IFNλ signaling in neutrophils enhances the pathogenesis of Bordetella pertussis infection.

Interferon lambda (IFN-λ) plays diverse roles in bacterial infections. Previously we showed that IFN-λ is induced in the lungs of B. pertussis-infected adult mice and exacerbates inflammation. Here, we report that mice lacking the IFN-λ receptor (IFNLR1) specifically on neutrophils (MRP8creIFNLR1fl/fl mice) exhibit reduced lung bacterial load and inflammation compared to WT mice during B. pertussis infection. In B. pertussis-infected wild type (WT) mice, lung type I and III IFN responses were higher than in MRP8creIFNLR1fl/fl mice, correlating with increased lung inflammatory pathology. There was an increased proportion of IFN-γ-producing neutrophils in the lungs of MRP8creIFNLR1fl/fl mice compared to WT mice. IFNLR1-/- neutrophils incubated with B. pertussis exhibited higher killing compared to WT neutrophils. Treatment of WT neutrophils with IFN-λ further decreased their bacterial killing capacity and treatment of WT mice with IFN-λ increased lung bacterial loads. Contributing to the differential killing, we found that IFNLR1-/- neutrophils exhibit higher levels of reactive oxygen species, myeloperoxidase [1], matrix metalloproteinase-9 (MMP9) activity, neutrophil extracellular traps (NETs), and IFN-γ secretion than WT neutrophils, and inhibiting NADPH oxidase inhibited bacterial killing in IFNLR1-/- neutrophils. B. pertussis induced IFN-λ secretion and IFNLR1 gene expression in mouse and human neutrophils and this was dependent on the bacterial virulence protein pertussis toxin (PTX). PTX enhanced bacterial loads in WT but not in MRP8creIFNLR1fl/fl or IFNLR1-/- mice. Thus, PTX disrupts neutrophil function by enhancing type III IFN signaling, which prevents neutrophils from effectively clearing B. pertussis during infection, leading to higher bacterial loads and exacerbation of lung inflammation.

Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice.

Many respiratory infections are selectively injurious to infants, yet the etiology of age-associated susceptibility is unknown. One such bacterial pathogen is Bordetella pertussis. In adult mice, innate interferon γ (IFN-γ) is produced by natural killer (NK) cells and restricts infection to the respiratory tract. In contrast, infant pertussis resembles disease in NK cell- and IFN-γ-deficient adult mice that experience disseminated lethal infection. We hypothesized that infants exhibit age-associated deficits in NK cell frequency, maturation, and responsiveness to B. pertussis, associated with low IFN-γ levels. To delineate mechanisms behind age-dependent susceptibility, we compared infant and adult mouse models of infection. Infection in infant mice resulted in impaired upregulation of IFN-γ and substantial bacterial dissemination. B. pertussis-infected infant mice displayed fewer pulmonary NK cells than adult mice. Furthermore, the NK cells in the infant mouse lungs had an immature phenotype, and the infant lung showed no upregulation of the IFN-γ-inducing cytokine IL-12p70. Adoptive transfer of adult NK cells into infants, or treatment with exogenous IFN-γ, significantly reduced bacterial dissemination. These data indicate that the lack of NK cell-produced IFN-γ significantly contributes to infant fulminant pertussis and could be the basis for other pathogen-induced, age-dependent respiratory diseases.

DNA-Dependent Interferon Induction and Lung Inflammation in Bordetella pertussis Infection.

Pertussis, caused by Bordetella pertussis, is a resurgent respiratory disease but the molecular mechanisms underlying pathogenesis are poorly understood. We recently showed the importance of type I and type III interferon (IFN) induction and signaling for the development of lung inflammation in B. pertussis-infected mouse models. Classically, these IFNs are induced by signaling through a variety of pattern recognition receptors (PRRs) on host cells. Here, we found that the PRR signaling adaptor molecules MyD88 and TRIF contribute to IFN induction and lung inflammatory pathology during B. pertussis infection. However, the PRRs Toll-like receptors (TLR) 3 and TLR4, which signal through TRIF and MyD88, respectively, played no role in IFN induction. Instead, the DNA-sensing PRRs, TLR9 and STING, were important for induction of type I/III IFN and promotion of inflammatory pathology, indicating that DNA is a major inducer of lung IFN responses in B. pertussis infection. These results increase our understanding of this host-pathogen interaction and identify potential targets for host-directed therapies to reduce B. pertussis-mediated pathology.

