Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins

Abstract

The gastrointestinal tract is constantly challenged by foreign antigens and commensal bacteria but nonetheless is able to maintain a state of immunological quiescence. Recent advances have highlighted the importance of active suppression by regulatory lymphocytes and immunosuppressive cytokines in controlling mucosal immunity. Failures of these mechanisms contribute to the development of inflammatory bowel disease, but how these regulatory networks are established remains unclear. Here, we demonstrate key roles for alphav integrins in regulation of mucosal immunity. We report that deletion of alphav in the immune system causes severe colitis, autoimmunity, and cancer. Mice lacking immune cell alphav have fewer regulatory T (Treg) cells in the colon and corresponding increases in activated T cells and T cell cytokine production, leading to colitis. Using conditional gene targeting, we demonstrate that this is specifically attributable to loss of alphav from myeloid cells. Furthermore, we show that gut-associated macrophages and dendritic cells fail both to remove apoptotic cells efficiently and to induce Treg cells. Our results identify a vital role for myeloid alphav integrins in generating mucosal Treg cells and emphasize the importance of antigen-presenting cells in establishing immune tolerance.

Fig. 1.

Deletion of αv integrins in endothelial and hematopoietic cells in αv-tie2 mice. (a and b) Small intestine from tie2-CRE transgenic mice crossed to LacZ reporter mice, stained for LacZ activity. (a) CRE-mediated deletion in blood vessels and immune organs, indicated by an arrowhead and arrow, respectively. (b) Sections of stained tissue show CRE-mediated deletion in endothelial cells in vessels from control and reporter mice (Upper) and a single blood vessel from reporter mice at higher magnification (Lower). (c) PCR across exon 4 of αv gene in DNA from sorted lung endothelial cells (CD31⁺ cells) or splenocytes from mice carrying one floxed and one nonfloxed allele for αv show that the floxed allele is lost when mice also carry the tie2-CRE transgene. (d) FACS analysis of macrophages and total splenocytes from control (αv ^flox/+; tie2-CRE⁺) or αv-tie2 mice stained for surface expression of αv. Red lines show staining with isotype control antibody, and blue-filled histograms show staining for αv.

Fig. 2.

αv-tie2 mice develop colitis. (a–c) Weight (a), survival (b), and colitis score (c) of αv-tie2 (blue bars/blue line) and littermate control (open bars/black line) mice. Weight and colitis scores are expressed as mean ± SEM (n = 3–13 mice per group; males only used for weight; similar results seen for females). *, P < 0.01. (d) Acute intestinal obstruction in αv-tie2 mouse (20 weeks of age). Arrows show points of constriction in small intestine. (e) Cecum and colon from αv-tie2 mice (40 weeks) are enlarged compared with those from control mice. The cecum is inflamed (arrows), and the colon is thickened (arrowheads). (f) Colons of 20-week-old αv-tie2 mice have thickened mucosal and submucosal layers with transmural inflammation. Infiltrates are predominantly lymphocytes and monocytes. (g and h) Ulcer (#) (g) and tumor (*) (h) in colons of 40-week-old αv-tie2 mice.

Fig. 3.

Immune cell activation in αv-tie2 mice. (a) Total cell numbers isolated from peritoneal cavity (PerC), peripheral lymph nodes (pLN) and mLN, Peyer's patch (PP), and spleen of 12-week-old αv-tie2 and control mice. (b) FACS analysis of spleen cells from 12-week-old control and αv-tie2 mice, gated on CD4⁺ T cells, shows higher proportions of activated (CD44^high L-selectin^low) CD4⁺ cells in αv-tie2 mice. (c) Circulating cytokines from serum of αv-tie2 or control mice (aged 12 weeks). (d) Treg cells from indicated tissues from 8-week-old αv-tie2 and control mice. Data are expressed as percentage of CD4⁺ cells that are CD25⁺ FoxP3⁺. FACS data are representative of colon lymphocytes, gated on CD4⁺ cells. In all graphs, data are means ± SEM for at least three mice per group. *, P < 0.05.

Fig. 4.

Loss of αv from myeloid cells (but not lymphocytes) causes colitis. (a and b) Representative histology of colon (a), weight (b), and colitis score (b) of mice after bone marrow transplantation between 3-month-old αv-tie2 (KO) and control (WT) mice. Colitis was assessed a further 3 months after bone marrow transplantation. Note that transplantation of αv-deficient bone marrow (KO → WT) leads to colitis, whereas transplantation of control bone marrow is sufficient to reverse ongoing disease (WT → KO). (c and d) Representative histology of colon (c) and colitis score (d) from indicated αv-conditional knockout mice at 40–50 weeks of age. Deletion of αv from T cells (αv-lck) or from B cells (αv-CD19) does not lead to colitis, whereas deletion of αv from myeloid cells (αv-LysM) does. (e) Detail of colon (Upper) and mesentery (Lower) from 70 week-old αv-LysM mouse. Note ulceration in colon (*) and inflammation in mesentery. (f) Serum cytokine levels in 20-week-old αv-LysM mice. All graphs are mean ± SEM (n ≥ 4 mice per group). *, P < 0.05.

Fig. 5.

DCs from mLN of αv-tie2 mice have reduced ability to generate Treg cells. (a) Proportion of Treg cells generated from coculture of CD4⁺ CD25⁻ T cells with DCs purified from mLN or grown from bone marrow (bm) of control and αv-tie2 mice. (b) Proportion of DC subpopulations in mLN of 10-week-old control and αv-tie2 mice, expressed as percentage of CD11c^high cells. All graphs are mean ± SEM (n ≥ 3). *, P < 0.05.

Fig. 6.

αv integrins contribute to phagocytosis of apoptotic cells. (a) Phagocytosis of apoptotic cells (mouse thymocytes) by macrophages from control (con) or αv-tie2 mice (ko) or macrophages incubated with antibodies to αv integrins (αv), control Ig (Ig), RGD-containing peptides (RGD), or control peptide (RAD) (mean percentage phagocytic cells relative to untreated controls ± SEM; n ≥ 3). (b) Phagocytosis of apoptotic cells (human neutrophils) by macrophages from control or αv^flox/flox mice, untreated, or infected with adenovirus expressing CRE recombinase (MOI 100) (mean percentage phagocytic cells relative to untreated controls ± SEM; n ≥ 3). (c) Phagocytosis of apoptotic cells (mouse thymocytes) by DCs derived from bone marrow of control or αv-tie2 mice (mean percentage phagocytic cells ± SEM; n ≥ 3). (d) Phagocytosis of apoptotic cells (mouse thymocytes) instilled into the peritoneal cavity of control, αv-tie2, and αv-LysM mice. Data are percentage of recovered macrophages (F4/80⁺ cells) that had internalized apoptotic cells, expressed relative to control mice (mean ± SEM; n = 5). Phagocytosis was assessed by microscopy (a and b) or FACS (c and d). (e) Apoptotic cells (TUNEL⁺ cells) in colonic mucosae of precolitic 6-week-old αv-tie2 and control mice. Data are from analysis of two colon cross-sections per mouse and ≥3 mice per group (mean ± SEM). Shown are representative regions from control and αv-tie2 mice. (f) Autoantibodies in serum from control and αv-tie2 mice. (g) Antinuclear antibody (ANA) titers in serum from 45-week-old control and αv-tie2. Serum from autoimmune MRL-mp mice is included as a positive control. (h) Anti-dsDNA autoantibodies in serum of 70-week-old αv-LysM mice. In all graphs, *, P < 0.05; **, P < 0.01 (Student's t test for phagocytosis data, Mann–Whitney test for autoantibody titers).