determine the effect of α4 integrin deletion on T cell trafficking, we conducted competitive homing experiments. While α4+/+ T cells elicited moderate to severe colitis, colitis in the α4-/-→RAG-1-/- mice was remarkably attenuated. This correlated with a significant reduction in T cells, granulocytes, and monocytes in colons of these mice compared to the α4+/+→RAG-1-/- group. Analysis of cytokines produced by mononuclear cells isolated from colon LP in the α4-/-→RAG-1-/- mice showed reduced levels of IL-1β, IL-2, IL-17, and TNF-α but not IFN-γ or IL-12p70 compared to control. Single-cell analysis of CD4+ T cells by intracellular staining revealed comparable production of IFN-γ and IL-17 by α4-/- and α4+/+, suggesting that α4-/- T cells are not defective in their ability to become activated and secrete pro-inflammatory cytokines. Trafficking of α4-/- and control T cells to MLN was comparable to WT, while homing of α4-/- T cells to small intestine and colon was severely compromised. Finally, analysis of surface integrins in reconstituted mice revealed that following transmigration into colon LP, T cells downregulate α4β7 but maintain high levels of β1 integrin expression. Alpha4 integrins are important for intravascular recruitment of T cells to the intestine and induction of colitis. At the same time, our data suggested that β1 integrins may be important for their localization within the intestinal tissue. Further work will need to determine relative importance of these integrins in the ability of T cells to induce experimental colitis.