Human macrophage polarization shapes B. pertussis intracellular persistence.

We previously demonstrated that Bordetella pertussis, the etiologic agent of whooping cough, is able to survive inside human macrophages. The aim of this study was to examine the influence of macrophage polarization in the development of B. pertussis intracellular infections. To this end, primary human monocytes were differentiated into M1, M2a, or M2c macrophages and further infected with B. pertussis. Infected M1 macrophages showed a proinflammatory response evidenced by the production of TNF-α, IL-12p70, and IL-6. Conversely, infection of M2a and M2c macrophages did not induce TNF-α, IL-12p70, nor IL-6 at any time postinfection but showed a significant increase of M2 markers, such as CD206, CD163, and CD209. Interestingly, anti-inflammatory cytokines, like IL-10 and TGF-ß, were induced after infection in the 3 macrophage phenotypes. B. pertussis phagocytosis by M1 macrophages was lower than by M2 phenotypes, which may be ascribed to differences in the expression level of B. pertussis docking molecules on the surface of the different phenotypes. Intracellular bactericidal activity was found to be significantly higher in M1 than in M2a or M2c cells, but live bacteria were still detected within the 3 phenotypes at the late time points after infection. In summary, this study shows that intracellular B. pertussis is able to survive regardless of the macrophage activation program, but its intracellular survival proved higher in M2 compared with the M1 macrophages, being M2c the best candidate to develop into a niche of persistence for B. pertussis.

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of Bordetella pertussis.

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMß2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.

Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture.

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

Melanin Produced by Bordetella parapertussis Confers a Survival Advantage to the Bacterium during Host Infection.

Bordetella parapertussis causes respiratory infection in humans, with a mild pertussis (whooping cough)-like disease. The organism produces a brown pigment, the nature and biological significance of which have not been elucidated. Here, by screening a transposon library, we demonstrate that the gene encoding 4-hydroxyphenylpyruvate dioxygenase (HppD) is responsible for production of this pigment. Our results also indicate that the brown pigment produced by the bacterium is melanin, because HppD is involved in the biosynthesis of a type of melanin called pyomelanin, and homogentisic acid, the monomeric precursor of pyomelanin, was detected by high-performance liquid chromatography-mass spectrometry analyses. In an infection assay using macrophages, the hppD-deficient mutant was internalized by THP-1 macrophage-like cells, similar to the wild-type strain, but was less able to survive within the cells, indicating that melanin protects B. parapertussis from intracellular killing in macrophages. Mouse infection experiments also showed that the hppD-deficient mutant was eliminated from the respiratory tract more rapidly than the wild-type strain, although the initial colonization levels were comparable between the two strains. In addition, melanin production by B. parapertussis was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. Taken together, our findings indicate that melanin produced by B. parapertussis in a BvgAS-independent manner confers a survival advantage to the bacterium during host infection. IMPORTANCE In addition to the Gram-negative bacterium Bordetella pertussis, the etiological agent of pertussis, Bordetella parapertussis also causes respiratory infection in humans, with a mild pertussis-like disease. These bacteria are genetically closely related and share many virulence factors, including adhesins and toxins. However, B. parapertussis is clearly distinguished from B. pertussis by its brown pigment production, the bacteriological significance of which remains unclear. Here, we demonstrate that this pigment is melanin, which is known to be produced by a wide range of organisms from prokaryotes to humans and helps the organisms to survive under various environmental stress conditions. Our results show that melanin confers a survival advantage to B. parapertussis within human macrophages through its protective effect against reactive oxygen species and eventually contributes to respiratory infection of the bacterium in mice. This study proposes melanin as a virulence factor involved in the increased survival of B. parapertussis during host infection.

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein.

The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air-liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC- toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT- toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.

Reinvestigating the Coughing Rat Model of Pertussis To Understand Bordetella pertussis Pathogenesis.

Bordetella pertussis is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP, DTaP, and Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the reemergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. The emergence of genetically diverging strains, combined with waning aP vaccine efficacy, calls for reevaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged 7-week-old Sprague-Dawley rats with 108 viable Tohama 1 and D420 and measured the hallmark signs/symptoms of B. pertussis infection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole-body plethysmography. Onset of cough occurred between 2 and 4 days after B. pertussis challenge, averaging five coughs per 15 min, with peak coughing occurring at day 8 postinfection, averaging upward of 13 coughs per 15 min. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days postchallenge. The rats exhibited increased bronchial restriction following B. pertussis infection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-B. pertussis IgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences between B. pertussis strains.

Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) Contributes to Bordetella pertussis Inflammatory Pathology.

