NLRP6 aggravates graft-versus-host disease (GvHD) regardless of gut microbial composition

Graft-versus-host disease (GvHD) is a life-threatening complication following allogeneic hematopoietic cell transplantation (allo-HCT). The gastrointestinal tract is a main target organ in GvHD, and the severity of gastrointestinal GvHD (GI GvHD) is in part mediated by the host’s innate immunity and microbiome. NOD-like receptor family pyrin domain-containing 6 (NLRP6) has been shown to regulate innate immune responses and gastrointestinal homeostasis by providing protective resistance against enteric infections in synergy with the host microbiome.¹

Tomomi Toubai et al., from the University of Michigan, US, reported in Nature Microbiology their findings on the role of NLRP6 in GI GvHD, with the hypothesis that NLRP6 absence in the host would exacerbate GI GvHD.

Study design/methods  

Bone marrow transplant

Bone marrow transplants (BMT) were performed using B6 wild-type (WT) and B6 NLRP6^−/− murine models. Mice received 11 Gy total body irradiation on Day One before administration of 5 × 10⁶ bone marrow cells or 5 × 10⁶ whole-spleen cells from BALB/c mice on Day 0.

Cytokine production

• Measurement of cytokine production:

• 1cm colon and ileum sections were taken from WT B6 and NLRP6^−/− mice following allo-BMT

• ELISA assessment of IFN-γ, IL-17A, TNF-α, IL-6, IL-1β and IL-18 concentrations in the serum or cell culture supernatant were performed

Tumor model and histopathological assessment

• B6 WT or NLRP6^−/− mice were lethally irradiated and then infused with MBL-2 tumor cells, along with with CD90+ T cells and T cell-depleted bone marrow cells (from either B6 WT or NLRP6^−/− animals or MHC-mismatched BALB/c donors), at the same time as allo-HCT. Growth was monitored using bioluminescence imaging 21 days after allo-BMT

• Primary GvHD target organs underwent histopathological analysis: liver, gastrointestinal tract, lungs and skin using a semiquantitative scoring system implemented by a single pathologist

• Histopathological evaluation of epithelial damage in the tissue sections was performed using hematoxylin and eosin staining or MUC2 immunohistochemistry

• Survival and the degree of clinical GvHD were monitored after allo-HCT daily

Fecal microbiota transplantation

To assess host-microbiota difference:

• NLRP6^−/−and B6 WT littermates were placed under germ-free (GF) conditions
• The GF littermates were then colonized with the microbiome from specific pathogen-free (SPF) animals
• Microbial communities were left to reconstitute in the gastrointestinal tract for one month

Results

Absence of NLRP6 reduces the severity of GvHD

• The gastrointestinal tracts of the B6 NLRP6^−/− hosts showed significantly less severe GI GvHD-specific histopathological scores and exhibited a better survival rate after allo-BMT compared to the B6 WT hosts

• Goblet cell numbers (in both the small and the large intestines) were increased in B6 NLRP6^−/− recipients during the early phase of allo-BMT. Mucus production by the goblet cells is crucial for gut homeostasis preservation

• However, donor conventional T-cell and regulatory T-cell expansion, CD69 expression, interferon-γ (IFN-γ)-producing T cells, and pro-inflammatory cytokines’ (IFN-γ, tumor necrosis factor-α (TNF-α) and interleukin-17A (IL-17A)) serum levels did not significantly differ between the two mice strains following allo-BMT

Improved GvHD and OS in NLRP6^−/− mice is independent of the microbiome composition

• NLRP6^-/- mice displayed the signature of dysbiosis before and after allo-BMT, the severity of GI GvHD was reduced, and survival was improved

• Similarly, antibiotic-treated NLRP6^−/− mice maintained a reduced GI GvHD rate and had a higher survival rate than B6 WT mice

• The fecal transplant experiment showed that GvHD severity and risk of death after BMT continued to be reduced in the NLRP6^−/− hosts irrespective of stool graft type (SPF B6 WT stool versus SPF NLRP6^−/− stool). Similarly, B6 WT animals transplanted with SPF NLRP6^−/− stool showed similar mortality and GvHD severity compared to B6 WT animals colonized with SPF B6 WT stool

• After co-housing NLRP6^−/−and B6 WT mice for two weeks, there was no significant difference in fecal bacterial colonization between the two types of mice. Nevertheless, B6 WT animals still had higher mortality and increased severe GI GvHD after allo-BMT compared to NLRP6^-/- animals

Graft-versus-tumor effect is intact in NLRP6^-/- mice

• As GvHD is associated with graft-vs-tumor (GvT) effect, the authors investigated whether deficiency of NLRP6 affects the GvT effect, with the assumption that GvT would be preserved in NLRP6 ^-/-animals, due to the similar immune response

• As was expected, NLRP6 ^-/-animals showed equivalent GvT responses compared to B6 WT animals

Conclusion

B6 NLRP6 ^-/- animals showed significantly improved survival in comparison to the B6 WT animals after allo-BMT, which was independent of the microbiome composition and treatment with antibiotics. In contrast, allo-BMT transplanted B6 NLRP6 ^-/- animals demonstrated a similar inflammasome response and therapeutic GvT effect compared to transplanted B6 WT mice, indicating that loss of NLRP6 does not interfere with immune response processes. The results unveiled an unanticipated pathogenic role for NLRP6 in GI GvHD that is independent of the intestinal microbiome and immunity, and which provides a potentially novel target for therapeutic intervention.