Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice

Mark A Travis, Boris Reizis, Andrew C Melton, Emma Masteller, Qizhi Tang, John M Proctor, Yanli Wang, Xin Bernstein, Xiaozhu Huang, Louis F Reichardt, Jeffrey A Bluestone, Dean Sheppard, Mark A Travis, Boris Reizis, Andrew C Melton, Emma Masteller, Qizhi Tang, John M Proctor, Yanli Wang, Xin Bernstein, Xiaozhu Huang, Louis F Reichardt, Jeffrey A Bluestone, Dean Sheppard

Abstract

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta 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 alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.

Figures

Figure 1. (Vav1-cre)Itgb8 fl/fl mice develop age-related…
Figure 1. (Vav1-cre)Itgb8fl/fl mice develop age-related autoimmunity
a, Weight loss in control and (Vav1-cre)Itgb8fl/fl mice (white, control; black, (Vav1-cre)Itgb8fl/fl; n=7 per group; asterisk, P=0.011; double asterisk, P=0.0026). Error bars represent s.e.m. b, Lymphoid organs of 5-month-old mice. c, Haematoxylin- and eosin-stained sections of livers (10 months, original magnification ×200). Arrows show cellular infiltrates. d, Haematoxylin- and eosin-stained colon sections (9 months, original magnification ×50). Short arrows, epithelium (top arrow) and smooth muscle (bottom arrow); large arrows, cellular infiltrates (top panel) and large cyst (bottom panel). e, ELISA for anti-dsDNA and anti-ribonuclear protein (9–12 months, n=5, P=0.0013).
Figure 2. (Vav1-cre)Itgb8 fl/fl mice develop enhanced…
Figure 2. (Vav1-cre)Itgb8fl/fl mice develop enhanced numbers of activated/memory T cells expressing IL-4 and IFN-γ, and increased serum IgE, IgG1 and IgA levels
a, Activated/memory T cells from spleen (CD62LlowCD44high, 4–6-month-old mice) were analysed by flow cytometry. Representative flow cytometry plots and plotted mean values are shown (white, control; black, (Vav1-cre)Itgb8fl/fl; n=14; asterisk, P=2.7×10−11; double asterisk, P=4.9×10−5). b, IL-4 and IFN-γ levels in T cells from spleen were analysed by intracellular flow cytometry. Representative flow cytometry plots and plotted mean values are shown (white, control; black, (Vav1-cre)Itgb8fl/fl; n=9; asterisk, P=1.8×10−8; double asterisk, P=1.8×10−7; triple asterisk, P=0.00048). c, ELISA for IgE, IgG1 and IgA levels in sera (4–6-month-old mice; white, control; black, (Vav1-cre)Itgb8fl/fl; n=4; asterisk, P=0.0028; double asterisk, P=0.011; triple asterisk, P=0.016). All error bars represent s.e.m.
Figure 3. Mice lacking integrin β 8…
Figure 3. Mice lacking integrin β8 on dendritic cells develop an identical immune phenotype to mice lacking β8integrin on all leukocytes
a, Activated/memory T cells from spleen (CD62LlowCD44high, 4–6-week-old mice) were analysed by flow cytometry. Representative flow cytometry plots and plotted mean values are shown (white, control; black, Itgb8 conditional knockout; n=6 per group; asterisk, P<8.2×10−4; double asterisk, P<1.4×10−4). b, IL-4 and IFN-γ levels in T cells from spleen were analysed by intracellular flow cytometry. Representative flow cytometry plots and plotted mean values are shown (white, control; black, Itgb8 conditional knockout; n=6 per group; asterisk, P<0.0063; double asterisk, P=0.0055; triple asterisk, P=0.0045). c, ELISA for IgE, IgG1 and IgA levels in sera (4–6-week-old mice; white, control; black, Itgb8 conditional knockout; n=6; asterisk, P<0.0018; double asterisk, P<0.016; triple asterisk, P=0.0015). All error bars represent s.e.m.
Figure 4. β 8 -deficient dendritic cells…
Figure 4. β8-deficient dendritic cells fail to induce TR cells in vitro, and (CD11c-cre)Itgb8fl/fl mice have reduced proportions of TR cells in colonic tissue
a, b, Induction of TR cells (CD4+GFP–Foxp3+) by control or β8-deficient dendritic cells in the presence of control or anti-TGF-β antibody (a) or active TGF- β (b). Representative flow cytometry plots and mean data plots (expressed as percentage of CD4+ cells that expressed GFP–Foxp3) are shown (white, control dendritic cells; black, β8-deficient dendritic cells; n=5; asterisk, P=0.013). c, TGF-β activation by control or β8-deficient dendritic cells, detected using mink lung reporter cells (white, control dendritic cells; black, β8-deficient dendritic cells; n=6; asterisk, P=0.0006). d, TR cell proportions present in spleen or colonic lamina propria. Representative flow cytometry plots and mean data plots (expressed as percentage of CD4+ cells that expressed GFP–Foxp3) are shown (white, control; black, (CD11c-cre)Itgb8fl/fl; n=6; asterisk, P=0.012). NS, not significant. All error bars represent s.e.m.

