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Immune Response

Article Reference Dectin-1 is expressed in human lung and mediates the proinflammatory immune response to nontypeable Haemophilus influenzae.
The C-type lectin receptor Dectin-1 is expressed mainly on myeloid cells mediating the immune response targeting respiratory pathogens such as Aspergillus fumigatus and Mycobacterium tuberculosis. The pulmonary epithelium serves as an important interface for interactions between these pathogens and the respiratory tract. Therefore, we analyzed the expression pattern of Dectin-1 in the human lung. Immunohistochemically stained human lung sections from 17 out of 19 individuals were positive for Dectin-1, which was expressed mainly apically on bronchial and alveolar epithelium. Our results showed no correlation with chronic obstructive pulmonary disease (COPD) or the smoking habits of the patients. Nontypeable Haemophilus influenzae (NTHI), an important bacterial pathogen of the respiratory tract with significant importance in COPD, has also been proposed to be recognized by Dectin-1, suggesting a possible impact on the NTHI-dependent immune response in human airways. Therefore, the involvement of Dectin-1 in NTHI-triggered cytokine responses was investigated in primary normal human bronchial epithelial (NHBE) cells and in the A549 cell line stably transfected with Dectin-1. The presence of Dectin-1 significantly increased cytokine release in response to NTHI in NHBE and A549 cells. In addition, phosphorylation of the Dectin-1 hem-immunoreceptor tyrosine-based activation motif (hemITAM) was essential for the Dectin-1-triggered response to NTHI in A549 cells. In conclusion, in human airways, epithelium-expressed Dectin-1 may play a significant role in generating an NTHI-mediated, proinflammatory immune response.
Article Reference Co-colonization by Haemophilus influenzae with Streptococcus pneumoniae enhances pneumococcal-specific antibody response in young children.
Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mcat) are common bacterial pathogens of respiratory infections and common commensal microbes in the human nasopharynx (NP). The effect of interactions among theses bacteria during co-colonization of the NP on the host immune response has not been evaluated. The objective of this study was to assess the impact of co-colonization by Hi or Mcat on the systemic antibody response to vaccine protein candidate antigens of Spn and similarly the impact of co-colonization by Spn and Mcat on antibody responses to Hi vaccine protein candidate antigens.
Article Reference Children with chronic suppurative lung disease have a reduced capacity to synthesize interferon-gamma in vitro in response to non-typeable Haemophilus influenzae.
Chronic suppurative lung disease (CSLD) is characterized by the presence of a chronic wet or productive cough and recurrent lower respiratory infections. The aim of this study was to identify features of innate, cell-mediated and humoral immunity that may increase susceptibility to respiratory infections in children with CSLD. Because non-typeable Haemophilus influenzae (NTHi) is commonly isolated from the airways in CSLD, we examined immune responses to this organism in 80 age-stratified children with CSLD and compared their responses with 51 healthy control children. Cytokines involved in the generation and control of inflammation (IFN-γ, IL-13, IL-5, IL-10 at 72 hours and TNFα, IL-6, IL-10 at 24 hours) were measured in peripheral blood mononuclear cells challenged in vitro with live NTHi. We also measured circulating IgG subclass antibodies (IgG1 and IgG4) to two H. influenzae outer membrane proteins, P4 and P6. The most notable finding was that PBMC from children with CSLD produced significantly less IFN-γ in response to NTHi than healthy control children whereas mitogen-induced IFN-γ production was similar in both groups. Overall there were minor differences in innate and humoral immune responses between CSLD and control children. This study demonstrates that children with chronic suppurative lung disease have an altered systemic cell-mediated immune response to NTHi in vitro. This deficient IFN-γ response may contribute to increased susceptibility to NTHi infections and the pathogenesis of CSLD in children.
Article Reference Co-colonization by Haemophilus influenzae with Streptococcus pneumoniae enhances pneumococcal-specific antibody response in young children.
Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mcat) are common bacterial pathogens of respiratory infections and common commensal microbes in the human nasopharynx (NP). The effect of interactions among theses bacteria during co-colonization of the NP on the host immune response has not been evaluated. The objective of this study was to assess the impact of co-colonization by Hi or Mcat on the systemic antibody response to vaccine protein candidate antigens of Spn and similarly the impact of co-colonization by Spn and Mcat on antibody responses to Hi vaccine protein candidate antigens.
