Lymphocyte predominant Hodgkin lymphoma, antigen-driven after all?

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) was suggested as an entity separate from other types of Hodgkin lymphoma 40 years ago and recognized in the WHO classification in 2001. Based on its relatively benign course with late distant relapses, relation with lymph node hyperplasia with progressively transformed germinal centers, presence of clonal immunoglobulin gene rearrangements with somatic hypermutations and ongoing mutations, and relation with a number of inherited defects affecting the immune system, it has been suspected that NLPHL might be antigen-driven. Recent evidence has shown that cases of IgD-positive NLPHL are associated with infection by Moraxella catarrhalis, a common bacterium in the upper respiratory tract and in lymph nodes. This review summarizes the evidence for NLPHL as a B-cell lymphoma involving follicular T-lymphocytes normally found in germinal centers, its molecular features and relation to inherited immune defects, and its relation and differential diagnosis from similar entities. Finally, it discusses the evidence that in many cases a watch and wait policy might be a viable initial management strategy. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CEACAM1 inhibits Toll-like receptor 2-triggered antibacterial responses of human pulmonary epithelial cells.

Although Moraxella catarrhalis and Neisseria meningitidis are important human pathogens, they often colonize the human respiratory tract without causing overt clinical symptoms. Both pathogens express structurally unrelated proteins that share the ability to stimulate the adhesion molecule CEACAM1 expressed on human cells. Here we demonstrate that the interaction of CEACAM1 with ubiquitous surface protein A1 expressed on M. catarrhalis or with opacity-associated proteins on N. meningitidis resulted in reduced Toll-like receptor 2-initiated transcription factor NF-kappaB-dependent inflammatory responses of primary pulmonary epithelial cells. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results identify a CEACAM1-dependent immune-evasion strategy.

Short Leucine-Rich Proteoglycans Modulate Complement Activity and Increase Killing of the Respiratory Pathogen Moraxella catarrhalis.

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs.

PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis.

Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae, but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

Moraxella catarrhalis Evades Host Innate Immunity via Targeting Cartilage Oligomeric Matrix Protein.

Moraxella catarrhalis is a respiratory tract pathogen commonly causing otitis media in children and acute exacerbations in patients suffering from chronic obstructive pulmonary disease. Cartilage oligomeric matrix protein (COMP) functions as a structural component in cartilage, as well as a regulator of complement activity. Importantly, COMP is detected in resident macrophages and monocytes, alveolar fluid, and the endothelium of blood vessels in lung tissue. We show that the majority of clinical isolates of M. catarrhalis (n = 49), but not other tested bacterial pathogens, bind large amounts of COMP. COMP interacts directly with the ubiquitous surface protein A2 of M. catarrhalis. Binding of COMP correlates with survival of M. catarrhalis in human serum by inhibiting bactericidal activity of the complement membrane attack complex. Moreover, COMP inhibits phagocytic killing of M. catarrhalis by human neutrophils. We further observed that COMP reduces bacterial adhesion and uptake by human lung epithelial cells, thus protecting M. catarrhalis from intracellular killing by epithelial cells. Taken together, our findings uncover a novel mechanism that M. catarrhalis uses to evade host innate immunity.

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.

Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils.

Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) play both pro- and anti-inflammatory activities and are produced during infection and inflammation. Moraxella catarrhalis is one of the leading infectious agents responsible for inflammatory exacerbation in chronic obstructive pulmonary disease (COPD). Since the airway inflammation in COPD is connected with activation of both epithelial cells and accumulated neutrophils, in this study we determined the in vitro effects of neuropeptides on the inflammatory potential of these cells in response to M. catarrhalis outer membrane vesicle (OMV) stimulant. The various OMV-mediated proinflammatory effects were demonstrated. Next, using hBD-2-pGL4[luc2] plasmid with luciferase reporter gene, SP and CGRP were shown to inhibit the IL-1ß-dependent expression of potent neutrophil chemoattractant, hBD-2 defensin, in transfected A549 epithelial cells (type II alveolar cells) upon OMV stimulation. Both neuropeptides exerted antiapoptotic activity through rescuing a significant fraction of A549 cells from OMV-induced cell death and apoptosis. Finally, CGRP caused an impairment of specific but not azurophilic granule exocytosis from neutrophils as shown by evaluation of gelatinase-associated lipocalin (NGAL) or CD66b expression and elastase release, respectively. Concluding, these findings suggest that SP and CGRP mediate the dampening of proinflammatory action triggered by M. catarrhalis OMVs towards cells engaged in lung inflammation in vitro.

