Efficient colonic mucosal wound repair requires Trem2 signaling

Abstract

The colonic epithelial lining undergoes constant replacement, driven by epithelial stem cells in crypts of Lieberkühn. Stem cells lost because of damage or disease can be replaced by adjacent crypts that undergo fission. The close proximity of an extraordinary number of luminal microbes creates a challenge for this repair process; infection must be prevented while immune system activation and epithelial stem cell genetic damage must be minimized. To understand the factors that modulate crypt/stem cell replacement in the mouse colon, we developed an in vivo acute injury system analogous to punch biopsy of the skin. In contrast to epidermal stem cells, colonic epithelial progenitors did not migrate over the wound bed. Instead, their proliferative expansion was confined to crypts adjacent to wound beds and was delayed to the latter phase of healing. This increased epithelial proliferation was coincident with the infiltration of Trem2 expressing macrophages and increased expression of IL-4 and IL-13 in the wound bed. Interestingly, Trem2(-/-) mice displayed slow and incomplete wound healing of colonic mucosal injuries. We found the latter phase of healing in Trem2(-/-) mice showed a diminished burst of epithelial proliferation, increased expression of IFN-gamma and TNF-alpha, diminished expression of IL-4 and IL-13, and increased markers of classical macrophage activation. Ablation of these cytokines in injured WT and Trem2(-/-) mice demonstrated that their expression ultimately determined the rate and nature of wound healing. These studies show that Trem2 signaling is an important pathway to promote healing of wounds in the colon where stem cell replacement is necessary.

Fig. 1.

Mucosal regeneration of WT mice after colonic biopsy injury. (A) Serial endoscopic views of a biopsy injury site in a WT mouse colon. The yellow dotted lines depict the depressed area margin of the lesion at each time point. No depressed area was observed at day 6. The length of the major axis = 1.75 mm (day 0), = 1.5 mm (day 2), = 1.0 mm (day 4) and = 0.6 mm (day 6). (B) Plot of the average surface area of lesions ± SD relative to the original size of the wound (n = 20 lesions from 5 mice). (C) H+E-stained sections of WT mouse colons at various times post biopsy injury. At 6 h, fibrin, platelets, neutrophils, and red blood cells were extruded and attached to surface of the injured area (arrow). By day 1, clotted material was no longer present and in its place an epithelial cell monolayer had formed over the wound bed (arrowheads). Between days 2 and 4, the cell census of adjacent crypts was expanded. By day 6, crypts repopulated the wound bed. Bars = 100 μm. (D) Serial sections from C stained with BrdU to label cells in S-phase. Insets (yellow dashed boxes) show crypts adjacent to wound. Bars = 100 μm (Insets = 20 μm). (E) Quantification of epithelial cell proliferation (percentage of BrdU positive cells) in the three crypts adjacent to the wound (n = 12 lesions/group from 3 mice per group). At days 2 and 4 postinjury, epithelial cell proliferation was significantly increased as compared with day 0 (P < 0.01; blue asterisks). (F) BrdU stained sections of a WT mouse one day post biopsy injury showing two populations of BrdU negative surface epithelial cells emanating from a crypt (BrdU positive cells). (Scale bar: 20 μm.) (G) Section of WT injury stained with antisera directed against claudin-4 (red) and β-catenin (green). Only the WAE cells are claudin-4 positive (Inset). (Scale bars: 100 μm for main panel and 20 μm for the Inset.)

Fig. 2.

