Integrated Signaling Pathways Mediate Bordetella Immunomodulation, Persistence, and Transmission.

The mammalian immune system includes a sophisticated array of antimicrobial mechanisms. However, successful pathogens have developed subversive strategies to detect, modulate, and/or evade immune control and clearance. Independent disciplines study host immunology and bacterial pathogenesis, but interkingdom signaling between bacteria and host during natural infection remains poorly understood. An efficient natural host infection system has revealed complex communication between Bordetella spp. and mice, identified novel regulatory mechanisms, and demonstrated that bordetellae can respond to microenvironment and inflammatory status cues. Understanding these bacterial signaling pathways and their complex network that allows precisely timed expression of numerous immunomodulatory factors will serve as a paradigm for other organisms lacking such a powerful experimental infection system. VIDEO ABSTRACT.

Immunomodulation as a Novel Strategy for Prevention and Treatment of Bordetella spp. Infections.

Well-adapted pathogens have evolved to survive the many challenges of a robust immune response. Defending against all host antimicrobials simultaneously would be exceedingly difficult, if not impossible, so many co-evolved organisms utilize immunomodulatory tools to subvert, distract, and/or evade the host immune response. Bordetella spp. present many examples of the diversity of immunomodulators and an exceptional experimental system in which to study them. Recent advances in this experimental system suggest strategies for interventions that tweak immunity to disrupt bacterial immunomodulation, engaging more effective host immunity to better prevent and treat infections. Here we review advances in the understanding of respiratory pathogens, with special focus on Bordetella spp., and prospects for the use of immune-stimulatory interventions in the prevention and treatment of infection.

Enhancement of immune response against Bordetella spp. by disrupting immunomodulation.

Well-adapted pathogens must evade clearance by the host immune system and the study of how they do this has revealed myriad complex strategies and mechanisms. Classical bordetellae are very closely related subspecies that are known to modulate adaptive immunity in a variety of ways, permitting them to either persist for life or repeatedly infect the same host. Exploring the hypothesis that exposure to immune cells would cause bordetellae to induce expression of important immunomodulatory mechanisms, we identified a putative regulator of an immunomodulatory pathway. The deletion of btrS in B. bronchiseptica did not affect colonization or initial growth in the respiratory tract of mice, its natural host, but did increase activation of the inflammasome pathway, and recruitment of inflammatory cells. The mutant lacking btrS recruited many more B and T cells into the lungs, where they rapidly formed highly organized and distinctive Bronchial Associated Lymphoid Tissue (BALT) not induced by any wild type Bordetella species, and a much more rapid and strong antibody response than observed with any of these species. Immunity induced by the mutant was measurably more robust in all respiratory organs, providing completely sterilizing immunity that protected against challenge infections for many months. Moreover, the mutant induced sterilizing immunity against infection with other classical bordetellae, including B. pertussis and B. parapertussis, something the current vaccines do not provide. These findings reveal profound immunomodulation by bordetellae and demonstrate that by disrupting it much more robust protective immunity can be generated, providing a pathway to greatly improve vaccines and preventive treatments against these important pathogens.

Emergence of Bordetella holmesii as a Causative Agent of Whooping Cough, Barcelona, Spain.

We describe the detection of Bordetella holmesii as a cause of whooping cough in Spain. Prevalence was 3.9% in 2015, doubling to 8.8% in 2016. This emergence raises concern regarding the contribution of B. holmesii to the reemergence of whooping cough and the effectiveness of the pertussis vaccine.

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model.

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii-specific serum antibodies might play an important role in protection. Immuno-proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA-like protein of B. holmesii. An aBH vaccine prepared from a BipA-like protein-deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA-like protein plays an important role in the protective efficacy of aBH vaccine.

Lymphoid-Tissue-Resident Commensal Bacteria Promote Members of the IL-10 Cytokine Family to Establish Mutualism.

Physical separation between the mammalian immune system and commensal bacteria is necessary to limit chronic inflammation. However, selective species of commensal bacteria can reside within intestinal lymphoid tissues of healthy mammals. Here, we demonstrate that lymphoid-tissue-resident commensal bacteria (LRC) colonized murine dendritic cells and modulated their cytokine production. In germ-free and antibiotic-treated mice, LRCs colonized intestinal lymphoid tissues and induced multiple members of the IL-10 cytokine family, including dendritic-cell-derived IL-10 and group 3 innate lymphoid cell (ILC3)-derived IL-22. Notably, IL-10 limited the development of pro-inflammatory Th17 cell responses, and IL-22 production enhanced LRC colonization in the steady state. Furthermore, LRC colonization protected mice from lethal intestinal damage in an IL-10-IL-10R-dependent manner. Collectively, our data reveal a unique host-commensal-bacteria dialog whereby selective subsets of commensal bacteria interact with dendritic cells to facilitate tissue-specific responses that are mutually beneficial for both the host and the microbe.

