Two main subsets of B cells, B1 and B2, are major contributors to intestinal homeostasis, mainly through secretion of IgA. The role of IgM in the control of intestinal microbiota, however, is not understood and until now has been mostly ignored. Using a mouse model of pneumonic tularemia, we have recently shown that IgM directed against the LPS of the Gram-negative bacterium Francisella tularensis (Ft) plays a protective role in the early phase of the infection. These antibodies are rapidly produced in a IL-1?-dependent fashion by B1 B cells. Our preliminary results also show that anti-Ft LPS IgM is present in the small intestine and is cross- reactive with components of the intestinal microbiota, though not with several Gram-negative pathogens. Finally, we have evidence that lung infection with Ft causes a shift in the composition of the intestinal microbiota. Taken together, these results suggest a previously unknown mechanism through which pulmonary infections can potentially alter the composition of the intestinal microbiota. Our hypothesis is that Ft-specific IgM generated in the early phase of the infection is cross-reactive with intestinal commensals and causes a rapid shift in the composition of the microbiota. We will test this novel concept in the following specific aims.
Aim1. How does Ft lung infection change the intestinal microbiota? 1A. Intestinal microbiota diversity will be examined before and after Ft lung infection, immunization with purified Ft LPS, or passive transfer of Ft- immune serum. We will determine whether the microbiota change is transitory or long lasting, and whether it depends on IL-1?. 1B. Mouse strains that lack secretory IgM, or the polymeric Ig receptor, or activation- induced cytidine deaminase (AID) will be used to test the hypothesis that IgM is necessary and sufficient for the microbiota changes associated to Ft infection/vaccination.
Aim2. Which B1 B cell subset contributes to the generation of Ft-specific and microbiota-cross-reactive IgM? 2A. Adoptive transfer of B1a, B1b, marginal zone B cells, or follicular B cells into Rag1-/- mice will clarify which of these B cell subsets is responsible for production of the Ft-specific and microbiota cross-reactive IgM and whether different subsets generate each specificities. These experiments will also reveal the role of each B cells subsets in the homeostatic regulation of intestinal microbiota. 2B. Adoptive transfer of B1 cells deficient in the integrin ?4?7 will determine whether microbiota changes require homing of cells to the intestinal lamina propria. The successful completion of the proposed research will identify the role of IgM and B1 cell subsets in the regulation of gut microbial diversity and will uncover a novel mechanism through which infections may lead to dysbiosis.
Infections cause changes in the intestinal flora that may affect development of immunity and determine increased susceptibility to certain diseases. The successful completion of the proposed research will uncover a novel mechanism through which infections may cause changes in the composition of intestinal microbial diversity and will identify the role of IgM and B1 cell subsets in these events. These novel concepts would represent a paradigm shift in our understanding of microbial homeostasis and will potentially promote the development of novel vaccination strategies.
