Persistence Of Nontypeable Haemophilus Influenzae Research Articles

Pathogenesis of nontypeable Haemophilus influenzae infections in chronic suppurative lung disease.

The respiratory tract antimicrobial defense system is a multilayered defense mechanism that relies upon mucociliary clearance and components of both the innate and adaptive immune systems to protect the lungs from inhaled or aspirated microorganisms. One of these potential pathogens, nontypeable Haemophilus influenzae (NTHi), adopts several, multifaceted redundant strategies to successfully colonize the lower airways and establish a persistent infection. NTHi can impair mucociliary clearance, express multiple multifunctional adhesins for various cell types within the respiratory tract and evade host defenses by surviving within and between cells, forming biofilms, increasing antigenic drift, secreting proteases and antioxidants, and by host-pathogen cross-talk, impair macrophage and neutrophil function. NTHi is recognized as an important pathogen in several chronic lower respiratory disorders, such as protracted bacterial bronchitis, bronchiectasis, cystic fibrosis, and primary ciliary dyskinesia. The persistence of NTHi in human airways, including its capacity to form biofilms, results in chronic infection and inflammation, which can ultimately injure airway wall structures. The complex nature of the molecular pathogenetic mechanisms employed by NTHi is incompletely understood but improved understanding of its pathobiology will be important for developing effective therapies and vaccines, especially given the marked genetic heterogeneity of NTHi and its possession of phase-variable genes. Currently, no vaccine candidates are ready for large phase III clinical trials.

Nontypeable Haemophilusinfluenzae infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma.

Nontypeable Haemophilus influenzae (NTHi) is a respiratory tract pathobiont that chronically colonizes the airways of asthma patients and is associated with severe, neutrophilic disease phenotypes. The mechanism of NTHi airway persistence is not well understood, but accumulating evidence suggests NTHi can persist within host airway immune cells such as macrophages. We hypothesized that NTHi infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma. Bronchoalveolar lavage (BAL) samples from 25 severe asthma patients were assessed by fluorescence in situ hybridisation to quantify NTHi presence. Weighted gene correlation network analysis (WGCNA) was performed on RNASeq data from NTHi-infected monocyte-derived macrophages to identify transcriptomic networks associated with NTHi infection. NTHi was detected in 56% of BAL samples (NTHi+) and was associated with longer asthma duration (34 vs 22.5 years, p=.0436) and higher sputum neutrophil proportion (67% vs 25%, p=.0462). WGCNA identified a transcriptomic network of immune-related macrophage genes significantly associated with NTHi infection, including upregulation of T17 inflammatory mediators and neutrophil chemoattractants IL1B, IL8, IL23 and CCL20 (all p < .05). Macrophage network genes SGPP2 (p=.0221), IL1B (p=.0014) and GBP1 (p=.0477) were more highly expressed in NTHi+ BAL and moderately correlated with asthma duration (IL1B; rho=0.41, p=.041) and lower prebronchodilator FEV1/FVC% (GBP1; rho=-0.43, p=.046 and IL1B; rho=-0.42, p=.055). NTHi persistence with pulmonary macrophages may contribute to chronic airway inflammation and T17 responses in severe asthma, which can lead to decreased lung function and reduced steroid responsiveness. Identifying therapeutic strategies to reduce the burden of NTHi in asthma could improve patient outcomes.

Dual RNASeq Reveals NTHi-Macrophage Transcriptomic Changes During Intracellular Persistence.

Nontypeable Haemophilus influenzae (NTHi) is a pathobiont which chronically colonises the airway of individuals with chronic respiratory disease and is associated with poor clinical outcomes. It is unclear how NTHi persists in the airway, however accumulating evidence suggests that NTHi can invade and persist within macrophages. To better understand the mechanisms of NTHi persistence within macrophages, we developed an in vitro model of NTHi intracellular persistence using human monocyte-derived macrophages (MDM). Dual RNA Sequencing was used to assess MDM and NTHi transcriptomic regulation occurring simultaneously during NTHi persistence. Analysis of the macrophage response to NTHi identified temporally regulated transcriptomic profiles, with a specific ‘core’ profile displaying conserved expression of genes across time points. Gene list enrichment analysis identified enrichment of immune responses in the core gene set, with KEGG pathway analysis revealing specific enrichment of intracellular immune response pathways. NTHi persistence was facilitated by modulation of bacterial metabolic, stress response and ribosome pathways. Levels of NTHi genes bioC, mepM and dps were differentially expressed by intracellular NTHi compared to planktonic NTHi, indicating that the transcriptomic adaption was distinct between the two different NTHi lifestyles. Overall, this study provides crucial insights into the transcriptomic adaptations facilitating NTHi persistence within macrophages. Targeting these reported pathways with novel therapeutics to reduce NTHi burden in the airway could be an effective treatment strategy given the current antimicrobial resistance crisis and lack of NTHi vaccines.

