The role of the gut microbiota and its metabolites in acute vascular rejection

The gut microbiota influences development and control of the immune system. In the context of solid organ transplantation, the composition of this microbial community affects immune responses that cause rejection. My thesis project has used a mouse model of vascular rejection to examine how disruption of the gut microbiota with antibiotics early in life exacerbates immune responses that cause acute vascular rejection. The gut microbiota and its composition were disrupted in murine recipients of fully major histocompatibility complex (MHC) mismatched aortic interposition transplants using an antibiotic cocktail of ampicillin, metronidazole, neomycin sulphate and vancomycin in the drinking water. Initial experiments examined the effect of eliminating the gut microbiota by treating aortic transplant recipients with antibiotics for life. Transplants were performed at 8 – 12 weeks of age. Antibiotic treatment increased immune cell accumulation and medial degradation in aortic allografts, which are features of acute vascular rejection. Medial injury was specifically related to increased neutrophil accumulation early after transplantation. The effect of early life disruption of the gut microbiota on rejection was then assessed by treating mice with antibiotics for only the first three weeks of life. Early life disruption of the gut microbiota in this way altered the composition of the bacterial population by 8 – 12 weeks of age, as determined by 16S sequencing, but the total abundance of bacteria was not changed. This treatment also increased neutrophil accumulation and acute rejection of vascular grafts that were performed later in life at 8 – 12 weeks of age. Although these experiments established a role for the gut microbiota in controlling neutrophil responses in transplant rejection, the taxonomic and metagenomic changes in the microbiota that could be responsible for this effect remained poorly understood. Subsequent studies examined the composition of the gut microbiota using both 16S and metagenomic sequencing of DNA isolated from mouse stool, followed by contemporary bioinformatic approaches that permitted the inference and direct identification of genes related to metabolic reactions. Antibiotic treatment early in life (for only the first three weeks, hereafter referred to as antibiotic treated) resulted in persistent changes to the composition of the gut microbiota, including a decrease in the bacterial class Bacteroidia, and an increase in Clostridia. Shotgun sequencing determined that Akkermansia muciniphila, Lactobacillus murinus, and L. Johnsonii were virtually absent in antibiotic treated mice, all of which are species that produce the immunoregulatory metabolite acetate that can inhibit neutrophil activation. Cohousing antibiotic treated mice with untreated mice reversed these compositional changes. Antibiotic treatment also altered the abundance of genes encoding metabolic pathways, including a significant reduction in the abundance of several genes related to the metabolism of mucin that produces acetate as a by-product. Specifically, beta-N-acetylhexosaminidase is an enzyme that is responsible for an initial cleavage of mucin carbohydrates and was decreased in antibiotic treated mice, and this was rescued by cohousing. These observations suggested a role of acetate and the taxa that produce it in regulating neutrophil accumulation in vascular rejection. The role of acetate in controlling acute vascular rejection was then experimentally examined based on the implication of this metabolite from genomic analyses of the microbiome. Artery graft recipients were untreated, treated with antibiotics for the first 3 weeks of life, co-housed, or treated with antibiotics and then administered acetate. Normalizing the gut microbiota of antibiotic-treated mice by co-housing and providing exogenous acetate prevented the exacerbation of neutrophil responses and acute vascular rejection caused by early life disruption of the gut microbiota. All together, my findings show that dysbiosis of the gut microbiota caused by its disruption early in life may have consequences on rejection of solid organ transplants and identify bacterial species and acetate as immune regulatory components of this microbial community in this setting.