IntroductionBordetella are respiratory pathogens comprised of three classical Bordetella species: B. pertussis, B. parapertussis, and B. bronchiseptica. With recent surges in Bordetella spp. cases and antibiotics becoming less effective to combat infectious diseases, there is an imperative need for novel antimicrobial therapies. Our goal is to investigate the possible targets of host immunomodulatory mechanisms that can be exploited to promote clearance of Bordetella spp. infections. Vasoactive intestinal peptide (VIP) is a neuropeptide that promotes Th2 anti-inflammatory responses through VPAC1 and VPAC2 receptor binding and activation of downstream signaling cascades.MethodsWe used classical growth in vitro assays to evaluate the effects of VIP on Bordetella spp. growth and survival. Using the three classical Bordetella spp. in combination with different mouse strains we were able to evaluate the role of VIP/VPAC2 signaling in the infectious dose 50 and infection dynamics. Finally using the B. bronchiseptica murine model we determine the suitability of VPAC2 antagonists as possible therapy for Bordetella spp. infections.ResultsUnder the hypothesis that inhibition of VIP/VPAC2 signaling would promote clearance, we found that VPAC2-/- mice, lacking a functional VIP/VPAC2 axis, hinder the ability of the bacteria to colonize the lungs, resulting in decreased bacterial burden by all three classical Bordetella species. Moreover, treatment with VPAC2 antagonists decrease lung pathology, suggesting its potential use to prevent lung damage and dysfunction caused by infection. Our results indicate that the ability of Bordetella spp. to manipulate VIP/VPAC signaling pathway appears to be mediated by the type 3 secretion system (T3SS), suggesting that this might serve as a therapeutical target for other gram-negative bacteria.ConclusionTaken together, our findings uncover a novel mechanism of bacteria-host crosstalk that could provide a target for the future treatment for whooping cough as well as other infectious diseases caused primarily by persistent mucosal infections.

Bordetella spp. are respiratory pathogens equipped with immune evasion mechanisms. We previously characterized a Bordetella bronchiseptica mutant (RB50ΔbtrS) that fails to suppress host responses, leading to rapid clearance and long-lasting immunity against reinfection. This work revealed eosinophils as an exclusive requirement for RB50ΔbtrS clearance. We also show that RB50ΔbtrS promotes eosinophil-mediated B/T cell recruitment and inducible bronchus-associated lymphoid tissue (iBALT) formation, with eosinophils being present throughout iBALT for Th17 and immunoglobulin A (IgA) responses. Finally, we provide evidence that XCL1 is critical for iBALT formation but not maintenance, proposing a novel role for eosinophils as facilitators of adaptive immunity against B. bronchiseptica. RB50ΔbtrS being incapable of suppressing eosinophil effector functions illuminates active, bacterial targeting of eosinophils to achieve successful persistence and reinfection. Overall, our discoveries contribute to understanding cellular mechanisms for use in future vaccines and therapies against Bordetella spp. and extension to other mucosal pathogens.