Whooping cough (pertussis) is a severe pulmonary infectious disease caused by the bacteria Bordetella pertussis. Pertussis infects an estimated 24 million people annually, resulting in >150,000 deaths. The NIH placed pertussis on the list of emerging pathogens in 2015. Antibiotics are ineffective unless administered before the onset of the disease characteristic cough. Therefore, there is an urgent need for novel pertussis therapeutics. We have shown that sphingosine-1-phosphate receptor (S1PR) agonists reduce pertussis inflammation without increasing bacterial burden. Transcriptomic studies were performed to identify this mechanism and allow for the development of pertussis therapeutics that specifically target problematic inflammation without sacrificing bacterial control. These data suggested a role for triggering receptor expressed on myeloid cells-1 (TREM-1). TREM-1 cell surface receptor functions as an amplifier of inflammatory responses. Expression of TREM-1 is increased in response to bacterial infection of mucosal surfaces. In mice, B. pertussis infection results in Toll-like receptor 9 (TLR9)-dependent increased expression of TREM-1 and its associated cytokines. Interestingly, S1PR agonists dampen pulmonary inflammation and TREM-1 expression. Mice challenged intranasally with B. pertussis and treated with ligand-dependent (LP17) and ligand-independent (GF9) TREM-1 inhibitors showed no differences in bacterial burden and significantly reduced tumor necrosis factor-α (TNF-α) and C-C motif chemokine ligand 2 (CCL-2) expression compared to controls. Mice receiving TREM-1 inhibitors showed reduced pulmonary inflammation compared to controls, indicating that TREM-1 promotes inflammatory pathology, but not bacterial control, during pertussis infection. This implicates TREM-1 as a potential therapeutic target for the treatment of pertussis.

Cytokine expression patterns in hospitalized children with Bordetella pertussis, Rhinovirus or co-infection.

Mechanisms of interaction between Bordetella pertussis and other viral agents are yet to be fully explored. We studied the inflammatory cytokine expression patterns among children with both viral-bacterial infections. Nasopharyngeal aspirate (NPA) samples were taken from children, aged < 1 year, positive for Rhinovirus, Bordetella pertussis and for Rhinovirus and Bordetella pertussis. Forty cytokines were evaluated in NPA by using human cytokine protein arrays and a quantitative analysis was performed on significantly altered cytokines. Forty cytokines were evaluated in NPA by using human cytokine protein arrays and a quantitative analysis was performed on significantly altered cytokines. Our results show that co-infections display a different inflammatory pattern compared to single infections, suggesting that a chronic inflammation caused by one of the two pathogens could be the trigger for exacerbation in co-infections.

IL-17 mediates protective immunity against nasal infection with Bordetella pertussis by mobilizing neutrophils, especially Siglec-F+ neutrophils.

Understanding the mechanism of protective immunity in the nasal mucosae is central to the design of more effective vaccines that prevent nasal infection and transmission of Bordetella pertussis. We found significant infiltration of IL-17-secreting CD4+ tissue-resident memory T (TRM) cells and Siglec-F+ neutrophils into the nasal tissue during primary infection with B. pertussis. Il17A-/- mice had significantly higher bacterial load in the nasal mucosae, associated with significantly reduced infiltration of Siglec-F+ neutrophils. Re-infected convalescent mice rapidly cleared B. pertussis from the nasal cavity and this was associated with local expansion of IL-17-producing CD4+ TRM cells. Depletion of CD4 T cells from the nasal tissue during primary infection or after re-challenge of convalescent mice significantly delayed clearance of bacteria from the nasal mucosae. Protection was lost in Il17A-/- mice and this was associated with significantly less infiltration of Siglec-F+ neutrophils and antimicrobial peptide (AMP) production. Finally, depletion of neutrophils reduced the clearance of B. pertussis following re-challenge of convalescent mice. Our findings demonstrate that IL-17 plays a critical role in natural and acquired immunity to B. pertussis in the nasal mucosae and this effect is mediated by mobilizing neutrophils, especially Siglec-F+ neutrophils, which have high neutrophil extracellular trap (NET) activity.

Bordetella holmesii Lipopolysaccharide Hide and Seek Game with Pertussis: Structural Analysis of the O-Specific Polysaccharide and the Core Oligosaccharide of the Type Strain ATCC 51541.