Article Reference Impacts of peroxisome proliferator-activated receptor-γ activation on cigarette smoke-induced exacerbated response to bacteria.
Chronic obstructive pulmonary disease (COPD) is characterised by a state of chronic pulmonary inflammation punctuated by microbial exacerbations. Despite advances in treatment options, COPD remains difficult to manage. In this study, we investigated the potential of peroxisome proliferator-activated receptor (PPAR)γ activation as a new therapy against cigarette smoke-induced inflammation and its associated bacterial exacerbation. C57BL/6 mice were exposed to room air or cigarette smoke for either 4 days or 4 weeks and treated either prophylactically or therapeutically with rosiglitazone. The impact of rosiglitazone on cigarette smoke-induced exacerbated response to the bacterial pathogen nontypeable Haemophilus influenzae (NTHi) was studied using the therapeutic treatment protocol. We found that rosiglitazone was able to reduce cigarette smoke-induced neutrophilia both when administered prophylactically or therapeutically. Therapeutic intervention with rosiglitazone was also effective in preventing cigarette smoke-induced neutrophilia exacerbation following NTHi infection. Moreover, the anti-inflammatory effects of rosiglitazone did not lead to an increase in the pulmonary bacterial burden, unlike dexamethasone. Altogether, our data suggest that pharmacological activation of PPARγ may be an effective therapeutic approach to improve COPD management, as it is able to reduce cigarette smoke-induced inflammation and decrease the magnitude of bacterial exacerbations, without compromising the ability of the immune system to control the infection.
Article Reference Impaired innate immune alveolar macrophage response and the predilection for COPD exacerbations.
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.
Article Reference Haemophilus influenzae and the lung (Haemophilus and the lung).
Haemophilus influenzae is present as a commensal organism in the nasopharynx of most healthy adults from where it can spread to cause both systemic and respiratory tract infection. This bacterium is divided into typeable forms (such as type b) or nontypeable forms based on the presence or absence of a tough polysaccharide capsule. Respiratory disease is predominantly caused by the nontypeable forms (NTHi). Haemophilus influenzae has evolved a number of strategies to evade the host defense including the ability to invade into local tissue. Pathogenic properties of this bacterium as well as defects in host defense may result in the spread of this bacterium from the upper airway to the bronchi of the lung. This can result in airway inflammation and colonization particularly in chronic obstructive pulmonary disease. Treatment of respiratory tract infection with Haemophilus influenzae is often only partially successful with ongoing infection and inflammation. Improvement in patient outcome will be dependent on a better understanding of the pathogenesis and host immune response to this bacterium.
Article Reference Haemophilus influenzae reside in tonsills and use IgD binding as an evasion strategy.
Haemophilus influenzae (Hi) causes respiratory tract infections and is also considered as a commensal, particularly in pre-school children. Tonsils from patients (n=617) undergoing tonsillectomy due to chronical infection or hypertrophy were examined. We found that 51 % of tonsils were positive for Hi, and in 95 % of cases analysed in detail (n=39) Hi resided intracellularly in the core tonsillar tissue. Patients harboured several intracellular unique strains and the majority were non-typeable Hi (NTHi). Interestingly, the isolated NTHi bound soluble immunoglobulin (Ig) D at the constant heavy chain domain 1 as revealed by recombinant IgD/IgG chimeras. NTHi also interacted with B lymphocytes via the IgD B cell receptor resulting in internalization of bacteria, T-cell independent activation via Toll like receptor 9, and differentiation into non-NTHi specific IgM producing cells. Taken together, IgD-binding NTHi leads to an unspecific immune response and may support the bacteria to circumvent the host defense.
Article Reference Effects of cigarette smoke on Toll-like receptor (TLR) activation of chronic obstructive pulmonary disease (COPD) macrophages.
Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal innate immune response. We have investigated the changes in the innate immune response of COPD alveolar macrophages exposed to both cigarette smoke and Toll-like receptor (TLR) stimulation. COPD and control alveolar macrophages were exposed to cigarette smoke extract (CSE) followed by TLR-2, -4 and -5 ligands [Pam3CSK4, lipopolysaccharide (LPS) and phase I flagellin (FliC), respectively] or non-typeable Haemophilus influenzae (NTHi). CSE exposure suppressed TLR-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and regulated on activation, normal T cell expressed and secreted (RANTES) production in both COPD and control alveolar macrophages, but had no effect on interleukin 8 (CXCL8) production. Similarly, CSE suppressed NTHi-induced TNF-α but not NTHi-induced CXCL8 production in COPD alveolar macrophages. Gene expression analysis showed that CSE suppressed LPS-induced TNF-α transcription but not CXCL8 transcription in COPD alveolar macrophages. The dampening effect of CSE on LPS-induced cytokine production was associated with a reduction in p38, extracellular signal regulated kinase (ERK) and p65 activation. In conclusion, CSE caused a reduced innate immune response in COPD alveolar macrophages, with the exception of persistent CXCL8 production. This could be a mechanism by which alveolar macrophages promote neutrophil chemotaxis under conditions of oxidative stress and bacterial exposure.
Article Reference Kinetic analysis and evaluation of the mechanisms involved in the resolution of experimental nontypeable Haemophilus influenzae-induced otitis media after transcutaneous immunization.
Transcutaneous immunization (TCI) is a simple and needle-free method with which to induce protective immune responses. Using a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM), we examined the efficacy afforded by TCI with a novel chimeric immunogen called 'chimV4' which targets two critical adhesins expressed by NTHI, outer membrane protein P5 and the majority subunit of NTHI Type IV pilus, PilA. Experimental OM was first established in cohorts of animals, and then TCI performed via a therapeutic immunization regime by rubbing vaccine formulations on hydrated pinnae. The kinetics of resolution of established experimental disease was evaluated by clinically-relevant assessments of OM, bacterial culture of planktonic and adherent NTHI within the middle ear and gross examination of the relative amount of NTHI mucosal biofilms within the middle ear space. Within seven days after primary TCI, a significant reduction in the signs of OM, significantly fewer NTHI adherent to the middle ear mucosa and significant resolution of mucosal biofilms was detected in animals that received chimV4+ the adjuvant LT(R192G-L211A), compared to animals administered LT(R192G-L211A) alone or saline by TCI (p<0.05) with eradication of NTHI within an additional seven days. The mechanism for rapid disease resolution involved efflux of activated dermal dendritic cells from the pinnae after TCI, secretion of factors chemotactic for CD4(+) T-cells, induction of polyfunctional IFNγ- and IL-17-producing CD4(+) T-cells and secretion of host defense peptide within the middle ear. These data support TCI as a therapeutic intervention against experimental NTHI-induced OM and begin to elucidate the host response to immunization by this noninvasive regimen.
Article Reference The inflammasome adaptor ASC contributes to multiple innate immune processes in the resolution of otitis media.
This study was designed to understand the contribution of the inflammasome and IL-1β activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1β and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc(-/-)-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1β maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc(-/-) mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.
Article Reference Zinc oxide nanoparticles impair bacterial clearance by macrophages.
The extensive development of nanoparticles (NPs) and their widespread employment in daily life have led to an increase in environmental concentrations of substances that may pose a biohazard to humans. The aim of this work was to examine the effects of zinc oxide nanoparticles (ZnO-NPs) on the host's pulmonary immune system response to nontypeable Haemophilus influenzae (NTHi) infection.
Article Reference Lower airway colonization and inflammatory response in COPD: a focus on Haemophilus influenzae.
Bacterial infection of the lower respiratory tract in chronic obstructive pulmonary disease (COPD) patients is common both in stable patients and during acute exacerbations. The most frequent bacteria detected in COPD patients is Haemophilus influenzae, and it appears this organism is uniquely adapted to exploit immune deficiencies associated with COPD and to establish persistent infection in the lower respiratory tract. The presence of bacteria in the lower respiratory tract in stable COPD is termed colonization; however, there is increasing evidence that this is not an innocuous phenomenon but is associated with airway inflammation, increased symptoms, and increased risk for exacerbations. In this review, we discuss host immunity that offers protection against H. influenzae and how disturbance of these mechanisms, combined with pathogen mechanisms of immune evasion, promote persistence of H. influenzae in the lower airways in COPD. In addition, we examine the role of H. influenzae in COPD exacerbations, as well as interactions between H. influenzae and respiratory virus infections, and review the role of treatments and their effect on COPD outcomes. This review focuses predominantly on data derived from human studies but will refer to animal studies where they contribute to understanding the disease in humans.