ABSTRACT A common feature of pathogens is their ability to suppress host immune responses. Understanding the molecular mechanisms and the common pathways that bacteria utilize to block host immune signaling cascade might provide novel avenues for vaccine and therapeutic development. Preventable infectious diseases remain one of the major causes of morbidity and mortality worldwide and the current rise in antibiotic resistance is increasing this burden. Bordetella spp. are respiratory pathogens that cause the long-term illness known as whooping cough. Bordetella infections cause over 150,000 deaths each year, despite a vaccine being available. In our studies, we used the mouse pathogen B. bronchiseptica to investigate the pneumonic stage of disease, which very well mimics the fatal disease caused by B. pertussis . In our previous work we discovered a B. bronchiseptica mutant, RB50Δ btrS , that clears rapidly from the lungs of mice and generates protective immunity that lasts for at least 15 months post-challenge. Combining the mouse immunological tools and both bacteria, the wildtype RB50 and mutant RB50Δ btrS , which persists for up to 56 days and clears in 14-21 days, respectively, we investigated the mechanisms by which the wildtype B. bronchiseptica blocks host immune response to cause long term lung infection. Previous research indicated eosinophils as critical for rapid clearance of the mutant bacteria from the lungs. In vitro assays with eosinophils demonstrated that the RB50Δ btrS mutant strain promotes the secretion of pro-inflammatory signals. In contrast, infection of eosinophils with RB50 promoted the secretion of anti-inflammatory signals such as IL1RA. Interestingly, IL1RA was also increased in the lungs of mice infected with the wildtype but not with the mutant RB50 strain. Infection with RB50Δ bscN , which lacks a functional type 3 secretion system (T3SS), was sufficient to prevent IL1RA induction suggesting that the bacterial effector responsible for IL1RA upregulation is a substrate of the T3SS. Supplementation with IL1RA after infection with RB50 or RB50Δ btrS resulted in increased lung bacterial burden for both bacterial strains. However, more rapid clearance of RB50 was observed after infection of mice in which IL1RA was knocked out. Furthermore, anti-IL1RA antibody treatment promoted rapid clearance of not only RB50 but also the human pathogens B. pertussis and B. parapertussis . This suggests that IL1RA may be a promising therapeutic target to treat severe cases of whooping cough. Overall, this work demonstrates that Bordetella spp. induces IL1RA expression to promote persistence using the T3SS. Since other bacteria have also been shown to target IL1RA, this may be a conserved bacterial mechanism to promote host-immune suppression.

ABSTRACTA characteristic that differentiates pathogenic and opportunistic bacteria is that pathogens have been selected by their ability to suppress host inflammatory responses allowing colonization and persistence. Bordetella spp. are respiratory pathogens characterized for the arsenal of mechanisms they use to manipulate host immune responses. We have previously characterized a B. bronchiseptica mutant, RB50ΔbtrS, that is not able to suppress host immune responses, resulting not only in rapid clearance of the infection but also long-term lung sterilizing immunity against reinfection with the three classical Bordetella spp. Interestingly, this strong immune response requires eosinophils. In this work our results indicate that wildtype B. bronchiseptica, RB50, blocks eosinophil pro-inflammatory functions to prevent the rapid recruitment of B and T cells to the lung that results in iBALT formation. Moreover, eosinophils promote a TH17 microenvironment within the iBALT that might be responsible for the long-term robust protective immunity generated by infection with this mutant. Overall, this work provides a novel role for eosinophils as promoters of adaptive immune responses and protective immunity, while also indicating that bacteria actively manipulate those cells to promote long-term persistence and reinfection.

Eosinophils are granulocytes primarily associated with TH2 responses to parasites or immune hyper-reactive states, such as asthma, allergies, or eosinophilic esophagitis. However, it does not make sense from an evolutionary standpoint to maintain a cell type that is only specific for parasitic infections and that otherwise is somehow harmful to the host. In recent years, there has been a shift in the perception of these cells. Eosinophils have recently been recognized as regulators of immune homeostasis and suppressors of over-reactive pro-inflammatory responses by secreting specific molecules that dampen the immune response. Their role during parasitic infections has been well investigated, and their versatility during immune responses to helminths includes antigen presentation as well as modulation of T cell responses. Although it is known that eosinophils can present antigens during viral infections, there are still many mechanistic aspects of the involvement of eosinophils during viral infections that remain to be elucidated. However, are eosinophils able to respond to bacterial infections? Recent literature indicates that Helicobacter pylori triggers TH2 responses mediated by eosinophils; this promotes anti-inflammatory responses that might be involved in the long-term persistent infection caused by this pathogen. Apparently and on the contrary, in the respiratory tract, eosinophils promote TH17 pro-inflammatory responses during Bordetella bronchiseptica infection, and they are, in fact, critical for early clearance of bacteria from the respiratory tract. However, eosinophils are also intertwined with microbiota, and up to now, it is not clear if microbiota regulates eosinophils or vice versa, or how this connection influences immune responses. In this review, we highlight the current knowledge of eosinophils as regulators of pro and anti-inflammatory responses in the context of both infection and naïve conditions. We propose questions and future directions that might open novel research avenues in the future.