Common Cell Shape Evolution of Two Nasopharyngeal Pathogens.

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.

Virulence determinants of Moraxella catarrhalis: distribution and considerations for vaccine development.

Moraxella catarrhalis is a human-restricted opportunistic bacterial pathogen of the respiratory mucosa. It frequently colonizes the nasopharynx asymptomatically, but is also an important causative agent of otitis media (OM) in children, and plays a significant role in acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults. As the current treatment options for M. catarrhalis infection in OM and exacerbations of COPD are often ineffective, the development of an efficacious vaccine is warranted. However, no vaccine candidates for M. catarrhalis have progressed to clinical trials, and information regarding the distribution of M. catarrhalis virulence factors and vaccine candidates is inconsistent in the literature. It is largely unknown if virulence is associated with particular strains or subpopulations of M. catarrhalis, or if differences in clinical manifestation can be attributed to the heterogeneous expression of specific M. catarrhalis virulence factors in the circulating population. Further investigation of the distribution of M. catarrhalis virulence factors in the context of carriage and disease is required so that vaccine development may be targeted at relevant antigens that are conserved among disease-causing strains. The challenge of determining which of the proposed M. catarrhalis virulence factors are relevant to human disease is amplified by the lack of a standardized M. catarrhalis typing system to facilitate direct comparisons of worldwide isolates. Here we summarize and evaluate proposed relationships between M. catarrhalis subpopulations and specific virulence factors in the context of colonization and disease, as well as the current methods used to infer these associations.

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

BACKGROUND: During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. RESULTS: Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. CONCLUSIONS: Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Immunoinformatics Identifies a Lactoferrin Binding Protein A Peptide as a Promising Vaccine With a Global Protective Prospective Against Moraxella catarrhalis.

BACKGROUND: Moraxella catarrhalis is an established pathogen that is causing substantial infections to both children and adults. However, so far there is no effective vaccine to halt the spread of these infections. METHODS: Immunoinformatics tools were used to predict M. catarrhalis epitopes that could offer immunoprotection among major proportions of human populations worldwide. Mice were immunized with the best 3 peptides and then challenged with M. catarrhalis in the pulmonary clearance model. Finally, antibodies against these epitopes were detected in humans. RESULTS: Immunoinformatics analyses identified 44 epitopes that are predicted to be good major histocompatibility complex class II binders and at the same time show high population coverage worldwide. After intraperitoneal immunization of mice with the best 3 peptides, peptide A, derived from lactoferrin-binding protein A, showed superior activity in immunogenicity and in clearing M. catarrhalis from mouse lungs. Higher clearance was obtained by combining intraperitoneal and intranasal immunization. In the serum samples from children with otitis media infected with M. catarrhalis, antibody levels against peptide A were significantly lower than in samples from children without otitis media. CONCLUSIONS: Peptide A is the first promising peptide-based vaccine against M. catarrhalis Immunoinformatics predicts that it should have a global protection around the world.

Moraxella catarrhalis Binds Plasminogen To Evade Host Innate Immunity.

