More than 80% of mammalian antibody-secreting cells reside in the intestinal mucosa and secrete antibodies of the immunoglobulin A (IgA) isotype. IgA plays a prominent role in mediating intestinal homeostasis: IgA deficiency is common in humans and these individuals show increased disposition toward various intestinal pathologies including inflammatory bowel disease, celiac disease, and allergy. However, despite decades of research, the specificity of IgA remains elusive. IgA coats a fraction of the intestinal microbiota, yet the identities of the bacteria targeted by IgA remain unknown and it is unclear whether IgA targets all commensal bacteria or only a subset. Further, it is unknown what types of humoral responses are responsible for maintaining homeostasis with commensal bacteria. IgA may also target dietary antigens, but it is unclear whether most IgA is directed against dietary antigens, commensal microbiota, or other intestinal antigens. Lastly, specific antigens recognized by homeostatic IgA antibodies have not been described. Here we present extensive preliminary data characterizing the commensal bacteria targeted by IgA using a novel method involving bacterial flow cytometry and 16S rRNA gene sequencing, termed IgA-Seq. We further present a method for cloning and expression of recombinant monoclonal antibodies from single intestinal IgA-producing cells and rapid characterization of their reactivity against microbiota using IgA-Seq. Finally, we detail a plan for determining the antigenic targets of these antibodies. We propose to elucidate the specificity of homeostatic intestinal IgA with the following specific aims: 1) To characterize the commensal bacteria targeted by IgA; and 2) To characterize the specificities of single IgA antibodies. These data will be integrated to develop a working understanding of the commensal bacteria and specific bacterial and/or dietary antigens that drive homeostatic IgA responses. An understanding of the specificity of IgA will clarify hypotheses regarding its function, which remains largely unknown. Further, manipulation of the IgA response may represent a promising therapeutic approach to various microbiota- associated pathologies including inflammatory bowel disease, celiac disease, obesity, and diabetes, but cannot be achieved without a thorough understanding of its specificity. Lastly, our studies will generate a large panel of monoclonal antibodies specific for commensal microbiota that may allow manipulation and characterization of defined bacterial subsets in healthy or dysbiotic microbiota and may have applications as research, diagnostic, or therapeutic tools.
The proposed research will result in the generation of new tools and concepts to understand the specificity of intestinal immunoglobulin A (IgA), which is prominently involved in maintaining intestinal health. This research is relevant to public health because the results will facilitate development of new approaches to prevent, correct, or diagnose a variety of intestinal diseases including inflammatory bowel diseases, obesity, celiac disease, diabetes, allergy, and infection. Therefore, this work will directly support the NIH mission to seek fundamental knowledge that will help reduce the burden of disease and, specifically, prevent and treat intestinal disease.
|Bunker, Jeffrey J; Bendelac, Albert (2018) IgA Responses to Microbiota. Immunity 49:211-224|
|Bunker, Jeffrey J; Erickson, Steven A; Flynn, Theodore M et al. (2017) Natural polyreactive IgA antibodies coat the intestinal microbiota. Science 358:|
|Ho, Irvin Y; Bunker, Jeffrey J; Erickson, Steven A et al. (2016) Refined protocol for generating monoclonal antibodies from single human and murine B cells. J Immunol Methods 438:67-70|
|Bunker, Jeffrey J; Flynn, Theodore M; Koval, Jason C et al. (2015) Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria with Immunoglobulin A. Immunity 43:541-53|