Article Reference Transcriptome signature in young children with acute otitis media due to non-typeable Haemophilus influenzae.
Non-typeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in young children. In our recent paper in Microbes and Infection we described the transcriptome signature elicited from PBMCs at onset of AOM caused by Streptococcus pneumoniae. In the current study we found very different results with NTHi AOM infections; 5.1% of 29&emsp14;187 genes were differentially regulated by more than 2-fold at the onset of AOM compared with the pre-infection healthy state in the same children. Among the 1487 transcripts, 100 genes associated with the immune defense response were specifically analyzed. About half of the differentially regulated genes associated with antibacterial activity and the cell-mediated immune response were activated and half were suppressed. The important signatures for NTHi in children suggested that the balance of the immune response was toward suppression. Moreover, 90% of the genes associated with a pro-inflammatory cytokine response were down-regulated. The genes associated with the classic complement pathway were down-regulated, although the alternative complement pathway genes were up-regulated. These results provide the first human transcriptome data identifying gene expression in the immune response to be predominantly down-regulated at the onset of AOM due to NTHi.
Article Reference Cellular immune response in young children accounts for recurrent acute otitis media.
Acute otitis media (AOM) is a common disease in young children. Streptococcus pneumoniae (Spn) and Haemophilus influenzae (NTHi) are the two most common pathogens that cause AOM. Over the past 5 years, our group has been studying the immunologic profile of children that experience repeated AOM infections despite tympanocentesis drainage of middle ear fluid and individualized antibiotic treatment; we call these children stringently-defined otitis prone(sOP). Although protection against AOM is primarily mediated by ototpathogen-specific antibody, our recent studies suggest that suboptimal memory B and T cell responses and an immaturity in antigen-presenting cells may play a significant role in the propensity to recurrent AOM infections. This review focuses on the studies performed to define immunologic dysfunction in sOP children.
Article Reference COPD and asthma-associated Proteobacteria, but not commensal Prevotella spp.,promote TLR2-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 COPD and asthma patients (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 Haemophilus influenzae induced severe 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, Prevotella nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram-negative bacteria Prevotella 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. This article is protected by copyright. All rights reserved.
Article Reference The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis.
Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapA-deficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.
Article Reference Intranasal immunization with nontypeable Haemophilus influenzae outer membrane vesicles induces cross-protective immunity in mice.
Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.
Article Reference Moraxella catarrhalis activates murine macrophages through multiple toll like receptors and has reduced clearance in lungs from TLR4 mutant mice.
Moraxella catarrhalis is a gram negative bacterium and a leading causative agent of otitis media (OM) in children. Several recent reports have provided strong evidence for an association between toll like receptors and OM. It has been found that both Streptococcus pneumoniae and nontypeable Haemophilus influenzae activate host protective immune responses through toll like receptors (TLRs), however, the precise mechanism by which Moraxella catarrhalis initiates the host immune response is currently unknown. In this report, using murine macrophages generated from a series of knock-out mice, we have demonstrated that M. catarrhalis lipooligosaccharide (LOS) and either heat killed or live bacteria are recognized by one or more TLRs. LOS activates the host immune response through a membrane bound CD14-TLR4 complex, while both heat killed and live M.cat require recognition by multiple toll like receptors such as TLR2, TLR4 and TLR9 without the requirement of CD14. We have also shown that M.cat stimuli are capable of triggering the host innate immune response by both MyD88- and TRIF- dependent signaling pathways. We further showed that M.cat induced activation of mitogen activated protein kinase (MAPK) is essential in order to achieve optimal secretion of pro-inflammatory cytokine TNF-α. We finally showed that TLR4 mutant C3H/HeJ mice produce significantly lower levels of pro-inflammatory cytokines TNF-α and IL-6 in vivo, An increased bacterial loads at 12 and 24 hours (P<0.001) in their lungs upon challenge with live M.cat in an aerosol chamber compared to wild-type (WT) control mice. These data suggest that TLRs are crucial for an effective innate immune response induced by M.cat. The results of these studies contribute to an increased understanding of molecular mechanism and possible novel treatment strategies for diseases caused by M.cat by specifically targeting TLRs and their signaling pathways.
Article Reference ERK2-dependent activation of c-Jun is required for nontypeable Haemophilus influenzae-induced CXCL2 upregulation in inner ear fibrocytes.
The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.