Glycomacropeptide administration attenuates airway inflammation and remodeling associated to allergic asthma in rat.

OBJECTIVE: Glycomacropeptide (GMP) is a bioactive peptide derived from milk that has been reported to exhibit a range of anti-inflammatory and immunomodulatory properties. The aim of this study was to analyze the prophylactic effect of GMP administration on airway inflammation and remodeling in an experimental model of asthmatic rat. METHODS: Animals treated orally with or without GMP (500 mg/kg/day) were ovalbumin-sensitized and -nebulized and several indicators of Th2 response, airway structural changes and inflammatory cells recruitment were evaluated. RESULTS: Treatment with GMP prior and during asthma development resulted in reduction of allergen-specific IgE titers in serum and blood eosinophilia. Also, GMP substantially suppressed the recruitment of inflammatory cells to bronchoalveolar compartment. Histological studies demonstrated that GMP markedly inhibits eosinophils infiltration, goblet cells hyperplasia and collagen deposit in lung tissue. The latter effect was related with an inhibition in transforming growth factor-ß expression. In addition, expression of interleukin-5 and -13 were substantially inhibited in lung while that of interleukin-10 was increased. CONCLUSION: Our results suggest that administration of GMP may prevent the development of an excessive Th2 response in asthma and effectively ameliorates the progression of the disease.

Nuevo Calendario de Vacunación para España, 2016 (Parte 1) / Toward a new immunization schedule in Spain, 2016 (Part 1)

El calendario de vacunación en España es una herramienta dinámica de salud pública que ha ido incorporando cambios en función de la situación epidemiológica y la evidencia científica. La Ponencia del Programa y Registro de Vacunaciones, órgano científico-técnico del Consejo Interterritorial del Sistema Nacional de Salud, realiza evaluaciones y propone modificaciones que se incorporan en el calendario de vacunación de las comunidades autónomas (CCAA). Este artículo está dividido en dos partes y presenta la evaluación realizada para proponer un nuevo esquema de vacunación frente a difteria, tétanos, tosferina, poliomielitis, hepatitis B y enfermedad invasora por Haemophilus influenzae tipo b, centrándose esta primera parte en la exposición de motivos, el repaso a la política de vacunación en España y su impacto así como en la revisión de los calendarios de vacunación en países de nuestro entorno (AU)

The immunization schedule is a dynamic public health tool that has incorporated different changes over the years influenced by the epidemiologic situation and the scientific evidence. The Immunization Advisory Committee [Ponencia de Programa y Registro de Vacunaciones], as the Interterritorial Council scientific and technical advisory body, carries out assessments of different programmes and vaccines and proposes changes that after approval will be introduced in the Regions schedule. This article is divided into two parts presenting the rationale followed to propose a new schedule for the immunization against diphtheria, tetanus, pertussis, hepatitis B and invasive disease by Haemophilus influenzae type b. This first part is focused in the reasoning to undertake the assessment, the review of the immunization policy and the impact of immunization in Spain, as well as a review of the immunization schedules in similar countries (AU)

Bacterial Metabolism in the Host Environment: Pathogen Growth and Nutrient Assimilation in the Mammalian Upper Respiratory Tract.

Pathogens evolve in specific host niches and microenvironments that provide the physical and nutritional requirements conducive to their growth. In addition to using the host as a source of food, bacterial pathogens must avoid the immune response to their presence. The mammalian upper respiratory tract is a site that is exposed to the external environment, and is readily colonized by bacteria that live as resident flora or as pathogens. These bacteria can remain localized, descend to the lower respiratory tract, or traverse the epithelium to disseminate throughout the body. By virtue of their successful colonization of the respiratory epithelium, these bacteria obtain the nutrients needed for growth, either directly from host resources or from other microbes. This chapter describes the upper respiratory tract environment, including its tissue and mucosal structure, prokaryotic biota, and biochemical composition that would support microbial life. Neisseria meningitidis and the Bordetella species are discussed as examples of bacteria that have no known external reservoirs but have evolved to obligately colonize the mammalian upper respiratory tract.

Other Bordetellas, lessons for and from pertussis vaccines.