Several bacterial species recruit the complement regulators C4b-binding protein, factor H, and vitronectin, resulting in resistance against the bactericidal activity of human serum. It was recently demonstrated that bacteria also bind plasminogen, which is converted to plasmin that degrades C3b and C5. In this study, we found that a series of clinical isolates (n = 58) of the respiratory pathogen Moraxella catarrhalis, which is commonly isolated from preschool children and adults with chronic obstructive pulmonary disease (COPD), significantly binds human plasminogen. Ubiquitous surface protein A2 (UspA2) and hybrid UspA2 (UspA2H) were identified as the plasminogen-binding factors in the outer membrane proteome of Moraxella. Furthermore, expression of a series of truncated recombinant UspA2 and UspA2H proteins followed by a detailed analysis of protein-protein interactions suggested that the N-terminal head domains bound to the kringle domains of plasminogen. The binding affinity constant (KD) values of full-length UspA2(30-539) (amino acids 30 to 539 of UspA2) and full-length UspA2H(50-720) for immobilized plasminogen were 4.8 × 10(-8) M and 3.13 × 10(-8) M, respectively, as measured by biolayer interferometry. Plasminogen bound to intact M. catarrhalis or to recombinant UspA2/UspA2H was readily accessible for a urokinase plasminogen activator that converted the zymogen into active plasmin, as verified by the specific substrate S-2251 and a degradation assay with fibrinogen. Importantly, plasmin bound at the bacterial surface also degraded C3b and C5, which consequently may contribute to reduced bacterial killing. Our findings suggest that binding of plasminogen to M. catarrhalis may lead to increased virulence and, hence, more efficient colonization of the host.

Chronic obstructive pulmonary disease and asthma-associated Proteobacteria, but not commensal Prevotella spp., promote Toll-like receptor 2-independent lung inflammation and pathology.

Recent studies of healthy human airways have revealed colonization by a distinct commensal bacterial microbiota containing Gram-negative Prevotella spp. However, the immunological properties of these bacteria in the respiratory system remain unknown. Here we compare the innate respiratory immune response to three Gram-negative commensal Prevotella strains (Prevotella melaninogenica, Prevotella nanceiensis and Prevotella salivae) and three Gram-negative pathogenic Proteobacteria known to colonize lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma (Haemophilus influenzae B, non-typeable Haemophilus influenzae and Moraxella catarrhalis). The commensal Prevotella spp. and pathogenic Proteobacteria were found to exhibit intrinsic differences in innate inflammatory capacities on murine lung cells in vitro. In vivo in mice, non-typeable H. influenzae induced severe Toll-like receptor 2 (TLR2)-independent COPD-like inflammation characterized by predominant airway neutrophilia, expression of a neutrophilic cytokine/chemokine profile in lung tissue, and lung immunopathology. In comparison, P. nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram-negative bacteria P. nanceiensis was completely TLR2-dependent. These findings demonstrate weak inflammatory properties of Gram-negative airway commensal Prevotella spp. that may make colonization by these bacteria tolerable by the respiratory immune system.

Moraxella catarrhalis induces an immune response in the murine lung that is independent of human CEACAM5 expression and long-term smoke exposure.

In patients with chronic obstructive pulmonary disease (COPD), Moraxella catarrhalis infection of the lower airways is associated with chronic colonization and inflammation during stable disease and acute exacerbations. Chronic smoke exposure induces chronic inflammation and impairs mucociliary clearance, thus contributing to bacterial colonization of the lower airways in COPD patients. The human-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, expressed in human airways, has been shown to contribute to epithelial colonization of CEACAM-binding pathogens. To investigate the impact of CEACAM5 expression on pulmonary M. catarrhalis colonization, we infected mice transgenic for human CEACAM5 (hCEACAM5) and wild type mice intratracheally with M. catarrhalis with or without preceding smoke exposure and analyzed bacterial colonization and local and systemic inflammation. Our results show that airway infection with M. catarrhalis accelerated acute local but not systemic inflammation, albeit independent of hCEACAM5 expression. Long-term smoke exposure alone or prior to M. catarrhalis infection did not contribute to increased local or systemic inflammation. No difference was found in pulmonary clearance of M. catarrhalis in hCEACAM5-transgenic mice compared with wild-type mice. Smoke exposure neither altered time nor extent of persistence of M. catarrhalis in the lungs of both genotypes. In conclusion, M. catarrhalis induced a local acute immune response in murine airways. Neither hCEACAM5 expression nor chronic smoke exposure nor a combination of both was sufficient as prerequisites for the establishment of chronic M. catarrhalis colonization. Our results demonstrate the difficulties in mirroring conditions of chronic airways colonization of M. catarrhalis in a murine model.

