Nico Marr

Attenuation of Respiratory Syncytial Virus-Induced and RIG-I-Dependent Type I IFN Responses in Human Neonates and Very Young Children

Newborn infants, including those born at term without congenital disorders, are at high risk of severe disease from respiratory syncytial virus (RSV) infection. Indeed, our current local surveillance data demonstrate that approximately half of children hospitalized with RSV were ≤3 mo old, and 74% were born at term. Informed by this clinical epidemiology, we investigated antiviral innate immune responses in early life, with the goal of identifying immunological factors underlying the susceptibility of infants and young children to severe viral lower respiratory tract infections. We compared RSV-induced innate cytokine production in blood mononuclear cells from neonates, young children aged 12–59 mo, and healthy adults. RSV-induced IFN-α production was primarily mediated by plasmacytoid dendritic cells (pDCs), and was significantly lower in term infants and young children < 5 y of age than in adults (p < 0.01). RSV-induced IFN-α production in human pDCs proceeded independently of endosomal TLRs, and human pDCs from healthy adult donors produced IFN-α in a retinoic acid–inducible gene I protein (RIG-I)–dependent manner. Of interest, young age and premature birth were independently associated with attenuated RIG-I–dependent IFN-α responses (p < 0.01). In contrast to IFN-α production, proinflammatory IL-6 responses to RSV were mediated by monocytes, appeared less dependent on RIG-I, and were significantly impaired only among preterm infants, not in term infants and young children. Our results suggest that human pDCs are less functional in early life, which may contribute to the increased susceptibility of infants and young children to severe RSV disease.

Glucosamine Found as a Substituent of Both Phosphate Groups in Bordetella Lipid A Backbones: Role of a BvgAS-Activated ArnT Ortholog

Endotoxins are amphipathic lipopolysaccharides (LPSs), major constituents of the outer membrane of gram-negative bacteria. They consist of a lipid region, covalently linked to a core oligosaccharide, to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of the biological activities of endotoxins have been associated with the lipid moiety of the molecule: unique to gram-negative bacteria, LPS is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous with respect to both the numbers and the lengths of fatty acids and the natures of substituents on the phosphate groups when present. The variants can significantly affect host immune responses. Nine species in the Bordetella genus have been described, and the fine LPS structures of seven of them have been published. In this report, lipids A from Bordetella pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described for bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. Reverse transcriptase PCR of this locus showed that it is Bvg regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

Bordetella pertussis Autotransporter Vag8 Binds Human C1 Esterase Inhibitor and Confers Serum Resistance

Bordetella pertussis employs numerous strategies to evade the immune system, including the ability to resist killing via complement. Previously we have shown that B. pertussis binds a complement regulatory protein, C1 esterase inhibitor (C1inh) to its surface in a Bvg-regulated manner (i.e. during its virulence phase), but the B. pertussis factor was not identified. Here we set out to identify the B. pertussis C1inh-binding factor. Using a serum overlay assay, we found that this factor migrates at approximately 100 kDa on an SDS-PAGE gel. To identify this factor, we isolated proteins of approximately 100 kDa from wild type strain BP338 and from BP347, an isogenic Bvg mutant that does not bind C1inh. Using mass spectrometry and bioinformatics, we identified the autotransporter protein Vag8 as the putative C1inh binding protein. To prove that Vag8 binds C1inh, vag8 was disrupted in two different B. pertussis strains, namely BP338 and 18–323, and the mutants were tested for their ability to bind C1inh in a surface-binding assay. Neither mutant strain was capable of binding C1inh, whereas a complemented strain successfully bound C1inh. In addition, the passenger domain of Vag8 was expressed and purified as a histidine-tagged fusion protein and tested for C1inh-binding in an ELISA assay. Whereas the purified Vag8 passenger bound C1inh, the passenger domain of BrkA (a related autotransporter protein) failed to do so. Finally, serum assays were conducted to compare wild type and vag8 mutants. We determined that vag8 mutants from both strains were more susceptible to killing compared to their isogenic wild type counterparts. In conclusion, we have discovered a novel role for the previously uncharacterized protein Vag8 in the immune evasion of B. pertussis. Vag8 binds C1inh to the surface of the bacterium and confers serum resistance.

Protective activity of the Bordetella pertussis BrkA autotransporter in the murine lung colonization model.

This study examined the vaccine potential of the autotransporter protein BrkA of Bordetella pertussis in the sublethal intranasal murine respiratory challenge model of infection. Five different acellular pertussis (Pa) vaccines, containing different pertussis-component antigens but all comprizing diphtheria (D) and tetanus (T) toxoids, were tested. A two-pertussis-component DTPa vaccine containing pertussis toxoid (PT) and filamentous hemagglutinin (FHA) induced only limited bacterial clearance. However, a three-pertussis-component DTPa vaccine containing PT, FHA and a recombinant BrkA protein (rBrkA) was found to be as efficacious in protecting mice against colonization by B. pertussis strains Tohama I and 18-323 as the commercial Infanrixtrade mark vaccine that also includes PT and FHA but pertactin (PRN) instead of rBrkA. Vaccination of mice with rBrkA as the only B. pertussis antigen did not protect against colonization by B. pertussis. We also demonstrated that BrkA is ubiquitously expressed by highly prevalent clinical isolates of B. pertussis and suggest that new acellular pertussis vaccine formulations that include BrkA have equivalent efficacy as currently available DTPa vaccines against B. pertussis infections.

Bordetella pertussis Binds Human C1 Esterase Inhibitor during the Virulent Phase, to Evade Complement-Mediated Killing

C1 esterase inhibitor (C1inh) is a major inhibitor of several pathways of inflammation in humans. In this study, we show that virulent-phase cultures of Bordetella pertussis, the etiological agent for whooping cough, but not other Bordetella species specifically recruit C1inh from human serum. Using a spontaneous mutant of B. pertussis that was deficient in C1inh binding, we demonstrate that the ability of B. pertussis to acquire high levels of human C1inh and wild-type levels of serum resistance are well correlated, suggesting that, in addition to and independent of BrkA expression, acquisition of C1inh is vital to B. pertussis resistance to complement-mediated killing.

Functional genetic variation in NFKBIA and susceptibility to childhood asthma, bronchiolitis, and bronchopulmonary dysplasia.

Respiratory diseases are the most frequent chronic illnesses in babies and children. Although a vigorous innate immune system is critical for maintaining lung health, a balanced response is essential to minimize damaging inflammation. We investigated the functional and clinical impact of human genetic variants in the promoter of NFKBIA, which encodes IκBα, the major negative regulator of NF-κB. In this study, we quantified the functional impact of NFKBIA promoter polymorphisms (rs3138053, rs2233406, and rs2233409) on promoter-driven protein expression, allele-specific and total NFKBIA mRNA expression, IκBα protein expression, and TLR responsiveness; mapped innate immune regulatory networks active during respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia; and genotyped and analyzed independent cohorts of children with respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Genetic variants in the promoter of NFKBIA influenced NFKBIA gene expression, IκBα protein expression, and TLR-mediated inflammatory responses. Using a systems biology approach, we demonstrated that NFKBIA/IκBα is a central hub in transcriptional responses of prevalent childhood lung diseases, including respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Finally, by examining independent pediatric lung disease cohorts, we established that this immunologically relevant genetic variation in the promoter of NFKBIA is associated with differential susceptibility to severe bronchiolitis following infection with respiratory syncytial virus, airway hyperresponsiveness, and severe bronchopulmonary dysplasia. These data highlight the importance of negative innate immune regulators, such as NFKBIA, in pediatric lung disease and begin to unravel common aspects in the genetic predisposition to bronchopulmonary dysplasia, bronchiolitis, and childhood asthma.