P187 Unravelling the contribution to the pro-inflammatory burden caused by the persistence of nontypeable haemophilus influenzae in cystic fibrosis

P187 Unravelling the contribution to the pro-inflammatory burden caused by the persistence of nontypeable haemophilus influenzae in cystic fibrosis

Immunoglobulin A Protease Variants Facilitate Intracellular Survival in Epithelial Cells By Nontypeable Haemophilus influenzae That Persist in the Human Respiratory Tract in Chronic Obstructive Pulmonary Disease.

Nontypeable Haemophilus influenzae (NTHi) persists in the airways in chronic obstructive pulmonary disease (COPD). NTHi expresses 4 immunoglobulin (Ig)A protease variants (A1, A2, B1, B2) with distinct cleavage specificities for human IgA1. Little is known about the different roles of IgA protease variants in NTHi infection. Twenty-six NTHi isolates from a 20-year longitudinal study of COPD were analyzed for IgA protease expression, survival in human respiratory epithelial cells, and cleavage of lysosomal-associated membrane protein 1 (LAMP1). IgA protease B1 and B2-expressing strains showed greater intracellular survival in host epithelial cells than strains expressing no IgA protease (P < .001) or IgA protease A1 or A2 (P < .001). Strains that lost IgA protease expression showed reduced survival in host cells compared with the same strain that expressed IgA protease B1 (P = .006) or B2 (P = .015). IgA proteases B1 and B2 cleave LAMP1. Passage of strains through host cells selected for expression of IgA proteases B1 and B2 but not A1. IgA proteases B1 and B2 cleave LAMP1 and mediate intracellular survival in respiratory epithelial cells. Intracellular persistence of NTHi selects for expression of IgA proteases B1 and B2. The variants of NTHi IgA proteases play distinct roles in pathogenesis of infection.

SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus

Non-typeable Haemophilus influenzae (NTHI) is a common commensal bacterium that resides in the human upper respiratory tract of healthy individuals. NTHI is also a known causative agent of multiple diseases including sinusitis, otitis media, as well as exacerbates disease severity of patients with cystic fibrosis and chronic obstructive pulmonary disease. We have previously shown that the Sap transporter mediates resistance to host antimicrobial peptides (AMPs) and import of the iron-containing compound heme. Here, we analyzed the contribution of the Sap structural ATPase protein, SapF, in these essential functions. In contrast to SapD, SapF was dispensable for NTHI survival when exposed to AMPs in vitro. SapF was responsible for heme utilization and recovery of depleted internal heme-iron stores. Further, a loss of SapF resulted in morphological plasticity and enhanced community development and biofilm architecture, suggesting the potential role of heme-iron availability in coordinating the complexity of NTHI biofilm architecture. SapF was required for colonization of the nasopharynx and acute infection of the middle ear, as SapF deficiency correlated with a statistically significant decrease in NTHI persistence in vivo. These data suggest that SapF is required for proper heme utilization which directly impacts NTHI survival. Thus, these studies further support a role for the Sap complex in the transport of multiple substrates and further defines substrate specificity for the two ATPase subunits. Given the multiple essential functions provided by the Sap transporter, this complex could prove to be an effective therapeutic target for the treatment of NTHI diseases.