Roles and Effects of Interferon Lambda Signaling in the Context of Bacterial Infections.

Type III interferon, or interferon lambda (IFNλ), was discovered 20 years ago and has been studied primarily for its role in combatting viral infections. However, it is also induced in response to certain bacterial infections but its roles and effects in this context are relatively poorly understood. In this mini review, we discuss the roles of IFNλ signaling in bacterial infections, highlighting its deleterious or protective effects for different infections. We also discuss a couple of recent studies showing that some bacteria possess defense mechanisms against the effects of IFNλ. We hope that this review will spur further investigation into the roles of IFNλ in the context of bacterial infections and will promote considerations of its therapeutic potential for these infections.

What Causes the Cough in Whooping Cough?

What causes the cough in whooping cough (pertussis) has been a longstanding question in the field but has been difficult to answer because of the perceived lack of convenient small animal models. Y. Hiramatsu, K. Suzuki, T. Nishida, N. Onoda, et al. (mBio 13:e01397-21, 2022, https://doi.org/10.1128/mbio.03197-21) used a mouse model and cellular studies to investigate bacterial and host factors that contribute to cough production during Bordetella pertussis infection. In elegant studies, they found that the bacterial factors pertussis toxin, lipooligosaccharide, and Vag8 function cooperatively to produce cough. These factors induce production of host bradykinin, a known cough inducer that sensitizes the ion channel TRPV1 on neurons, and they investigated host signaling pathways altered by the bacterial factors that exacerbate cough responses. This is a highly significant and important finding that not only elucidates mechanisms underlying the pathophysiology of the severe cough, but also may reveal potential novel therapeutic approaches to treat individuals suffering from the debilitating effects of cough in pertussis.

Pertussis Toxin Promotes Pulmonary Hypertension in an Infant Mouse Model of Bordetella pertussis Infection.

Pertussis, caused by Bordetella pertussis, is a reemerging disease that can produce severe disease manifestations in infants, including pulmonary hypertension (PH). B. pertussis-induced PH is a major risk factor for infection-induced death, but the molecular mechanisms promoting PH are unknown and there is no effective treatment. We examined B. pertussis-induced PH in infant and adult mouse models of pertussis by Fulton index, right heart catheterization, or Doppler echocardiogram. Our results demonstrate that B. pertussis-induced PH is age related and dependent on the expression of pertussis toxin by the bacterium. Hence, pertussis toxin-targeting treatments may ameliorate PH and fatal infant infection.

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.

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo.

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.

Highlights of the 12th International Bordetella Symposium.

To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.

Age-Dependent Effects of Type I and Type III IFNs in the Pathogenesis of Bordetella pertussis Infection and Disease.

Type I and III IFNs play diverse roles in bacterial infections, being protective for some but deleterious for others. Using RNA-sequencing transcriptomics we investigated lung gene expression responses to Bordetella pertussis infection in adult mice, revealing that type I and III IFN pathways may play an important role in promoting inflammatory responses. In B. pertussis-infected mice, lung type I/III IFN responses correlated with increased proinflammatory cytokine expression and with lung inflammatory pathology. In mutant mice with increased type I IFN receptor (IFNAR) signaling, B. pertussis infection exacerbated lung inflammatory pathology, whereas knockout mice with defects in type I IFN signaling had lower levels of lung inflammation than wild-type mice. Curiously, B. pertussis-infected IFNAR1 knockout mice had wild-type levels of lung inflammatory pathology. However, in response to infection these mice had increased levels of type III IFN expression, neutralization of which reduced lung inflammation. In support of this finding, B. pertussis-infected mice with a knockout mutation in the type III IFN receptor (IFNLR1) and double IFNAR1/IFNLR1 knockout mutant mice had reduced lung inflammatory pathology compared with that in wild-type mice, indicating that type III IFN exacerbates lung inflammation. In marked contrast, infant mice did not upregulate type I or III IFNs in response to B. pertussis infection and were protected from lethal infection by increased type I IFN signaling. These results indicate age-dependent effects of type I/III IFN signaling during B. pertussis infection and suggest that these pathways represent targets for therapeutic intervention in pertussis.

