Mast cells disrupt epithelial barrier function during enteric nematode infection

Abstract

We have investigated the influence of mast cells on the barrier function of intestinal epithelium during nematode infection. Trichinella spiralis infection induces a strong type 2 cytokine-mediated inflammation, resulting in a critical mucosal mastocytosis that is known to mediate expulsion of the parasites from the intestine. The host response to infection is also characterized by an increase in mucosal leakiness. We show here that intestinal epithelial permeability is markedly elevated during infection, with kinetics that mirror the adaptive immune response to primary and secondary infection. Furthermore, we have identified degradation of the tight junction protein, occludin, thereby providing a mechanism for increased paracellular permeability during helminth infection. We further demonstrate by using anti-c-kit antibody and IL-9 transgenic mice that mast cells are directly responsible for increasing epithelial paracellular permeability and that mice deficient in a mast cell-specific protease fail to increase intestinal permeability and fail to expel their parasite burden. These results provide the mechanism whereby mucosal mast cells mediate parasite expulsion from the intestine.

Fig. 1.

Kinetics of intestinal permeability during primary T. spiralis infection. Permeability of jejunum sections to mannitol was measured on days (d) 0, 7, 10, and 21 postinfection; *, P < 0.003 d0 vs. d10, Λ, P < 0.003 d10 vs. d21 (a). Parasite numbers in the jejunum were counted on days 6 and 10 postinfection, *, P < 0.005 (b).

Fig. 2.

Abrogation of mucosal mast cells reduces intestinal permeability. Mast cells were absent in jejuna from d9 postinfected mice treated with anti-c-kit (aB) compared with control mice treated with rat IgG (aA), magnification ×200. Numbers of mast cells in 20 VCUs from infected mice treated with rat IgG or anti-c-kit *, P < 0.005 (bA). mMCP-1 was absent in sera of infected mice treated with anti-c-kit compared with control mice **, P < 0.003 (bB). Worm burdens in jejunum from anti-c-kit-treated mice were compared with control mice on day 9 postinfection; *, P < 0.05 (c). Increased intestinal permeability at day 9 postinfection was blocked by anti-c-kit treatment. *, P < 0.05 (d).

Fig. 3.

IL-9 transgenic mice overexpress mucosal mast cells and enhance intestinal permeability. Intestinal permeability was compared between IL-9 transgenic (tg) mice and WT (wt) mice at days 0 and 4 postinfection. Λ, P < 0.002 wt vs. IL-9tg d0; *, P < 0.001 IL9tg d0 vs. d4 postinfection; ¶, P < 0.002 wt vs. IL-9tg d4 postinfection (a). Anti-c-kit antibody blocked mucosal mastocytosis in IL-9tg mice at d4 postinfection (bB) compared with infected control IgG-treated mice (bA), magnification ×200. Numbers of mast cells per 20 VCUs were counted in anti-c-kit-treated IL-9 tg mice compared with control IgG-treated mice; *, P < 0.03 (c). Anti-c-kit blocked enhanced mucosal permeability at d0 and d3 postinfection compared with IL-9 tg mice treated with control IgG antibodies, *, P < 0.03 control IgG vs. anti-c-kit d0; Λ, P < 0.05 control IgG vs. anti-c-kit d3 (d). Anti-c-kit antibody delayed worm expulsion compared with mice treated with control antibody; *, P < 0.02 (e).

Fig. 4.

Intestinal permeability is enhanced during secondary T. spiralis infection. Mast cell numbers were greater in jejuna of secondary infected mice on day 3 postinfection (aB) than in primary infected animals (aA). On day 11 postinfection mast cell numbers were greater in primary infected mice (aC) than in secondary infected mice (aD), magnification ×200. Mast cells were counted in 20 VCUs from primary and secondary infected mice on days 3 and 11 postinfection. **, P < 0.002 prim vs. sec d3; ΛΛ, P < 0.003 prim vs. sec d11 (bB). Sera levels of mMCP-1, *, P < 0.001 prim vs. sec d3; Λ, P < 0.001 prim vs. sec d11 (bA). Permeability was greater in secondary infected mice on day 3 postinfection compared with primarily infected mice but decreased by day 11 postinfection. *, P < 0.02 prim vs. sec d3; Λ, P < 0.05 prim vs. sec d11; ¶, P < 0.03 sec d3 vs. d11 (c). Worm burden of primary infected mice compared with secondary infected mice at d3 postinfection. *, P < 0.05 (d).

Fig. 5.

Delayed worm expulsion and reduced intestinal permeability of mMCP-1-deficient mice. Levels of mMCP-1 were measured in sera of WT and mMCP-1-deficient mice (mMCP-1-/-) at d0 and d15 postinfection. *, P < 0.02 -/- vs. wt d15 postinfection (a). Permeability of jejunum at d0 and d15 postinfection. *, P < 0.005 (b) and worm burdens at d15 postinfection. *, P < 0.04 (c) were compared between WT and mMCP-1-/- mice.

Fig. 6.

TJ expression by jejunal epithelial cells from naïve or d9 post-T. spiralis-infected mice. Shown are TJ proteins in epithelium of intestinal villi visualized by confocal microscopy. (a) Dual staining of occludin (O) and desmoplakin (Dp) shows that both localize to the plasma membrane of naïve enterocytes. (b) Occludin (green) is internalized by enterocytes from d9 postinfected (p.i.) mice whereas desmoplakin (red) remains within the plasma membrane. (cA) Representative Western blot of occludin expression in epithelial tissue from naïve mice compared with d9 postinfection mice. (cB) A Coomassie blue-stained gel shows equal loading of total protein. (cC) Four Western blots of occludin from naïve or infected mice were analyzed by densitometry and the mean density of the 66-kDa band ± SEM is shown. *, P < 0.05. (d–g) Jejunal villi from naïve or infected mice were stained for ZO-1 (d and e) or claudin-1 (f and g).