The Bordetella genus comprises nine species of which Bordetella pertussis and B. parapertussis are isolated from humans and are the most studied Bordetella species since they cause whooping cough. They both originate from B. bronchiseptica, which infects several mammals and immune compromised humans, but the intensive use of pertussis vaccines induced changes in B. pertussis and B. parapertussis populations. B. petrii and B. holmesii are other species of unknown reservoir and transmission pattern that have been described in humans. It is still unknown whether these species are pathogens for humans or only opportunistic bacteria but biological diagnosis has confirmed the presence of B. holmesii in human respiratory samples while B. petrii and the four other species have little implications for public health.

Horizontally acquired divergent O-antigen contributes to escape from cross-immunity in the classical bordetellae.

BACKGROUND: Horizontal gene transfer (HGT) allows for rapid spread of genetic material between species, increasing genetic and phenotypic diversity. Although HGT contributes to adaptation and is widespread in many bacteria, others show little HGT. This study builds on previous work to analyze the evolutionary mechanisms contributing to variation within the locus encoding a prominent antigen of the classical bordetellae. RESULTS: We observed amongst classical bordetellae discrete regions of the lipopolysaccharide O-antigen locus with higher sequence diversity than the genome average. Regions of this locus had less than 50% sequence similarity, low dN/dS ratios and lower GC content compared to the genome average. Additionally, phylogenetic tree topologies based on genome-wide SNPs were incongruent with those based on genes within these variable regions, suggesting portions of the O-antigen locus may have been horizontally transferred. Furthermore, several predicted recombination breakpoints correspond with the ends of these variable regions. To examine the evolutionary forces that might have selected for this rare example of HGT in bordetellae, we compared in vitro and in vivo phenotypes associated with different O-antigen types. Antibodies against O1- and O2-serotypes were poorly cross-reactive, and did not efficiently kill or mediate clearance of alternative O-type bacteria, while a distinct and poorly immunogenic O-antigen offered no protection against colonization. CONCLUSIONS: This study suggests that O-antigen variation was introduced to the classical bordetellae via HGT through recombination. Additionally, genetic variation may be maintained within the O-antigen locus because it can provide escape from immunity to different O-antigen types, potentially allowing for the circulation of different Bordetella strains within the same host population.

Adaptability and persistence of the emerging pathogen Bordetella petrii.

The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient's antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient's serum. Finally, we characterize one strain that was poorly recognized by the patient's antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.

Epidemiologic and laboratory features of a large outbreak of pertussis-like illnesses associated with cocirculating Bordetella holmesii and Bordetella pertussis--Ohio, 2010-2011.

BACKGROUND: During 9 May 2010-7 May 2011, an outbreak of pertussis-like illness (incidence, 80 cases per 100 000 persons) occurred in Franklin County, Ohio. The majority of cases were identified by IS481-directed polymerase chain reaction (PCR), which does not differentiate among Bordetella species. We sought to determine outbreak etiology and epidemiologic characteristics. METHODS: We obtained demographic, clinical, and vaccination-related data from the Ohio Disease Reporting System and Impact Statewide Immunization Information System. We tested sera from 14 patients for anti-pertussis toxin (PT) antibodies and used species-specific PCR on 298 nasopharyngeal specimens. RESULTS: Reported cases totaled 918. IS481 results were available for 10 serologically tested patients; 5 of 10 had discordant anti-PT antibody and IS481 results, suggestive of Bordetella holmesii, which lacks PT and harbors IS481. We identified specific Bordetella species in 164 of 298 specimens tested with multitarget PCR; B. holmesii and Bordetella pertussis were exclusively detected among 48 (29%) and 112 (68%), respectively; both were detected in 4 (2%). Among 48 patients with B. holmesii infections, 63% were aged 11-18 years, compared with 35% of 112 patients with B. pertussis infections (P = .001). Symptoms were similar among B. holmesii- and B. pertussis-infected patients. Adolescent pertussis ("Tdap") booster vaccinations were more effective against B. pertussis than B. holmesii (effectiveness: 67% and 36%, respectively; 95% confidence intervals, 38%-82% and -33% to 69%, respectively). CONCLUSIONS: We report the first documented mixed outbreak of B. pertussis and B. holmesii infections. Bordetella holmesii particularly affected adolescents. Although laboratory capacity limitations might inhibit routine use of multitarget PCR for clinical diagnosis, focused testing and enhanced surveillance might improve understanding the burden of B. holmesii infection.

Lack of cross-protection against Bordetella holmesii after pertussis vaccination.