References

    1. Shull MM, et al. Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease. Nature. 1992;359:693–699.
    1. Marie JC, Liggitt D, Rudensky AY. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-β receptor. Immunity. 2006;25:441–454.
    1. Li MO, Sanjabi S, Flavell RA. Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity. 2006;25:455–471.
    1. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFβ activation. J. Cell Sci. 2003;116:217–224.
    1. Huang XZ, et al. Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin. J. Cell Biol. 1996;133:921–928.
    1. Munger JS, et al. The integrin αvβ6 binds and activates latent TGFβ1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 1999;96:319–328.
    1. Mu D, et al. The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1. J. Cell Biol. 2002;157:493–507.
    1. Yang Z, et al. Absence of integrin-mediated TGFβ1 activation in vivo recapitulates the phenotype of TGFβ1-null mice. J. Cell Biol. 2007;176:787–793.
    1. Zhu J, et al. β8 integrins are required for vascular morphogenesis in mouse embryos. Development. 2002;129:2891–2903.
    1. Proctor JM, Zang K, Wang D, Wang R, Reichardt LF. Vascular development of the brain requires β8 integrin expression in the neuroepithelium. J. Neurosci. 2005;25:9940–9948.
    1. de Boer J, et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 2003;33:314–325.
    1. Gorelik L, Flavell RA. Abrogation of TGFβ signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity. 2000;12:171–181.
    1. Kim BG, et al. Smad4 signalling in T cells is required for suppression of gastrointestinal cancer. Nature. 2006;441:1015–1019.
    1. Bohr UR, et al. Prevalence and spread of enterohepatic Helicobacter species in mice reared in a specific-pathogen-free animal facility. J. Clin. Microbiol. 2006;44:738–742.
    1. Taylor NS, Xu S, Nambiar P, Dewhirst FE, Fox JG. Enterohepatic Helicobacter species are prevalent in mice obtained from commercial and academic institutions in Asia, Europe, and North America. J. Clin. Microbiol. 2007;45:2166–2172.
    1. Whary MT, Fox JG. Detection, eradication, and research implications of Helicobacter infections in laboratory rodents. Lab Anim. (NY) 2006;35:25–36.
    1. Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. J. Clin. Invest. 2007;117:514–521.
    1. Lee PP, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity. 2001;15:763–774.
    1. Caton ML, Smith-Raska MR, Reizis B. Notch-RBP-J signaling controls the homeostasis of CD8− dendritic cells in the spleen. J. Exp. Med. 2007;204:1653–1664.
    1. Sakaguchi S, et al. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 2006;212:8–27.
    1. Chen W, et al. Conversion of peripheral CD4+CD25− naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 2003;198:1875–1886.
    1. Nakamura K, et al. TGF-β1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J. Immunol. 2004;172:834–842.
    1. Fahlen L, et al. T cells that cannot respond to TGF-β escape control by CD4+CD25+ regulatory T cells. J. Exp. Med. 2005;201:737–746.
    1. Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF-β1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J. Exp. Med. 2005;201:1061–1067.
    1. Fontenot JD, et al. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity. 2005;22:329–341.
    1. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nature Rev. Immunol. 2003;3:253–257.
    1. Bluestone JA, Tang Q. How do CD4+CD25+ regulatory T cells control autoimmunity? Curr. Opin. Immunol. 2005;17:638–642.
    1. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-β regulation of immune responses. Annu. Rev. Immunol. 2006;24:99–146.
    1. Lefrancois L, Lycke N. Isolation of mouse small intestine intraepithelial lymphocytes, Peyer’s patch, and lamina propria cells. Curr. Protocols Immunol. 2001 Unit 3.19 doi:10.1002/0471142735.im0319s17.
    1. Abe M, et al. An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal. Biochem. 1994;216:276–284.

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