NLRP6 in Host Target Tissues Exacerbates Graft-Versus-Host Disease

Intestinal epithelial cells play an important role in the maintenance of host microbiota as well as intestinal homeostasis by expression of pattern recognition receptors (PRRs). NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed on the intestinal epithelium and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in intestinal infection and in preventing dysbiosis of gut normal microbiota thorough the secretion of IL-18 and mucus. In addition, NLRP6 knock out (NLRP6^-/-) mice show greater severity and mortality of inflammatory bowel diseases (IBD). Given its unique functions in GI tract and in immune cells, we determined its role in the severity of GI GVHD by testing the hypothesis that the absence of the NLRP6 in hosts would enhance GVHD.

 We utilized the well-characterized MHC disparate BALB/c→B6 model of GVHD. B6 wild type (WT) and B6 NLRP6^-/- animals were lethally irradiated (10Gy) and transplanted with 5x10⁶ BM and 3x10⁶ splenic T-cells from either syngeneic WT-B6 or allogeneic BALB/c donors. Contrary to the hypothesis and in contrast to IBD model, 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 antigen mismatched C3H.sw into B6 model for eliminating strain dependent artifacts. 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 CD11b⁺ cells in the gut (day21) was observed in both the allo-groups. The macrophages and the dendritic cells from the WT and NLRP6^-/- B6 animals showed similar allogeneic T cell proliferation in an MLR. Next, to dissect the cause for reducing GVHD mortality and to determine whether NLRP6 expression in host hematopoietic derived cells is critical, we generated [B6→B6Ly5.2] and [NLRP6^-/-B6→B6Ly5.2] chimeras and utilized them as hosts in 2^nd allo-BMT and found that both the chimeras demonstrated similar severity and mortality. These data collectively demonstrate that the reduction in GI GVHD, observed in the absence of NLRP6, is independent of NLRP6 expression in host hematopoietic derived cells.

Because the absence of NLRP6 could alter endogenous microbiota colonization, we co-housed the NLRP6^-/- and WT B6 for 3 weeks and then utilized them as hosts in allo-BMT. Co-housed B6 NLRP6^-/- animals still showed significantly improved survival and decreased GVHD severity when compared to WT B6 animals (p<0.05), 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 of GVHD is due to the absence of NLRP6 on the host non-hematopoietic cells such as the intestinal epithelium. To this end, we generated [B6→B6Ly5.2] and [B6Ly5.2→NLRP6^-/-] chimeras and utilized them as hosts. The allogeneic [B6Ly5.2→NLRP6^-/-] animals demonstrated significantly improved survival compared to the allogeneic [B6→B6Ly5.2] recipient animals (p<0.05). Furthermore, allo- NLRP6 ^-/- animals showed significantly less barrier permeability and increased level of Claudin 3 and EphB3 which are critical in the repair process in intestinal epithelium (p<0.05). Interestingly, serum level of IL-18, an inflammasome-dependent cytokine, and the expression of anti (bcl-2 or bcl-xl)- or pro- apoptotic genes (Bax or Bak) in epithelium of NLRP6^-/- animals after allo-BMT was equivalent to WT-B6 animals. These data suggest that the absence of NLRP6 in host non-hematopoietic cells, likely the intestinal epithelial cells, is crucial for protection from GVHD.

Our results indicate that NLRP6 has various functions and has differential effects in GVHD pathogenesis in contrast to IBD where NLRP6 has protective effect.