Non-Typeable Haemophilus influenzae Invasion and Persistence in the Human Respiratory Tract

Non-typeable Haemophilus influenzae (NTHI) is an opportunistic bacterial pathogen of the human respiratory tract and is a leading cause of respiratory infections in children and adults. NTHI is considered to be an extracellular pathogen, but has consistently been observed within and between human respiratory epithelial cells and macrophages, in vitro and ex vivo. Until recently, few studies have examined the internalization, trafficking, and fate of NTHI in host cells. It is important to clarify this interaction because of a possible correlation between intracellular NTHI and symptomatic infection, and because NTHI infections frequently persist and recur despite antibiotic therapy and the development of bactericidal antibodies, suggesting a possible intracellular state or reservoir for NTHI. How does NTHI enter host cells? Can NTHI survive intracellularly and, if so, for how long? Strides have been made in the identification of host receptors, signaling, endocytosis, and trafficking pathways involved in the entry and persistence of NTHI in the respiratory tract.

Impaired Phagocytosis of NontypeableHaemophilus influenzaeby Human Alveolar Macrophages in Chronic Obstructive Pulmonary Disease

Interactions of nontypeable Haemophilus influenzae (NTHI) with human alveolar macrophages are implicated in the persistence of NTHI in chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-induced macrophage responses are poorly understood. We hypothesized that immunologic responses of alveolar macrophages to NTHI are impaired in COPD. Blood and alveolar macrophages--obtained from ex-smokers with COPD (n = 14), ex-smokers without COPD (n = 15), and nonsmokers (n = 9)--were incubated with 3 distinct NTHI strains obtained from patients with COPD. Phagocytosis of 3H-NTHI, expressed as a percentage of the mean total radioactivity, and of intracellular viability, assessed as a percentage of viable cell-associated NTHI, were measured. Alveolar macrophages from donors with COPD, compared with those from donors without COPD, had impaired phagocytosis (median [interquartile range]) for each NTHI strain: 14P13H5, 0.26 (0.08-0.61) versus 1.36 (0.69-1.95); 6P5H1, 0.92 (0.32-1.82) versus 1.90 (1.32-2.68); and 14P14H1, 0.79 (0.23-1.32) versus 2.13 (1.13-2.40) (P < or = .01 for each). However, phagocytosis of all NTHI strains by blood macrophages from donors with COPD was indistinguishable from that of blood macrophages from donors without COPD and from nonsmokers. The intracellular killing of NTHI was not impaired in alveolar macrophages from donors with COPD. These results support a paradigm of impaired phagocytosis by alveolar macrophages, but not blood macrophages, in COPD and provide an immunologic basis for persistence of NTHI in the airways of adults with COPD.

Sequence stability of the gene encoding outer membrane protein P2 of nontypeable Haemophilus influenzae in the human respiratory tract.

Nontypeable Haemophilus influenzae (NTHI) is an important cause of lower respiratory tract infections in patients with chronic obstructive pulmonary disease. Recent findings suggest that the major outer membrane protein P2 should be reconsidered as a vaccine candidate for NTHI. A P2-based vaccine would require a relative degree of sequence stability of the gene encoding P2 (ompP2) during colonization. To characterize the sequence stability of ompP2 during colonization of the human respiratory tract, ompP2 genes from 13 sets of isolates that persisted in patients with chronic obstructive pulmonary disease (mean colonization, 7 months) were sequenced. In 9 sets of isolates, ompP2 did not change. Sequence changes were noted in 4 sets of isolates. Most of these changes occurred within areas of repetitive DNA, suggesting that this type of DNA has a role in antigenic variation of P2. The sequence of ompP2 is relatively stable during persistence of NTHI in the human host.

Persistence of nontypeable Haemophilus influenzae in adenoid macrophages: a putative colonization mechanism.

That nontypeable H. influenzae (NTHI) can reside intracellularly in human adenoid tissue has been suggested by use of in situ hybridization of a fluorescein labelled 16S rRNA-targeted oligonucleotide probe (FISH). Adenoid tissues from 43 children operated on in a clinically infection-free interval were investigated. FISH revealed H. influenzae in macrophage-like cells, located subepithelially in the crypts in all 43 adenoids. Furthermore, H. influenzae was detected in 22/22 adenoids using immunohistochemistry with the monoclonal antibody MAHI-3 recognizing a conserved H. influenzae LPS inner-core region. FISH and staining with monoclonal antibodies against immunophenotypic markers were performed simultaneously in order to characterize the cellular interrelations in this microenvironment. The findings of widespread presence of H. influenzae in cells of which some strongly expressed the CD14 marker of the monocyte/macrophage lineage may correspond to an important aspect of the colonization mechanisms whereby NTHI persists in the nasopharynx of children.