Deciphering the genetic basis of Moraxella catarrhalis complement resistance: a critical role for the disulphide bond formation system.

The complement system is an important innate defence mechanism, and the ability to resist complement-mediated killing is considered a key virulence trait of the respiratory tract pathogen M. catarrhalis. We studied the molecular basis of complement resistance by transcriptional profiling and Tn-seq, a genome-wide negative-selection screenings technology. Exposure of M. catarrhalis to human serum resulted in increased expression of 84 genes and reduced expression of 134 genes, among which genes encoding ABC transporter systems and surface proteins UspA1 and McaP. By subjecting a ∼ 15 800 transposon mutant library to serum, mutants of 53 genes were negatively selected, including the key complement-resistance factor uspA2H. Validation with directed mutants confirmed Tn-seq phenotypes of uspA2H and 11 newly identified genes, with mutants of MCR_0424, olpA, MCR_1483, and dsbB most severely attenuated. Detailed analysis showed that both components of the disulphide bond formation (DSB) system, DsbB and DsbA, were required for complement-resistance in multiple isolates, and fulfil a critical role in evasion of IgG-dependent classical pathway-mediated killing. Lipooligosaccharide (LOS) structure and membrane stability were severely affected in ΔdsbA strains, suggesting a pivotal role for the DSB system in LOS structure safeguarding and membrane stability maintenance.

The role and regulation of Moraxella catarrhalis-induced human beta-defensin 3 expression in human pulmonary epithelial cells.

BACKGROUND: Bacterial colonisation with Moraxella catarrhalis may partly sustain chronic inflammation in the lower airways of patients with chronic obstructive pulmonary disease (COPD). In addition, this bacterium causes infectious exacerbations of COPD, which often necessitate treatment with antibiotics. Antimicrobial peptides are the body's own antibiotic substances with bactericidal and bacteriostatic, as well as immunomodulatory function. In particular, human beta-defensin 3 (hBD-3) exerts an antimicrobial effect against an extraordinarily broad spectrum of pathogens. We therefore investigated the role of hBD-3 in infections of pulmonary epithelial cells with M. catarrhalis. METHODS: The antimicrobial activity of hBD-3 vs. M. catarrhalis was evaluated in an antimicrobial susceptibility assay. We analyzed hBD-3 secretion of M. catarrhalis-infected pulmonary epithelial cells using ELISA. The role of M. catarrhalis-specific virulence factors, toll-like receptors (TLR) 2 and 4, MAPK pathways, and transcription factors AP-1 and NF-κB in the induction and regulation of hBD-3 expression were explored with specific inhibitors, small interference RNA, Western Blot, and chromatin immunoprecipitation (ChIP) assays. RESULTS: HBD-3 exhibited a strong bactericidal effect against M. catarrhalis. M. catarrhalis induced hBD-3 expression in pulmonary epithelial cells, which was dependent on M. catarrhalis membranous lipoolygosaccharide (LOS), while the surface proteins UspA1 and UspA2 were not involved. Gene silencing of TLR2, but not TLR4, led to a reduced hBD-3 secretion after stimulation with M. catarrhalis or M. catarrhalis LOS. Inhibition of MAPKs ERK1/2 and JNK, but not p38, reduced hBD-3 secretion. HBD-3 expression was mediated through the recruitment of AP-1 to the hBD-3 gene promoter and was independent of NF-κB. CONCLUSION: The immune response of pulmonary epithelial cells towards M. catarrhalis involves secretion of hBD-3, which has a bactericidal effect against this pathogen. Binding of M. catarrhalis virulence factor LOS to TLR2 causes an ERK1/2- and JNK-dependent induction of AP-1-related transcription of the hBD-3 gene, resulting in the production and secretion of hBD-3.

