Nicholas. H. Carbonetti

Serious pertussis infections disproportionately affect infants but the biological basis for this age-dependent susceptibility remains unclear. Infant mouse models recapitulate features of severe human infant pertussis. We investigated the role of interferon lambda (IFN-λ), a key regulator of mucosal immunity, in Bordetella pertussis infection of infant mice. While infected adult mice upregulate lung IFN-λ, infant mice inoculated at P7 fail to upregulate IFN-λ and succumb to infection. We hypothesized that failure to produce IFN-λ during infection represents a critical immunological deficit in infant mice, and that restoring IFN-λ signaling would improve survival outcomes. Whereas wild-type mice gained complete protection from lethal B. pertussis infection by P10, mice lacking the IFN-λ receptor component IFNLR1 did not achieve full protection until P21. Loss of IFNLR1 was associated with enhanced bacterial dissemination to systemic organs. Infant mice possessed a functional IFN-λ receptor in the lungs but failed to upregulate IFN-λ during infection, and supplementing IFN-λ exogenously extended survival. RNA sequencing of lung tissue from infected and uninfected wild-type and IFNLR1-deficient mice inoculated at different ages identified an immune transcriptional framework distinguishing susceptible from resistant animals at a systems level and revealed IFN-λ signaling as a critical driver of immunological maturation in the infant lung. Infected infant IFNLR1-deficient mice had dysregulated immune cell recruitment to the lungs, indicating a quantitatively expanded but qualitatively impaired response. These findings demonstrate that IFN-λ affects immune maturation accounting for a critical window of age-dependent resistance to lethal pertussis with novel therapeutic possibilities for human infants with this disease.

Bordetella pertussis , the etiologic agent of whooping cough, remains a serious public health concern despite widespread vaccination. Improved therapeutics and vaccines are urgently needed to treat and prevent pertussis disease. Host recognition of bacterial peptidoglycan (PGN), including B. pertussis extracellular PGN fragment tracheal cytotoxin (TCT), shapes the immune response to infection. Peptidoglycan recognition proteins (PGLYRPs) are a conserved family of innate immune molecules which bind bacterial PGN. While they function as immune signaling receptors in arthropods (termed PGRPs in arthropods), PGLYRPs in mammals have thus far been primarily recognized for their bactericidal activity. Previously thought to function only as antimicrobial peptides in mammals, the immune modulatory roles of this family of peptidoglycan recognition proteins are beginning to gain greater appreciation. Peptidoglycan recognition protein 1 (PGLYRP1) is a secreted antimicrobial protein. However, its role in mammalian host defenses and immune signaling during infection with Gram-negative pathogens, such as B. pertussis , remain largely unknown. Here, we identify a dual role for PGLYRP1 in modulating host immune responses to B. pertussis . Using knockout mice, single-cell and bulk transcriptomics and functional assays, we show that PGLYRP1 contributes to host antibacterial responses to B. pertussis . PGLYRP1 also dampens inflammatory responses and paradoxically inhibits bacterial killing later in infection. Mechanistically, PGLYRP1 enhances nucleotide oligomerization domain (NOD)-1 signaling in response to TCT while suppressing NOD2- and triggering receptor expressed on myeloid cells-1 (TREM-1)-mediated inflammatory pathways. TCT-bound PGLYRP1 selectively impairs TREM-1 activation compared to PGNs from other bacteria, revealing a novel bacterial immune evasion strategy. These findings demonstrate that B. pertussis co-opts PGLYRP1 to temper inflammation and alter immune signaling, revealing a novel immune evasion mechanism of manipulating the availability and structure of their exogenous peptidoglycan, revealing implications for host-pathogen evolution, vaccine design and host-directed therapeutics.

Type I interferons (IFNs) play complex roles during bacterial infections. We previously found that type I IFNs were induced in Bordetella pertussis -infected adult mice but not in infant mice, a potentially relevant clinical dichotomy, since pertussis can be fatal in human infants. We investigated the role of type I IFNs and their cross-regulation with type III IFNs (IFN-λ) in B. pertussis infection across developmental stages. In contrast to global IFNAR1 knockout adult mice, in which lung inflammation was equivalent to that in wild-type mice, myeloid cell-specific deficiency of the type I IFN receptor protein IFNAR1 (LysM ^Cre IFNAR1 ^fl/fl ) resulted in significantly reduced lung inflammation and pro-inflammatory cytokine production, despite elevated pulmonary IFN-λ levels. Mechanistically, we found that, in contrast to WT macrophages, IFNAR1-deficient macrophages produced IFN-λ in response to B. pertussis or pertussis toxin, a process dependent on the G protein-coupled receptor lysophosphatidic acid receptor 1 (LPAR1). IFNAR1 deficiency did not affect type I IFN expression or killing capacity by macrophages and neutrophils. In striking contrast to WT infant mice, which developed resistance to lethal B. pertussis infection by postnatal day 10 (P10), LysM ^Cre IFNAR1 ^fl/fl infant mice remained highly susceptible to lethal infection through P21, exhibiting increased lung bacterial burden and inflammation, as well as increased bacterial dissemination compared to WT infant mice. These findings reveal a critical age- and cell-specific interplay between type I and III IFNs during B. pertussis infection and highlight a novel LPAR1-dependent pathway for IFN-λ induction in the absence of type I IFN signaling.

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 (MRP8^creIFNLR1^fl/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 MRP8^creIFNLR1^fl/fl mice, correlating with increased lung inflammatory pathology. There was an increased proportion of IFN-γ-producing neutrophils in the lungs of MRP8^creIFNLR1^fl/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 MRP8^creIFNLR1^fl/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.

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-gamma (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 suffer 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.

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.

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 (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.

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.

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.