Luminal-applied flagellin is internalized by polarized intestinal epithelial cells and elicits immune responses via the TLR5 dependent mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.     

M-Cells Contribute to the Entry of an Oral Vaccine but Are Not Essential for the Subsequent Induction of Protective Immunity against Francisella tularensis

M-cells (microfold cells) are thought to be a primary conduit of intestinal antigen trafficking. Using an established neutralizing anti-RANKL (Receptor Activator of NF-κB Ligand) antibody treatment to transiently deplete M-cells in vivo, we sought to determine whether intestinal M-cells were required for the effective induction of protective immunity following oral vaccination with ΔiglB (a defined live attenuated Francisella novicida mutant). M-cell depleted, ΔiglB-vaccinated mice exhibited increased (but not significant) morbidity and mortality following a subsequent homotypic or heterotypic pulmonary F. tularensis challenge. No significant differences in splenic IFN-γ, IL-2, or IL-17 or serum antibody (IgG1, IgG2a, IgA) production were observed compared to non-depleted, ΔiglB-vaccinated animals suggesting complementary mechanisms for ΔiglB entry. Thus, we examined other possible routes of gastrointestinal antigen sampling following oral vaccination and found that ΔiglB co-localized to villus goblet cells and enterocytes. These results provide insight into the role of M-cells and complementary pathways in intestinal antigen trafficking that may be involved in the generation of optimal immunity following oral vaccination.     

Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein.

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IkappaBalpha loss poststimulation and NF-kappaB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-gamma-primed DLD-1 cells stimulated with 1 microg/ml of 6HIS flag induced high levels of NO (60 +/- 0.95 microM) comparable to the combination of IL-1beta and IFN-gamma (77 +/- 1.2) or purified native SD flag (66.3 +/- 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IkappaBalpha degradation, NF-kappaB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1-2/ECH/CD2-1 also induced high levels of TNF-alpha (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.     

Immunomodulatory and Antibacterial Effects of Cystatin 9 against Francisella tularensis

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.     

Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction

Gram-negative sepsis is mediated by the actions of proinflammatory genes induced in response to microbes and their products. We report that flagellin, the monomeric subunit of flagella, is a potent proinflammatory species released by Salmonella. Flagellin (1 microgram/ml) induces IkappaBalpha degradation, NF-kappaB nuclear translocation, and inducible NO synthase expression in cultured intestinal epithelial cells (IEC). Aflagellic Salmonella mutants do not induce NF-kappaB activation or NO production by cultured IEC. Antiserum to flagellin blocks NO production in IEC induced by medium conditioned by a variety of motile Gram-negative enteric pathogens (Escherichia coli, Salmonella muenchen, Serratia marcescens, Proteus mirabilis, and Proteus vulgaris). Flagellin, when injected systemically (approximately 10 microgram/mouse), induces systemic inflammation characterized by the systemic expression of a range of proinflammatory cytokines and chemokines and of inducible NO synthase. At higher doses (approximately 300 microgram/mouse), flagellin induces shock, characterized by hypotension, reduced vascular contractility in mice, and death. The effects of flagellin do not diminish in C3H/HeJ LPS-resistant mice, indicating that the Toll-like receptor-4 receptor is not involved in flagellin's actions. In LPS-resistant mice, i.p. injection of S. dublin flagellin or medium conditioned by wild-type S. dublin induces serum IFN-gamma and TNF-alpha, whereas medium conditioned by aflagellic mutants has no effect. Flagellin can be detected in the blood of rats with septic shock induced by live bacteria at approximately 1 microg/ml. We propose that flagellin released by Gram-negative pathogens may contribute to the inflammatory response by an LPS- and Toll-like receptor-4-independent pathway.     

Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives.