Girish S. Kirimanjeswara

Type I IFN Signaling Constrains IL-17A/F Secretion by γδ T Cells during Bacterial Infections

Recognition of intracellular bacteria by macrophages leads to secretion of type I IFNs. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. In this study, we investigated the role of type I IFNs in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1−/−) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A+ γδ T cell population, indicating that type I IFNs negatively regulate the number of IL-17A+ γδ T cells during infection. Furthermore, IL-17A–deficient mice contained fewer neutrophils compared with wild-type mice during infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1−/− mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent F. tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A+ γδ T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1−/− mice to infection with F. novicida and other intracellular bacteria.

Modeling Systems-Level Regulation of Host Immune Responses

Many pathogens are able to manipulate the signaling pathways responsible for the generation of host immune responses. Here we examine and model a respiratory infection system in which disruption of host immune functions or of bacterial factors changes the dynamics of the infection. We synthesize the network of interactions between host immune components and two closely related bacteria in the genus Bordetellae. We incorporate existing experimental information on the timing of immune regulatory events into a discrete dynamic model, and verify the model by comparing the effects of simulated disruptions to the experimental outcome of knockout mutations. Our model indicates that the infection time course of both Bordetellae can be separated into three distinct phases based on the most active immune processes. We compare and discuss the effect of the species-specific virulence factors on disrupting the immune response during their infection of naive, antibody-treated, diseased, or convalescent hosts. Our model offers predictions regarding cytokine regulation, key immune components, and clearance of secondary infections; we experimentally validate two of these predictions. This type of modeling provides new insights into the virulence, pathogenesis, and host adaptation of disease-causing microorganisms and allows systems-level analysis that is not always possible using traditional methods.

Pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of Bordetella pertussis

Whooping cough is considered a childhood disease, although there is growing evidence that children are infected by adult carriers. Additionally, increasing numbers of vaccinated adults are being diagnosed with Bordetella pertussis disease. Thus it is critical to understand how B. pertussis remains endemic even in highly vaccinated or immune populations. Here we used the mouse model to examine the nature of sterilizing immunity to B. pertussis. Antibodies were necessary to control infection but did not rapidly clear B. pertussis from the lungs. However, antibodies affected B. pertussis after a delay of at least a week by a mechanism that involved neutrophils and Fc receptors, suggesting that neutrophils phagocytose and clear antibody-opsonized bacteria via Fc receptors. B. pertussis blocked migration of neutrophils and inhibited their recruitment to the lungs during the first week of infection by a pertussis toxin-dependent (PTx-dependent) mechanism; a PTx mutant of B. pertussis induced rapid neutrophil recruitment and was rapidly cleared from the lungs by adoptively transferred antibodies. Depletion of neutrophils abrogated the defects of the PTx mutant. Together these results indicate that PTx inhibits neutrophil recruitment, which consequently allows B. pertussis to avoid rapid antibody-mediated clearance and therefore successfully infect immune hosts.

Humoral and Cell-Mediated Immunity to the Intracellular Pathogen Francisella tularensis

Summary: Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell‐mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell‐derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell‐mediated immune responses to this pathogen.

Role of Antibodies in Immunity to Bordetella Infections

ABSTRACT The persistence of Bordetellapertussis and B. parapertussis within vaccinated populations and the reemergence of associated disease highlight the need to better understand protective immunity. The present study examined host immunity to bordetellae and addressed potential concerns about the mouse model by using a comparative approach including the closely related mouse pathogen B. bronchiseptica. As previously observed with B. pertussis, all three organisms persisted throughout the respiratory tracts of B-cell-deficient mice, indicating that B cells are required for bacterial clearance. However, adoptively transferred antibodies rapidly cleared B. bronchiseptica but not human pathogens. These results obtained with the mouse model are consistent with human clinical observations, including the lack of correlation between antibody titers and protection, as well as the limited efficacy of intravenous immunoglobulin treatments against human disease. Together, this evidence suggests that the mouse model accurately reflects substantial differences between immunities to these organisms. Although both B. pertussis and B. parapertussis are more closely related to B. bronchiseptica than they are to each other, they share the ability to resist rapid clearance from the lower respiratory tract by adoptively transferred antibodies, an adaptation that correlates with their emergence as human pathogens that circulate within vaccinated populations.

Adaptation of Francisella tularensis to the Mammalian Environment Is Governed by Cues Which Can Be Mimicked In Vitro

ABSTRACT The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacteriums infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

An Improved Vaccine for Prevention of Respiratory Tularemia Caused by Francisella tularensis SchuS4 Strain

Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB(Ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB(Ft) mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB(Ft) vaccinated group as compared to the LVS vaccinated mice. sodB(Ft) vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB(Ft) vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB(Ft) mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS.

Mucosal Immunopathogenesis of Francisella tularensis

Abstract:  Respiratory infection with Francisella tularensis is the deadliest form of disease and represents the most likely route to be used by bioterrorists. Although mucosal surfaces represent the first line of defense against respiratory tularemia, and in fact, against the great majority of human pathogens, little is known about protective immunity at these sites. The objective of this chapter is to review recent data examining the importance of various pulmonary immune mechanisms in defense against F. tularensis infection and to evaluate potential strategies for induction of protective lung immunity. Aerosol and intranasal mouse infection models have yielded essentially equivalent results and have implicated an important role for Th1‐type immune responses in protection, including IFN‐γ, TNF‐α, and IL‐12. The cells responsible for protection in the lung are not well‐characterized but NK cells are an early target for activation after infection although it appears that CD8 T cells might be most critical for host resistance. In addition, it is becoming increasingly clear that antibodies can provide prophylactic and therapeutic protection against pulmonary infection but only in the presence of active cell‐mediated immunity. In fact, in vitro exposure of resting macrophages to antibody‐coated bacteria in the absence of IFN‐γ can actually enhance infection. Although various immune mechanisms can be shown to be important for protection against attenuated strains such as LVS, the real challenge for the future is to design efficacious approaches to prevent disease by highly virulent strains such as SchuS4.

The Complex Mechanism of Antibody-Mediated Clearance of Bordetella from the Lungs Requires TLR4

Although the antibacterial effects of Abs are well studied in in vitro systems, the in vivo effects of Abs cannot always be accurately predicted. Complicated cross-talk between different effector functions of Abs and various arms of the immune system can affect their activities in vivo. Using the mouse respiratory pathogen Bordetella bronchiseptica, we examined the mechanisms of Ab-mediated clearance of bacteria from the respiratory tract. Interestingly, although TLR4 was not necessary for protective immunity following infection, it was required for rapid bacterial clearance in mice that were vaccinated or adoptively transferred Abs. TLR4 was important for the rapid recruitment of neutrophils that are necessary for Ab-mediated bacterial clearance via a mechanism that requires both FcγR and CR3. These data are consistent with a model in which TLR4-mediated inflammatory responses aid in the recruitment of neutrophils, which phagocytose Ab- and complement-opsonized bacteria via FcγRs and CR3. Although pattern recognition receptors are known to be involved in innate immunity and the generation of adaptive immunity, their contributions to specific adaptive immune functions should be considered in ongoing efforts to improve vaccine-induced protective immunity.

Antibody‐mediated bacterial clearance from the lower respiratory tract of mice requires complement component C3

To assess the contribution of complement to respiratory immunity in the context of a natural bacterial infection, we used mice genetically deficient in complement components and the murine pathogen Bordetella bronchiseptica. Complement component C3 was not required for the control of bacterial infection or for the generation of infection‐induced protective immunity. However, C3‐deficient (C3–/–) mice were severely defective, compared to wild type, in vaccine‐induced protective immunity. Adoptively transferred immune serum from convalescent wild‐type or C3–/– animals rapidly cleared B. bronchiseptica from the lungs of wild‐type mice but did not affect its growth in C3–/– mice, indicating that the defect is not in the generation of protective immunity, but in its function. Immune serum was effective in C5‐deficient mice but had little effect in the lungs of mice lacking either Fcγ receptors (FcγR) or CR3, suggesting bacterial clearance is not via direct complement‐mediated lysis. Together, these data indicate that complement is required for antibody‐mediated clearance of Bordetella and suggest the mechanism involves C3 opsonization of bacteria for phagocytosis that is both CR3‐ and FcγR‐dependent.