Detection of antibody responses against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis proteins in children with community-acquired pneumonia: effects of combining pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness.

We evaluated the effects of combining different numbers of pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness on the detection of IgG responses against eight Streptococcus pneumoniae proteins, three Haemophilus influenzae proteins, and five Moraxella catarrhalis proteins in 690 children aged <5 years with pneumonia. Serological tests were performed on acute and convalescent serum samples with a multiplexed bead-based immunoassay. The median sampling interval was 19 days, the median age was 26.7 months, and the median duration of illness was 5 days. The rate of antibody responses was 15.4 % for at least one pneumococcal antigen, 5.8 % for H. influenzae, and 2.3 % for M. catarrhalis. The rate of antibody responses against each pneumococcal antigen varied from 3.5 to 7.1 %. By multivariate analysis, pre-existing antibody levels showed a negative association with the detection of antibody responses against pneumococcal and H. influenzae antigens; the sampling interval was positively associated with the detection of antibody responses against pneumococcal and H. influenzae antigens. A sampling interval of 3 weeks was the optimal cut-off for the detection of antibody responses against pneumococcal and H. influenzae proteins. Duration of illness was negatively associated with antibody responses against PspA. Age did not influence antibody responses against the investigated antigens. In conclusion, serological assays using combinations of different pneumococcal proteins detect a higher rate of antibody responses against S. pneumoniae compared to assays using a single pneumococcal protein. Pre-existing antibody levels and sampling interval influence the detection of antibody responses against pneumococcal and H. influenzae proteins. These factors should be considered when determining pneumonia etiology by serological methods in children.

Substrate binding protein SBP2 of a putative ABC transporter as a novel vaccine antigen of Moraxella catarrhalis.

Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-µg and 50-µg doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis.

Impaired innate immune alveolar macrophage response and the predilection for COPD exacerbations.

BACKGROUND: Alveolar macrophages (AM) in COPD have fundamentally impaired responsiveness to Toll-like receptor 2 (TLR2) and TLR4 ligands of non-typeable Haemophilus influenzae (NTHI). However, the contribution of innate immune dysfunction to exacerbations of COPD is unexplored. We hypothesised that impaired innate AM responses in COPD extend beyond NTHI to other pathogens and are linked with COPD exacerbations and severity. METHODS: AMs, obtained by bronchoalveolar lavage from 88 volunteers with stable-to-moderate COPD, were incubated with respiratory pathogens (NTHI, Moraxella catarrhalis (MC), Streptococcus pneumoniae (SP) and TLR ligands lipopolysaccharide, Pam3Cys) and elicited IL-8 and TNF-α were measured by microsphere flow cytometry. NF-κB nuclear translocation was measured by colorimetric assay. AM TLR2 and TLR4 expression was determined by immunolabeling and quantitation of mean fluorescent indices. Participants were monitored prospectively for occurrence of COPD exacerbations for 1 year following bronchoscopy. Non-parametric analyses were used to compare exacerbation-prone and non-exacerbation-prone individuals. RESULTS: 29 subjects had at least one exacerbation in the follow-up period (exacerbation-prone) and 59 remained exacerbation-free (non-exacerbation-prone). AMs of exacerbation-prone COPD donors were more refractory to cytokine induction by NTHI (p=0.02), MC (p=0.045) and SP (p=0.046), and to TLR2 (p=0.07) and TLR4 (p=0.028) ligands, and had diminished NF-κB nuclear activation, compared with non-exacerbation-prone counterparts. AMs of exacerbation-prone subjects were more refractory to TLR2 upregulation by MC and SP (p=0.04 each). CONCLUSIONS: Our results support a paradigm of impaired innate responses of COPD AMs to respiratory pathogens, mediated by impaired TLR responses, underlying a propensity for exacerbations in COPD.