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P776 Dysbiosis in Nlrp6/Asc-deficient mice does not result from inflammasome deficiency

Mamantopoulos M.1,2, Ronchi F.3, Van Hauwermeiren F.2,4, Vieira-Silva S.5,6, Yilmaz B.3, Martens L.2,4, Saeys Y.2,4, Drexler S.7, Yazdi A.8, Raes J.5,6, Lamkanfi M.2,4, McCoy K.3, Wullaert A.*2,4

1Ghent University, Department of Internal Medicine, Ghent, Belgium 2VIB Inflammation Research Center, Ghent, Belgium 3Universitätsklinik Bern Inselspital, Department of Gastroenterology, Bern, Switzerland 4Ghent University, Ghent, Belgium 5KU Leuven, Rega Institute, Department of Microbiology and Immunobiology, Leuven, Belgium 6VIB Center for the Biology of Disease, Leuven, Belgium 7Biozentrum, University of Basel, Basel, Switzerland 8Department of Dermatology, University of Tübingen, Tübingen, Germany

Background

Shifts in intestinal microbial composition, termed dysbiosis, have been associated with Inflammatory Bowel Diseases (IBD). Genetic mouse models lacking Nlrp6 or Asc, which were proposed to regulate intestinal inflammasome activation, were reported with dysbiosis when compared to unrelated C57BL/6 controls, which conferred increased susceptibility to colon inflammation [1]. The Nlrp6/ASC inflammasome therefore was proposed as a host-encoded machinery that determines gut microbiota composition and thereby regulates intestinal health, suggesting that Nlrp6/ASC inflammasome deregulation may be a causal factor in eliciting dysbiosis-associated IBD.

Methods

Using unrelated separately housed wild-type mice as controls is prone to non-genetic factors such as housing conditions and maternal inheritance influencing the gut microbiota. We therefore subjected littermate and ex-germfree mice from distinct breeding schemes and housing conditions to faecal 16S sequencing in order to thoroughly delineate the contributions of host genetics, housing conditions and maternal inheritance to the gut microbiota composition in Nlrp6- or Asc-deficient mice.

Results

When comparing separately housed offspring from unrelated homozygous C57BL/6 and Nlrp6KO breeding couples, Nlrp6KO mice did not display overall gut microbial dysbiosis but did show statistically significant alterations in a number of individual bacterial taxa. In addition, cage and mother co-variates had bigger effects on the gut microbiota composition than the host genetic Nlrp6 status. Therefore, to minimise differential maternal inheritance as a confounding factor we next generated Nlrp6 and Asc+/− mice and inter-crossed those to obtain littermates of the three distinct +/+, +/− and −/− genotypes. Upon weaning, we housed these littermates separately according to their +/+, +/− and −/− genotype, thus avoiding coprophagy-mediated balancing of the gut microbiota. This tightly controlled experimental set-up allowed investigating whether the host Nlrp6 or Asc genetic status exerts an impact on the gut microbiota composition. In this controlled experimental set-up, Nlrp6−/− and Asc−/− mice did not display gut microbial dysbiosis when compared with wild-type littermates and none of the individual bacterial taxa differences in gut microbial alterations observed in Nlrp6KO vs C57BL/6 mice could be reproduced in these littermates.

Conclusion

These results show that the Nlrp6/Asc host genetic status does not influence the composition of the faecal microbiota and suggest that prior dysbiosis findings in inflammasome-deficient mice may result rather from differential maternal inheritance and/or long-term separate housing.

References:

[1] Elinav E, Strowig T, Kau AL, Henao-Mejia J, Thaiss CA, Booth CJ, Peaper DR, Bertin J, Eisenbarth SC, Gordon JI, Flavell RA (2011), NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell