Loss of integrin avb8 on dendritic cells causes autoimmunity and colitis in mice

The cytokine transforming growth factor-b (TGF-b) is an important negative regulator of adaptive immunity. TGF-b is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-b activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-b-activating integrin avb8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of avb8 on dendritic cells, as mice lacking avb8 principally on dendritic cells develop identical immunological abnormalities as mice lacking avb8 on all leukocytes, whereas mice lacking avb8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking avb8 fail to induce regulatory T cells (TR cells) in vitro, an effect that depends on TGF-b activity. Furthermore, mice lacking avb8 on dendritic cells have reduced proportions of TR cells in colonic tissue. These results suggest that avb8-mediated TGF-b activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of avb8 on dendritic cells to induce and/or maintain tissue TR cells. The critical pathways for activation of latent TGF-b at specific sites in vivo remain controversial. One important mechanism for activation of two of the three mammalian TGF-b isoforms (TGF-b1 and TGF-b3) is through interaction with members of the integrin receptor family. The in vivo importance of TGF-b activation by integrins has been recently demonstrated in mice with a mutated integrinbinding motif in TGF-b1 (ref. 8). These mice completely phenocopy Tgfb1 mice, which die from multi-organ inflammatory disease, showing that integrin-mediated TGF-b activation is vital to maintain immune homeostasis. However, the integrin(s) responsible for TGFb activation in the immune system in vivo, where these integrins are expressed, and the mechanisms by which integrin-mediated TGF-b activation maintains immune homeostasis remain a mystery. The integrin avb6 has previously been shown to activate TGF-b (ref. 6), but this integrin is restricted to a subset of epithelial cells and is not expressed on immune cells. Furthermore, the inflammatory phenotype of mice lacking avb6 is mild compared to mice lacking TGF-b1 or TGF-b3 (ref. 5). The avb8 integrin can also activate TGF-b (ref. 7). Mice completely lacking avb8 die before or shortly after birth from defects in brain vascular development, so tissue-specific deletion was required to determine whether avb8-mediated TGF-b activation has a function in regulating adaptive immunity. Polymerase chain reaction with reverse transcription (RT–PCR) analysis revealed that b8 was expressed in total splenocytes, CD4 1 T cells and dendritic cells (Supplementary Fig. 1a) but was present at very low or undetectable levels in macrophages, CD8 T cells, natural killer cells and B cells. To assess the in vivo significance of avb8 expression in the immune system, we produced mice with conditional loss of b8 on leukocytes by crossing mice homozygous for a floxed b8 (Itgb8) allele with mice expressing Cre recombinase under the control of the Vav1 promoter (hereafter called (Vav1-cre)Itgb8). Quantitative (q)RT–PCR revealed efficient knockdown (.98%) of Itgb8 messenger RNA expression in CD4 T cells and dendritic cells from (Vav1-cre)Itgb8 mice (Supplementary Fig. 1b). (Vav1-cre)Itgb8 mice were phenotypically normal until 4–5 months of age, when they began to develop a progressive wasting disorder (Fig. 1a). (Vav1-cre)Itgb8 mice also developed splenomegaly, massive enlargement of mesenteric lymph nodes (Fig. 1b) and accumulations of mononuclear cells adjacent to portal triads of the liver (Fig. 1c). By 10 months of age, all surviving (Vav1cre)Itgb8 mice developed severe colitis, characterized by cellular infiltration of the colonic wall with eosinophils and plasma cells, and formation of colonic cysts (Fig. 1d). These mice also developed high levels of auto-antibodies directed against double-stranded DNA and ribonuclear proteins (Fig. 1e). These findings are remarkably similar to phenotypes described for mice with a partial loss of TGF-b signalling in T cells as a result of expression of a dominant negative TGF-b receptor, and for mice lacking the key TGF-b signalling protein Smad4 in T cells. Mice lacking Smad4 in T cells also had increased tumorigenesis, a finding we did not observe. PCR of stool samples revealed the presence of the common intestinal bacteria Helicobacter hepaticus and Helicobacter ganmani from all control and experimental mice tested. These organisms, endemic in our facility and in over 85% of mouse research colonies worldwide, are not pathogenic in most strains of mice, but have been reported to cause colitis and hepatic inflammation in some immunosuppressed strains. We therefore cannot exclude the possibility that the presence of these organisms, or other unmeasured microbial flora, contribute to the severity of colonic and/or hepatic pathology in (Vav1-cre)Itgb8 mice. Such a response could be relevant to inflammatory bowel diseases in humans, where abnormal responses to normally non-pathogenic intestinal flora have been suggested to have a role. Mice with impaired T-cell responsiveness to TGF-b were also shown to have increased numbers of activated peripheral T cells, increased circulating levels of IgA and IgG1, and increased numbers of T cells that produce interleukin-4 (IL-4) and/or interferon-c (IFNc). (Vav1-cre)Itgb8 mice (4–6 months old) also had enhanced numbers of activated/memory CD4 and CD8 T cells (Fig. 2a), and increased numbers of CD4 T cells producing IFN-c and IL-4, and CD8 cells producing IFN-c (Fig. 2b and Supplementary Fig. 2).