Fc gamma receptor signaling in mast cells links microbial stimulation to mucosal immune inflammation in the intestine

Abstract

Microbes and microbial products are closely associated with the pathogenesis of inflammatory bowel disease (IBD); however, the mechanisms behind this connection remain unclear. It has been previously reported that flagellin-specific antibodies are increased in IBD patient sera. As mastocytosis is one of the pathological features of IBD, we hypothesized that flagellin-specific immune responses might activate mast cells that then contribute to the initiation and maintenance of intestinal inflammation. Thirty-two colonic biopsy samples were collected from IBD patients. A flagellin/flagellin-specific IgG/Fc gamma receptor I complex was identified on biopsied mast cells using both immunohistochemistry and co-immunoprecipitation experiments; this complex was shown to co-localize on the surfaces of mast cells in the colonic mucosa of patients with IBD. In addition, an ex vivo study showed flagellin-IgG was able to bind to human mast cells. These cells were found to be sensitized to flagellin-specific IgG; re-exposure to flagellin induced the mast cells to release inflammatory mediators. An animal model of IBD was then used to examine flagellin-specific immune responses in the intestine. Mice could be sensitized to flagellin, and repeated challenges with flagellin induced an IBD-like T helper 1 pattern of intestinal inflammation that could be inhibited by pretreatment with anti-Fc gamma receptor I antibodies. Therefore, flagellin-specific immune responses activate mast cells in the intestine and play important roles in the pathogenesis of intestinal immune inflammation.

Figure 1

Immune staining of flagellin immune complex and FcγRI on the surface of mast cells. A: Colonic biopsy samples were obtained from patients with inflammatory bowel disease (IBD) (Aa, Ab) or normal controls (Ac, Ad). Consecutive sections were cut; a and b were from an IBD sample; c and d were from a control sample. Co-staining was performed with antibodies against tryptase and FcγRI (Aa and Ac) or tryptase and flagellin (Ab and Ad). Tryptase immune positive products were stained in red; FcγRI or flagellin was stained in blue. B: A confocal image of human mononuclear cell-derived mast cells (moMCs). The positive staining includes: Ba, flagellin; Bb, flagellin-specific IgG; Bc, FcγRI; Bd, merged by Ba and Bb; Be, merged by Ba, Bb and Bc. Bf is a negative control image.

Figure 2

Co-immunoprecipitation of flagellin, IgG and FcγRI. Proteins were extracted from intestinal samples (A) or cultured cells (B) and analyzed by co-immunoprecipitation. Antibodies used for precipitation were: lane 1, anti-flagellin; lane 2, anti-FcγRI; lane 3, anti-TLR5. Antibodies used for blotting are presented above each gel. IBD, samples were taken from IBD patients. Normal, samples were taken from normal intestinal tissue. Sensitized, cells exposed to flagellin-specific IgG and then exposed to flagellin in culture. Naïve, naïve cells.

Figure 3

Re-exposure to specific antigen flagellin activates sensitized moMCs. Flagellin-IgG sensitized moMCs were re-exposed to specific antigen flagellin; levels of histamine and TNF-α in supernatants were determined by ELISA. Bars indicate levels of histamine (A) and TNF-α (B). Naïve, moMCs were treated with media alone. F+IgG, moMCs were sensitized with flagellin-IgG and challenged with flagellin. F or IgG, moMCs were treated with flagellin alone or IgG alone. αTLR5, moMCs were treated with anti-TLR5 antibody. RNAi, moMCs were transfected with FcγRI siRNA at 300 nmol/L (RNAi1) or 100 nmol/L (RNAi2), or control siRNA (RNAi3). 48/80, moMCs were treated with 48/80 compound (5 μg/ml). Data are expressed as the means ± SD, *P < 0.05, compared with naïve cells.

Figure 4

Flagellin-specific immune response induces inflammation in mouse intestine. Mice were sensitized and challenged with flagellin. Intestinal segments were observed with light and electron microscopy. A–C: Representative images of colonic histology (×200) from mice sensitized to and challenged with flagellin (A), or mice sensitized to flagellin, pretreated with anti-FcγRI antibody, and then challenged with flagellin (B, ×100), or from naïve mice (C, ×100). D: Bars indicate the inflammatory scores in the intestine. E: Bars indicate the weight loss of the experimental mice that is presented as the percentage of the original body weight., *P < 0.05, compared with mice treated with saline. Balb, BALB/c mice. Wv1, W/Wv mice. Wv2, W/Wv mice were reconstituted with mast cells. Ab, pretreated with anti-FcγRI antibody. iAb, pretreated with isotype antibody. F+IgG, mice were sensitized and challenged with flagellin.

Figure 5

Mast cell activation in the intestinal mucosa. Colonic sections were observed under a light microscope. A: Bars indicate numbers of mast cell and mononuclear cells in colonic tissue sections that are expressed as the means ± SD from 20 fields (×200) from each mouse., *P < 0.05, compared with naïve BALB/c mice. Ba–Bc: Photomicrographs show mast cell staining in colonic sections (stained by toludine blue) from naïve mouse (Ba), sensitized mice (Bb), and W^v/W^v mice (Bc). Bd–Be: Electron photomicrographs show mast cell degranulation (pointed by a solid arrow; Bd) from sensitized mice compared with normal mast cells with intact granules (Be, pointed by an open arrow) from naïve mice. Group legends are the same as that in Figure 4.

Figure 6

Flagellin-specific cellular response in the intestine and serum IgG. A–C: LPMCs were cultured in the presence of flagellin for 72 hours. Cellular immune response was observed by antigen-specific T cell proliferation. Bars indicate the [³H] incorporation in isolated LPMC (A), cytokine levels in culture supernatants (B) and positively stained IFN-γ⁺ or IL-4⁺ T cells (C, analyzed by fluorescence-activated cell sorting). D: Bars indicate the levels of serum flagellin-specific IgG that were assessed by ELISA. Data are presented as the means ± SD, *P < 0.05, compared with mice treated with naïve BALB/c mice.