NLRP6 Expression By the Hosts Enhances the Severity of Experimental Graft-Versus-Host Disease (GVHD)

Members of the NOD-like receptor (NLR) family play an important role in intestinal homeostasis. NLRP6, a NLR whose upstream agonist is not yet known, is highly expressed on the intestinal epithelium and certain immune cells. NLRP6 is an inflammasome that regulates IL-1β and IL-18 secretion. However, in contrast to other NLRs, it has also been shown to act as a regulator of intestinal inflammation and tumorigenesis either by alteration of gut microflora composition or through regulation macrophages and/ or the integrity of the epithelial barrier by goblet cells. Given its unique functions in GI tract, we determined its role in the severity of GI GVHD by testing the hypothesis that the absence of the NLRP6 would enhance GI GVHD severity. We utilized the well-characterized MHC disparate BALB/c→B6 model of GVHD. B6 wild type (WT) and B6 NLRP6^-/- animals were lethally irradiated (11Gy) and transplanted with 5x10⁶ BM and 3x10⁶ splenic T-cells from either syngeneic WT-B6 or allogeneic BALB/c donors. Contrary to the hypothesis, allogeneic B6 NLRP6^-/- animals showed significantly improved survival compared to the allo-B6 WT animals (p<0.01). The decrease in mortality was associated with reduced GVHD specific clinical severity and histopathology of the GI tract (p<0.05). Similar results were obtained in a MHC matched, minor disparate model.

Despite the reduction in GVHD mortality and GI damage, donor T-cells demonstrated equivalent expansion and cytokine secretion (IFNγ, IL-17 and TNFα) in both allogeneic WT and NLRP6^-/- animals (days 7, 14, and 21) both in the spleen and in the GI tract. Similar recruitment of donor derived monocytes in the gut (day21) was observed in both the allo-groups. The macrophages and the dendritic cells from the WT and NLRP6^-/- B6 animals induced similar in vitro proliferation and cytokine secretion of the allogeneic BALB/c T-cells. To further specifically evaluate the role of host hematopoietic derived immune cells in vivo, we generated [B6→B6Ly5.2] and [NLRP6^-/-B6→B6Ly5.2] chimeras (only the radiosensitive host hematopoietic cells are deficient for NLRP6) and utilized them as allogeneic recipients as above. Both the chimeras demonstrated similar severity and mortality. These data collectively demonstrate that the reduction in GI GVHD observed in the absence of NLRP6 in the host is unlikely due the direct effect on donor T-cells by the host hematopoietic derived cells.

Next, to test whether the alteration in the microbiota associated with NLRP6 deficiency was critical for protection from GVHD, we co-housed the NLRP6^-/- and WT B6 for 3 weeks and then utilized them as allo-recipients as above. Co-housed B6 NLRP6^-/- animals still showed significantly improved survival and decreased GVHD severity when compared to WT B6 animals, suggesting that alteration of microbiota is dispensable for protection from GVHD.

Because NLRP6 is highly expressed on the intestinal epithelium we next evaluated whether the reduction in GVHD is due to the absence of NLRP6 on the host non-hematopoietic cells such as the intestinal epithelial cells. To this end, we generated [B6→B6Ly5.2] [and [B6Ly5.2→NLRP6^-/-] BM chimeras and utilized them as allo-recipients. The allogeneic [B6Ly5.2→NLRP6^-/-] animals demonstrated significantly improved survival compared to the allogeneic [B6→B6Ly5.2] recipient animals (p<0.04). Furthermore, significantly less barrier permeability, determined by labeled dextran and greater staining of the LGR4+ intestinal cells was observed in the absence of NLRP6. These data indicate that the absence of NLRP6 in host non-hematopoietic cells, likely the intestinal epithelial cells, is critical for protection from GVHD.

To establish the mechanism through which these results are observed, we hypothesized that the GVHD protection is due to greater resistance of the intestinal epithelial cells in NLRP6 ^-/- to apoptosis following allo-BMT. We isolated intestinal epithelial cells from WT and NLRP6^-/- animals and determined the expression of pro- and anti-apoptotic genes. The expression of anti-apoptotic genes, Bcl and Bcl-XL was significantly increased without significant changes in the expression of pro-apoptotic genes in NLRP6^-/- animals. These results demonstrate a novel role and mechanism through which NLRP6 contributes to the pathogenesis of GVHD and suggest that targeting NLRP6 could be a novel strategy to mitigate GVHD.