Bacteroides fragilis outer membrane vesicles are used for secretion of a discrete subset of bacterial RNAs that stimulate an immune response in colonic epithelial cells.

Alterations in the diversity and function of the gut microbiome are associated with changes in the host physiology, including inflammation. A critical component of the inflammatory response system are receptors capable of sensing foreign nucleic acids (e.g. small RNAs) that are carried as cargo in bacterial outer membrane vesicles (OMVs). The mechanisms by which human extracellular RNAs elicit immune responses have been well established, while the contribution of bacterial sRNA to host physiology remain unclear. We hypothesize that pathogenic and commensal microbes use OMV-associated small RNA species to differentially affect host inflammatory responses. First, we profiled the small RNA contents of purified OMVs from a commensal strain (NTBF) and a pathogenic strain (ETBF) of Bacteroides fragilis. To distinguish the differences in the sRNA profiles of both strains and their OMVs, we conducted small RNA-seq and identified enrichment of discrete sRNA species in OMVs that were also differentially expressed between the two strains. This evidence led us to investigate the differential effects of these OMVs upon intestinal epithelial cells. To understand the effects of OMVs on pattern recognition receptors, we treated Toll-like receptor (TLR) reporter cells with NTBF and ETBF vesicles. We observed activation of TLR2 in a dose-dependent manner, and activation of TLRs 3 and 7 at high doses of OMVs. Using Caco-2 and HT29 cells exposed to OMVs from each strain, we ran qPCR to test several pro- and anti-inflammatory cytokines. We observed that both strains upregulate the expression of IL-1β and TGFβ, but NTBF stimulates a greater IL-8 response compared to ETBF. These results indicate that bacteria may preferentially load small RNAs into vesicles that target host cells, which differentially affect host immune responses through RNA-sensing pathways. Overall, our data suggest a key function of bacterial small RNAs and their OMV vehicles in controlling host immune system.