Role of Major Toxin Virulence Factors in Pertussis Infection and Disease Pathogenesis.

Bordetella pertussis produces several toxins that affect host-pathogen interactions. Of these, the major toxins that contribute to pertussis infection and disease are pertussis toxin, adenylate cyclase toxin-hemolysin and tracheal cytotoxin. Pertussis toxin is a multi-subunit protein toxin that inhibits host G protein-coupled receptor signaling, causing a wide array of effects on the host. Adenylate cyclase toxin-hemolysin is a single polypeptide, containing an adenylate cyclase enzymatic domain coupled to a hemolysin domain, that primarily targets phagocytic cells to inhibit their antibacterial activities. Tracheal cytotoxin is a fragment of peptidoglycan released by B. pertussis that elicits damaging inflammatory responses in host cells. This chapter describes these three virulence factors of B. pertussis, summarizing background information and focusing on the role of each toxin in infection and disease pathogenesis, as well as their role in pertussis vaccination.

Association of Pertussis Toxin with Severe Pertussis Disease.

Pertussis, caused by respiratory tract infection with the bacterial pathogen Bordetella pertussis, has long been considered to be a toxin-mediated disease. Bacteria adhere and multiply extracellularly in the airways and release several toxins, which have a variety of effects on the host, both local and systemic. Predominant among these toxins is pertussis toxin (PT), a multi-subunit protein toxin that inhibits signaling through a subset of G protein-coupled receptors in mammalian cells. PT activity has been linked with severe and lethal pertussis disease in young infants and a detoxified version of PT is a common component of all licensed acellular pertussis vaccines. The role of PT in typical pertussis disease in other individuals is less clear, but significant evidence supporting its contribution to pathogenesis has been accumulated from animal model studies. In this review we discuss the evidence indicating a role for PT in pertussis disease, focusing on its contribution to severe pertussis in infants, modulation of immune and inflammatory responses to infection, and the characteristic paroxysmal cough of pertussis.

Peptidoglycan Recognition Protein 4 Suppresses Early Inflammatory Responses to Bordetella pertussis and Contributes to Sphingosine-1-Phosphate Receptor Agonist-Mediated Disease Attenuation.

Incidence of whooping cough (pertussis), a bacterial infection of the respiratory tract caused by the bacterium Bordetella pertussis, has reached levels not seen since the 1950s. Antibiotics fail to improve the course of disease unless administered early in infection. Therefore, there is an urgent need for the development of antipertussis therapeutics. Sphingosine-1-phosphate receptor (S1PR) agonists have been shown to reduce pulmonary inflammation during Bordetella pertussis infection in mouse models. However, the mechanisms by which S1PR agonists attenuate pertussis disease are unknown. We report the results of a transcriptome sequencing study examining pulmonary transcriptional responses in B. pertussis-infected mice treated with S1PR agonist AAL-R or vehicle control. This study identified peptidoglycan recognition protein 4 (PGLYRP4) as one of the most highly upregulated genes in the lungs of infected mice following S1PR agonism. PGLYRP4, a secreted, innate mediator of host defenses, was found to limit early inflammatory pathology in knockout mouse studies. Further, S1PR agonist AAL-R failed to attenuate pertussis disease in PGLYRP4 knockout (KO) mice. B. pertussis virulence factor tracheal cytotoxin (TCT), a secreted peptidoglycan breakdown product, induces host tissue damage. TCT-oversecreting strains were found to drive an early inflammatory response similar to that observed in PGLYRP4 KO mice. Further, TCT-oversecreting strains induced significantly greater pathology in PGLYRP4-deficient animals than their wild-type counterparts. Together, these data indicate that S1PR agonist-mediated protection against pertussis disease is PGLYRP4 dependent. Our data suggest PGLYRP4 functions, in part, by preventing TCT-induced airway damage.

Fatal Pertussis in the Neonatal Mouse Model Is Associated with Pertussis Toxin-Mediated Pathology beyond the Airways.