Substitution of the bordetella pertussis lipid a phosphate groups with glucosamine is required for robust nf-κb activation and release of proinflammatory cytokines in cells expressing human but not murine toll-like receptor 4-MD-2-CD14

ABSTRACT Bordetella pertussis endotoxin is a key modulator of the host immune response, mainly due to the role of its lipid A moiety in Toll-like receptor 4 (TLR4)-mediated signaling. We have previously demonstrated that the lipid A phosphate groups of B. pertussis BP338 can be substituted with glucosamine in a BvgAS-regulated manner. Here we examined the effect of this lipid A modification on the biological activity of B. pertussis endotoxin. We compared purified endotoxin and heat-killed B. pertussis BP338 whole cells that have modified lipid A phosphate groups to an isogenic mutant lacking this modification with respect to their capacities to induce the release of inflammatory cytokines by human and murine macrophages and to participate in the TLR4-mediated activation of NF-κB in transfected HEK-293 cells. We found inactivated B. pertussis cells to be stronger inducers of proinflammatory cytokines in THP-1-derived macrophages when lipid A was modified. Most notably, lack of lipid A modification abolished the ability of purified B. pertussis endotoxin to induce the release of inflammatory cytokines by human THP-1-derived macrophages but led to only slightly reduced inflammatory cytokine levels when stimulating murine (RAW 264.7) macrophages. Accordingly, upon stimulation of HEK-293 cells with inactivated bacteria and purified endotoxin, lack of lipid A modification led to impaired NF-κB activation only when human, and not when murine, TLR4-MD-2-CD14 was expressed. We speculate that in B. pertussis, lipid A modification has evolved to benefit the bacteria during human infection by modulating immune defenses rather than to evade innate immune recognition.

Role of human TLR4 in respiratory syncytial virus-induced NF-κB activation, viral entry and replication

TLRs play a key role in innate immune defenses. It was previously reported that purified respiratory syncytial virus (RSV) fusion protein elicits an inflammatory response in hematopoietic cells, which required expression of TLR4 and its co-receptor CD14. However, a biological role of TLR4 in immunity to RSV, as initially proposed, has remained inconclusive and controversial. Here, we directly assess the role of human TLR4 and its co-receptors in NF-κB activation, viral entry and replication using intact virions rather than purified RSV components. We used HEK 293 reporter cells that are highly permissive for RSV and that either express or a lack a functional human TLR4/MD-2/CD14 complex. We demonstrate that RSV-mediated NF-κB activation, viral entry and replication are independent of the expression of a functional human TLR4/MD-2/CD14 complex and that, in turn, human TLR4 activation by LPS remains unaffected in RSV-infected cells. Thus, although isolated viral compounds such as purified RSV F protein may bind TLR4 and/or CD14, a direct interaction between intact RSV particles and the human TLR4 receptor complex does not seem to play a biological role in RSV pathogenesis.

IL‐4Rα on CD4+ T cells plays a pathogenic role in respiratory syncytial virus reinfection in mice infected initially as neonates

RSV is the major cause of severe bronchiolitis in infants, and severe bronchiolitis as a result of RSV is associated with subsequent asthma development. A biased Th2 immune response is thought to be responsible for neonatal RSV pathogenesis; however, molecular mechanisms remain elusive. Our data demonstrate, for the first time, that IL‐4Rα is up‐regulated in vitro on human CD4+ T cells from cord blood following RSV stimulation and in vivo on mouse pulmonary CD4+ T cells upon reinfection of mice, initially infected as neonates. Th cell‐specific deletion of Il4ra attenuated Th2 responses and abolished the immunopathophysiology upon reinfection, including airway hyper‐reactivity, eosinophilia, and mucus hyperproduction in mice infected initially as neonates. These findings support a pathogenic role for IL‐4Rα on Th cells following RSV reinfection of mice initially infected as neonates; more importantly, our data from human cells suggest that the same mechanism occurs in humans.

Minor Modifications to the Phosphate Groups and the C3′ Acyl Chain Length of Lipid A in Two Bordetella pertussis Strains, BP338 and 18-323, Independently Affect Toll-like Receptor 4 Protein Activation

Background: Lipid A activation of TLR4 shapes immunity to Gram-negative bacteria. Results: In Bordetella pertussis lipid A, the genetic basis for longer C3′ acyl chains and glucosamine modification of the phosphate groups was identified; each variation independently increased TLR4 activation. Conclusion: Minor changes in the penta-acylated B. pertussis lipid A affect TLR4 activation. Significance: This aids our understanding how lipid A species interact with TLR4. Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4′-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3′ acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399–BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3′ acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.