Trem2 is required for proper colon mucosal healing. (A) Plot of Trem2 mRNA expression (mean ± SD) at times postinjury. WT and Myd88^−/− mice showed similar patterns of Trem2 mRNA up-regulation that peaked two days after biopsy (n = 12 lesions/group from 4 mice per group, P < 0.01; asterisks indicate statistically significant differences for WT in blue and Myd88 in red versus their own 0 h baselines). (B) Double-label immunohistochemistry from WT and Trem2^−/− mice showing expression of Trem2 protein (red) and F4/80-positive macrophages (green) located in the wound bed two days after biopsy. (C) Serial endoscopic views of a biopsy injury site from an adult Trem2^−/− mouse colon. The yellow dotted lines depict the margin of the lesion at each time point. A depressed area remained at day 6 and persisted through day 10. The length of the major axis = 1.5 mm (day 0), = 1.0 mm (day 6), and = 0.8 mm (day 10). (D) H+E-stained sections of Trem2^−/− mouse colons at times postbiopsy injury. The surface of the wound bed was covered by epithelial cells by day 2 in Trem2^−/− mice. Stromal cells persisted in the wound bed (asterisk) at day 6. (E) Serial sections from (C) stained with BrdU (Insets of crypts adjacent to the wound bed in yellow dashed boxes). (F) Plot of square root (to generate linear graphs) of the mean (± SD) surface area of lesions in Trem2^−/− mice (orange) and WT (blue) mice showed healing was delayed in the absence of Trem2. These healing curves are statistically significantly different (see Table S1 for description). (G) Quantification of percentage BrdU incorporation in three crypts adjacent to injured areas (WT and Trem2^−/− are compared at each time point, n = 12 lesions/group from 3 mice per group, blue asterisk indicates P < 0.01 by student's t test). (H) Plots of Gr-1 positive neutrophils at 6 h postinjury (Left) and F4/80-positive macrophages at day 2 postbiopsy injury (Right) in WT, Myd88^−/−, and Trem2^−/− mice (n = 16 lesions/group from 4 mice per group). (Scale bars: 10 μm in B; 100 μm in D and E; and 20 μm in E Insets.

Fig. 3.

IL-4 and Il-13 are required for proper biopsy wound healing. (A) Plots of mRNA expression (mean ± SD) for Th1 and Th2 cytokines in biopsy injured WT and Trem2^−/− mice. For each mouse genotype, comparisons between individual time points post injury were compared with uninjured areas (0 h). Statistically significant differences (P < 0.01) are denoted by either blue asterisks (WT) or red asterisks (Trem2^−/−). (B and C) Administration of neutralizing antibodies for TNF-α and IFN-γ accelerated the healing in Trem2^−/− mice. Administration of neutralizing antibodies for IL-4 and IL-13 delayed wound healing in WT mice. For both experiments, the healing curves were statistically different (P < 0.001) as determined by generalized estimating equations, n = 12 lesions from 3 mice per group (see Table S1 for description and p values). (D) Plots of mean (± SD) fold-change of relative mRNA expression as determined by qRT-PCR analysis of wound beds procured at day 2 post injury (baseline of expression is 0h for each genotype) for WT, Myd88^−/−, Trem2^−/− mice (n = 12 lesions/group from 3 mice per group). Statistically significant differences (P < 0.01) as determined by Student's t tests between groups are indicated by an orange asterisk.

Fig. 4.

Trem2 sits at a focal point for the promotion of wound repair. Trem2 expression peaks in wound associated macrophages (blue circle) at day 2 post injury at a time when the WAE cell layer is formed and IL-4 and IL-13 also increase. IL-4 and IL-13 are known to increase Trem2 expression on macrophages (24). Our hypothesis is that the WAE cells form a barrier that limits exposure to bacterial products (either none or low levels). At low levels of TLR antigens, Trem2 is known to inhibit Myd88-mediated TLR signaling (23, 24). At higher levels of TLR antigens, Trem2 expression is inhibited (24), thus relieving the inhibition of TLR signaling that we hypothesize leads to an increase in proinflammatory cytokines in macrophages and/or other wound bed cells. Key unanswered questions are the nature of the interaction of the WAE cells and macrophage activation, the precise functional sources of IL-4 and IL-13 in this system and the role of the activated macrophage products on colonic epithelial progenitor proliferation and thus wound healing (all indicated by asterisks in diagram).