In microbiome research, many mechanistic insights are enabled through the use of animal models. A new paper co-led by Andy Wullaert in Belgium and Kathy McCoy in Switzerland addresses a major question about the use of animal models and the conclusions drawn from experiments that are not rigorously designed.
Although it is well known that gut microbiota is important in defining susceptibility to multiple human diseases, including inflammatory bowel disease, the use of germ-free (GF) and gnotobiotic animals has led to the notion that the Nlrp6-ASC inflammasome is a hallmark of host innate immunity that shapes gut microbiota composition. Such a notion results from studies reporting dysbiosis in knock-out mice lacking key components of the inflammasome, which were compared with non-littermate wild type rodents.
The inflammasome is an enzymatic protein complex composed of NOD-like receptor (NLR) molecules that, upon binding with their ligand, will oligomerize and recruit ASC adaptor proteins to form filaments. The latter will bind and activate pro-caspase-1, which after dimerization and auto-cleavage, displays caspase-1 enzymatic activities. It then results in processing of pro-IL1ß and pro-IL-18 to activate these pro-inflammatory cytokines.
Works published previously in Cell in 2011 and 2015 have studied the differences in gut microbiota from mice deficient in the Nlrp/ASC pathway. Indeed, the fecal microbiota from ASC-deficient mice was very different from that of non-littermate wild type animals. Similar observations were made in mice lacking NLRP6; thus, the conclusion drawn from these results was that the inflammasome, i.e. innate immunity, was shaping the gut microbiota composition. Interestingly, these inflammasome-deficient mice displayed intestinal hyperplasia, and were more susceptible to dextran sulfate sodium (DSS)-induced colitis. Moreover, upon conventionalization, germ-free Nlrp6-KO mice showed dysbiosis when compared with non-littermate GF-control mice. The observed dysbiosis in inflammasome-deficient mice was therefore considered to be responsible for susceptibility to colitis, but also for liver steatosis and obesity.
However, in the microbiome field and especially in animal models, factors other than genetics may explain gut microbiota differences. These include cage effects, maternal inheritance, presence of unexpected pathogens or pathobionts, as well as factors related to the animal care personnel. In the present study, the two labs decided to use littermate controlled ex-GF rodents. They elegantly showed that variation in gut microbiota between separately housed inflammasome-deficient and non-littermate control mice seemed mainly explained by mother and cage covariates. Then, they crossed Nlrp6 -/- mice with control C57BL/6 mice. They obtained Nlrp6 -/+ mice that were crossed together to obtain Nlrp6 -/- and Nlrp6 +/+ littermates that were separated after birth. They observed that separate housing of littermates with two different genetic backgrounds did not reveal any significant difference concerning gut microbiota composition, even after one year of breeding or a second generation. Furthermore, they did not find any difference in susceptibility to colitis. Similar findings were obtained in littermate mice lacking ASC or not.
This interesting and provocative piece of work underlines the need for thoroughly designed animal experiments, and questions many of the conclusions drawn from studies published in the past on genetically raised animals—in addition to the inflammasome story.
Elinav E, Strowig T, Kau AL, et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell. 2011; 145:745-57.
Henao-Mejia J, Elinav E, Jin C, et al. Inflammasome mediated dysbiosis regulates progression of NAFLD and obesity. Nature. 2012; 482:179–85.
Levy M, Thaiss CA, Zeevi D, et al. Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling. Cell. 2015; 163:1428-43.
Mamantopoulos M, Ronchi F, Van Hauwermeiren F, et al. Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition. Immunity. 2017; 47(2):339–348.e4.
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