Whooping cough is a highly contagious disease caused predominantly by Bordetella pertussis, but it also comprises of a pertussis-like illness caused by B. holmesii. The virulence factors of B. holmesii and their role in the pathogenesis remain unknown. Lipopolysaccharide is the main surface antigen of all Bordetellae. Data on the structural features of the lipopolysaccharide (LPS) of B. holmesii are scarce. The poly- and oligosaccharide components released by mild acidic hydrolysis of the LPS were separated and investigated by 1H and 13C NMR spectroscopy, mass spectrometry, and chemical methods. The structures of the O-specific polysaccharide and the core oligosaccharide of B. holmesii ATCC 51541 have been identified for the first time. The novel pentasaccharide repeating unit of the B. holmesii O-specific polysaccharide has the following structure: {→2)-α-l-Rhap-(1→6)-α-d-Glcp-(1→4)-[ß-d-GlcpNAc-(1→3]-α-d-Galp-(1→3)-α-d-GlcpNAc-(1→}n. The SDS-PAGE and serological cross-reactivities of the B. holmesii LPS suggested the similarity between the core oligosaccharides of B. holmesii ATCC 51541 and B. pertussis strain 606. The main oligosaccharide fraction contained a nonasaccharide. The comparative analysis of the NMR spectra of B. holmesii core oligosaccharide fraction with this of the B. pertussis strain 606 indicated that the investigated core oligosaccharides were identical.

Effect of FHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and anatomical site.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.

Toward an Alum Free Mono-Component Monovalent Pertussis Vaccine: A Cytokine Response Assay.

BACKGROUND: Current evidence indicates the resurgence of whooping cough despite high coverage of whole-cell (wP) and acellular (aP) pertussis vaccines. OBJECTIVE: To investigate the cytokine response to a genetically inactivated protein containing the S1 subunit of pertussis toxin (PTS1) with and without the Listeriolysin O (LLO-PTS1), in comparison with current wP and aP vaccines in the mice model. METHODS: Thirty-six female NMRI mice aged 8 to 12 weeks (25 ± 5 g) were divided into six groups, including control (n=6) and five treated groups (n=6/each). Treated groups received intraperitoneal injection of recombinant PTS1, recombinant fusion LLO-PTS1, aP, wP, and sham (phosphate-buffered saline), whereas the control group did not receive anything. After 60 days, the serum levels of IFN-γ, IL-4, and IL-17 cytokines were evaluated by ELISA method. RESULTS: Our findings showed LLO-PTS1 significantly increased IL-17 and IL-4 cytokines compared with wP and aP vaccines. IFN-γ failed to increase substantially in the LLO-PTS1 group compared to others, but it was non-inferior to standard vaccines. CONCLUSION: Our alum free mono-component monovalent recombinant fusion protein (LLO-PTS1) could bear the capacity to stimulate the release of IFN-γ similar to wP and aP vaccines in the mouse model. Besides, it showed better results in stimulating the release of IL-17 and IL-4 response. This study can be regarded as a platform for further probes in booster pertussis vaccine development.

Anti-FIM and Anti-FHA Antibodies Inhibit Bordetella pertussis Growth and Reduce Epithelial Cell Inflammation Through Bacterial Aggregation.

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction of B. pertussis with A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Our in vitro results indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survival in vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction of B. pertussis with host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such as in vitro potency assays.

Early Protection against Pertussis Induced by Live Attenuated Bordetella pertussis BPZE1 Depends on TLR4.

Pertussis is a severe respiratory disease mainly caused by Bordetella pertussis Despite wide global vaccination coverage with efficacious pertussis vaccines, it remains one of the least well-controlled vaccine-preventable diseases, illustrating the shortcomings of the current vaccines. We have developed the live attenuated nasal pertussis vaccine BPZE1, currently undergoing clinical evaluation in human phase 2 trials. We have previously shown that in mice, BPZE1 provides strong and long-lasting protection against B. pertussis challenge by inducing potent Ab and T cell responses as well as secretory IgA and IL-17-producing resident memory T lymphocytes in the nasal cavity. In this study, we show that BPZE1 induces protection in mice against B. pertussis within days after vaccination, at a time when Ab and T cell responses were not detectable. Early protection was independent of T and B cell responses, as demonstrated by the use of SCID mice. Instead, it was due to TLR4-dependent signaling through the MyD88-dependent pathway of the innate immune response, as demonstrated in experiments with TLR4-deficient and MyD88-knockout mice. TLR2-dependent signaling did not play a major role in early protection. In addition, this study also shows that even at high doses, BPZE1 is safe in the severely immunocompromised MyD88-deficient mice, whereas virulent B. pertussis caused a severe pathological condition and death in these mice, even at a low dose. Finally, coadministration of virulent B. pertussis with BPZE1 did not cause exacerbated outgrowth of the virulent strain, thereby adding to the safety profile of this live vaccine candidate.

Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages.