In infants, Bordetella pertussis can cause severe disease, manifested as pronounced leukocytosis, pulmonary hypertension, and even death. The exact cause of death remains unknown, and no effective therapies for treating fulminant pertussis exist. In this study, a neonatal mouse model of critical pertussis is characterized, and a central role for pertussis toxin (PT) is described. PT promoted colonization, leukocytosis, T cell phenotypic changes, systemic pathology, and death in neonatal but not adult mice. Surprisingly, PT inhibited lung inflammatory pathology in neonates, a result which contrasts dramatically with observed PT-promoted pathology in adult mice. Infection with a PT-deficient strain induced severe pulmonary inflammation but not mortality in neonatal mice, suggesting that death in these mice was not associated with impaired lung function. Dissemination of infection beyond the lungs was also detected in neonatal mice, which may contribute to the observed systemic effects of PT. We propose that it is the systemic activity of pertussis toxin and not pulmonary pathology that promotes mortality in critical pertussis. In addition, we observed transmission of infection between neonatal mice, the first report of B. pertussis transmission in mice. This model will be a valuable tool to investigate causes of pertussis pathogenesis and identify potential therapies for critical pertussis.

Reduction of Pertussis Inflammatory Pathology by Therapeutic Treatment With Sphingosine-1-Phosphate Receptor Ligands by a Pertussis Toxin-Insensitive Mechanism.

Recent data have demonstrated the potential of sphingosine 1-phosphate (S1P) receptor (S1PR) agonism in the treatment of infectious diseases. A previous study used a murine model of Bordetella pertussis infection to demonstrate that treatment with the S1PR agonist AAL-R reduces pulmonary inflammation during infection. In the current study, we showed that this effect is mediated via the S1PR1 on LysM+ (myeloid) cells. Signaling via this receptor results in reduced lung inflammation and cellular recruitment as well as reduced morbidity and mortality in a neonatal mouse model of disease. Despite the fact that S1PRs are pertussis toxin-sensitive G protein-coupled receptors, the effects of AAL-R were pertussis toxin insensitive in our model. Furthermore, our data demonstrate that S1PR agonist administration may be effective at therapeutic time points. These results indicate a role for S1P signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis.

Highlights of the 11th International Bordetella Symposium: from Basic Biology to Vaccine Development.

Pertussis is a severe respiratory disease caused by infection with the bacterial pathogen Bordetella pertussis The disease affects individuals of all ages but is particularly severe and sometimes fatal in unvaccinated young infants. Other Bordetella species cause diseases in humans, animals, and birds. Scientific, clinical, public health, vaccine company, and regulatory agency experts on these pathogens and diseases gathered in Buenos Aires, Argentina from 5 to 8 April 2016 for the 11th International Bordetella Symposium to discuss recent advances in our understanding of the biology of these organisms, the diseases they cause, and the development of new vaccines and other strategies to prevent these diseases. Highlights of the meeting included pertussis epidemiology in developing nations, genomic analysis of Bordetella biology and evolution, regulation of virulence factor expression, new model systems to study Bordetella biology and disease, effects of different vaccines on immune responses, maternal immunization as a strategy to prevent newborn disease, and novel vaccine development for pertussis. In addition, the group approved the formation of an International Bordetella Society to promote research and information exchange on bordetellae and to organize future meetings. A new Bordetella.org website will also be developed to facilitate these goals.

Pertussis leukocytosis: mechanisms, clinical relevance and treatment.

The significant and sometimes dramatic rise in the number of circulating white blood cells (leukocytosis) in infants suffering from pertussis (whooping cough) has been recognized for over a century. Although pertussis is a disease that afflicts people of all ages, it can be particularly severe in young infants, and these are the individuals in whom leukocytosis is most pronounced. Very high levels of leukocytosis are associated with poor outcome in infants hospitalized with pertussis and modern treatments are often aimed at reducing the number of leukocytes. Pertussis leukocytosis is caused by pertussis toxin, a soluble protein toxin released by Bordetella pertussis during infection, but the exact mechanisms by which this occurs are still unclear. In this minireview, I discuss the history of clinical and experimental findings on pertussis leukocytosis, possible contributing mechanisms causing this condition and treatments aimed at reducing leukocytosis in hospitalized infants. Since recent studies have detailed significant associations between specific levels of pertussis leukocytosis and fatal outcome, this is a timely review that may stimulate new thinking on how to understand and combat this problem.

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1ß and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

Intracellular disassembly and activity of pertussis toxin require interaction with ATP.

The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.