Bordetella holmesii, a species closely related to B. pertussis, has been reported sporadically as a cause of whooping cough-like symptoms. To investigate whether B. pertussis-induced immunity is protective against infection with B. holmesii, we conducted an analysis using 11 human respiratory B. holmesii isolates collected during 2005-2009 from a highly B. pertussis-vaccinated population in Massachusetts. Neither whole-cell (wP) nor acellular (aP) B. pertussis vaccination conferred protection against these B. holmesii isolates in mice. Although T-cell responses induced by wP or aP cross-reacted with B. holmesii, vaccine-induced antibodies failed to efficiently bind B. holmesii. B. holmesii-specific antibodies provided in addition to wP were sufficient to rapidly reduce B. holmesii numbers in mouse lungs. Our findings suggest the established presence of B. holmesii in Massachusetts and that failure to induce cross-reactive antibodies may explain poor vaccine-induced cross-protection.

Septic arthritis caused by Bordetella holmesii in an adolescent with chronic haemolytic anaemia.

We describe a case of septic arthritis caused by Bordetella holmesii in a 15-year-old boy with chronic haemolytic anaemia. B. holmesii was identified by 16S rRNA gene sequence analysis. The patient outcome was favourable. To our knowledge, this is the first case of B. holmesii septic arthritis in an asplenic patient.

Chlamydia pneumoniae infection induced allergic airway sensitization is controlled by regulatory T-cells and plasmacytoid dendritic cells.

Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.

Expression of beta-defensins in the canine respiratory tract and antimicrobial activity against Bordetella bronchiseptica.

Beta-defensins are cationic peptides which form part of the innate immune response of the respiratory epithelium. Due to their antimicrobial properties and immunostimulatory activity, beta-defensins are potential tools for the treatment and prevention of respiratory disease. In dogs, infectious respiratory disease is a common problem, particularly in housed animals. This study aimed to assess the presence of four beta-defensins in the canine respiratory tract and to use quantitative real-time PCR to determine mRNA levels following microbial challenge. Three beta-defensins, CBD1, CBD103 and CBD108, were detected in respiratory cells. All three defensins were also readily expressed in skin samples, while their expression in lymphoid tissues and the kidney was low and inconsistent. Treatment of primary tracheal epithelial cells with lipopolysaccharide (LPS) or infection with canine respiratory coronavirus led to decreased expression of CBD103 and CBD108, while cells infected with canine parainfluenza virus had lower levels of CBD1 and CBD108. Furthermore CBD103 was demonstrated to have antimicrobial activity against the respiratory pathogen Bordetella bronchiseptica.

Oral administration of beta-1,3/1,6-glucan to dogs temporally changes total and antigen-specific IgA and IgM.

The effect of oral administration of beta-1,3/1,6-glucans from Saccharomyces cerevisiae on humoral immunity in domestic dogs is not known. In this study, 15 beagle dogs were orally given MacroGard tablets, which contain 150 mg of this beta-glucan, daily for 4 weeks. At the end of this period, the total serum immunoglobulin A (IgA) level decreased significantly in the group treated with the glucan compared to that in the control group as well as compared to the concentrations before supplementation. In contrast, the total serum IgM level rose significantly, whereas no effect on the IgG level occurred. Similar changes were seen in Bordetella-specific IgA and IgM titers following vaccination during the supplementation period. The IgA concentration also became significantly lower in the saliva and tears of the glucan group than in the placebo group. The effects disappeared 1 week after the cessation of the supplementation. In conclusion, the results showed a temporary change in the isotype profile during glucan supplementation.

Bordetella evades the host immune system by inducing IL-10 through a type III effector, BopN.

The inflammatory response is one of several host alert mechanisms that recruit neutrophils from the circulation to the area of infection. We demonstrate that Bordetella, a bacterial pathogen, exploits an antiinflammatory cytokine, interleukin-10 (IL-10), to evade the host immune system. We identified a Bordetella effector, BopN, that is translocated into the host cell via the type III secretion system, where it induces enhanced production of IL-10. Interestingly, the BopN effector translocates itself into the nucleus and is involved in the down-regulation of mitogen-activated protein kinases. Using pharmacological blockade, we demonstrated that BopN-induced IL-10 production is mediated, at least in part, by its ability to block the extracellular signal-regulated kinase pathway. We also showed that BopN blocks nuclear translocation of nuclear factor kappaB p65 (NF-kappaBp65) but, in contrast, promotes nuclear translocation of NF-kappaBp50. A BopN-deficient strain was unable to induce IL-10 production in mice, resulting in the elimination of bacteria via neutrophil infiltration into the pulmonary alveoli. Furthermore, IL-10-deficient mice effectively eliminated wild-type as well as BopN mutant bacteria. Thus, Bordetella exploits BopN as a stealth strategy to shut off the host inflammatory reaction. These results explain the ability of Bordetella species to avoid induction of the inflammatory response.