Pertussis is a highly contagious respiratory infection caused by the bacterium Bordetella pertussis. Humans are the only known natural reservoir of B. pertussis. In mice, macrophages and NK cells have a key role in confining B. pertussis to the respiratory tract. However, the mechanisms underlying this process, particularly during human infections, remain unclear. Here we characterized the activation of human macrophages and NK cells in response to B. pertussis and unraveled the role of inflammasomes in this process. NLRP3 inflammasome activation by B. pertussis in human macrophage-like THP-1 cells and primary monocyte-derived macrophages (mo-MΦ) was shown by the visualization of ASC-speck formation, pyroptosis, and the secretion of caspase-mediated IL-1ß and IL-18. In contrast to macrophages, stimulation of human CD56+CD3- NK cells by B. pertussis alone did not result in activation of these cells. However, co-culture of B. pertussis-stimulated mo-MΦ and autologous NK cells resulted in high amounts of IFNγ secretion and an increased frequency of IL-2Rα+ and HLA-DR+ NK cells, indicating NK cell activation. This activation was significantly reduced upon inhibition of inflammasome activity or blocking of IL-18 in the mo-MΦ/NK cell co-culture. Furthermore, we observed increased secretion of proinflammatory cytokines in the B. pertussis-stimulated mo-MΦ/NK co-culture compared to the mo-MΦ single culture. Our results demonstrate that B. pertussis induces inflammasome activation in human macrophages and that the IL-18 produced by these cells is required for the activation of human NK cells, which in turn enhances the pro-inflammatory response to this pathogen. Our data provides a better understanding of the underlying mechanisms involved in the induction of innate immune responses against B. pertussis. These findings contribute to the knowledge required for the development of improved intervention strategies to control this highly contagious disease.

Bordetella pertussis Whole Cell Immunization, Unlike Acellular Immunization, Mimics Naïve Infection by Driving Hematopoietic Stem and Progenitor Cell Expansion in Mice.

Hematopoietic stem and progenitor cell (HSPC) compartments are altered to direct immune responses to infection. Their roles during immunization are not well-described. To elucidate mechanisms for waning immunity following immunization with acellular vaccines (ACVs) against Bordetella pertussis (Bp), we tested the hypothesis that immunization with Bp ACVs and whole cell vaccines (WCVs) differ in directing the HSPC characteristics and immune cell development patterns that ultimately contribute to the types and quantities of cells produced to fight infection. Our data demonstrate that compared to control and ACV-immunized CD-1 mice, immunization with an efficacious WCV drives expansion of hematopoietic multipotent progenitor cells (MPPs), increases circulating white blood cells (WBCs), and alters the size and composition of lymphoid organs. In addition to MPPs, common lymphoid progenitor (CLP) proportions increase in the bone marrow of WCV-immunized mice, while B220+ cell proportions decrease. Upon subsequent infection, increases in maturing B cell populations are striking in WCV-immunized mice. RNAseq analyses of HSPCs revealed that WCV and ACV-immunized mice vastly differ in developing VDJ gene segment diversity. Moreover, gene set enrichment analyses demonstrate WCV-immunized mice exhibit unique gene signatures that suggest roles for interferon (IFN) induced gene expression. Also observed in naïve infection, these IFN stimulated gene (ISG) signatures point toward roles in cell survival, cell cycle, autophagy, and antigen processing and presentation. Taken together, these findings underscore the impact of vaccine antigen and adjuvant content on skewing and/or priming HSPC populations for immune response.

Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory.

Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response.

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers.

The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

Bordetella Adenylate Cyclase-Hemolysin Toxins.

Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented.

CD71+ erythroid suppressor cells impair adaptive immunity against Bordetella pertussis.

Infant's immune system cannot control infection or respond to vaccination as efficiently as older individuals, a phenomenon that has been attributed to immunological immaturity. Recently, we challenged this notion and proposed the presence of actively immunosuppressive and physiologically enriched CD71+ erythroid cells in neonates. Here we utilized Bordetella pertussis, a common neonatal respiratory tract pathogen, as a proof of concept to investigate the role of these cells in adaptive immunity. We observed that CD71+ cells have distinctive immunosuppressive properties and prevent recruitment of immune cells to the mucosal site of infection. CD71+ cells ablation unleashed induction of B. pertussis-specific protective cytokines (IL-17 and IFN-γ) in the lungs and spleen upon re-infection or vaccination. We also found that CD71+ cells suppress systemic and mucosal B. pertussis-specific antibody responses. Enhanced antigen-specific adaptive immunity following CD71+ cells depletion increased resistance of mice to B. pertussis infection. Furthermore, we found that human cord blood CD71+ cells also suppress T and B cell functions in vitro. Collectively, these data provide important insight into the role of CD71+ erythroid cells in adaptive immunity. We anticipate our results will spark renewed investigation in modulating the function of these cells to enhance host defense to infections in newborns.