Haemophilus influenzae Infection Drives IL-17-Mediated Neutrophilic Allergic Airways Disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses

Modulation of responses in allergic airways disease by Haemophilus influenzae infection

Research Doctorate - Doctor of Philosophy (PhD)Asthma is a common chronic inflammatory disease of the airways that affects over 2.2 million people in Australia. Asthma is a heterogeneous inflammatory disease typically characterised by T helper lymphocyte type 2 (Th2)-mediated eosinophilic inflammation, exaggerated responses to innocuous stimuli, mucus hypersecretion leading to airways obstruction and airway remodelling. These physiological changes result in wheezing, chest tightness, and breathing difficulties. However, it has been established that eosinophilic inflammation is only present in 50% of asthmatic patients. Around 30% of non-eosinophilic asthmatics have neutrophilic rather than eosinophilic inflammation, which is a key feature of neutrophilic asthma. Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that is commonly found in the upper respiratory tract of about 75% of healthy individuals. It is normally asymptomatically carried in people, however it may cause otitis media and is a common cause of community-acquired pneumonia. NTHi has also been linked to a number of chronic airway diseases. It has been detected in patients with bronchiectasis, chronic bronchitis and is commonly associated with chronic obstructive pulmonary disease (COPD) exacerbations. It has also recently been associated with neutrophilic asthma, however, the role of NTHi in neutrophilic asthma has not been investigated. Using murine models of NTHi infection and allergic airways disease (AAD), we investigated the relationship between infection and AAD. We showed that NTHi infection induced features of neutrophilic asthma; reduced Th2-mediated eosinophilic inflammation, reduced airways hyper-responsiveness (AHR) compared to eosinophilic AAD, and importantly, significantly increased Th17 responses and neutrophilic inflammation. In the first study it was demonstrated that the combination of infection and AAD reduced the expression of MHC II and CD86 on dendritic cells (DCs), suggesting that infection induced changes in presentation of antigen to naïve T-cells and subsequent adaptive responses. Infection also induced Interleukin (IL)-17 production from innate cells and Th17 cells. Critically, we show that inhibiting IL-17 significantly reduced neutrophilic inflammation in the airways. This highlights the crucial role of IL-17 in infection-induced neutrophilic AAD. The second study showed that the induction of AAD during infection delayed bacterial clearance from the lungs compared to infection alone controls. In contrast to Th2-mediated eosinophilic inflammation, this model of infection-induced neutrophilic AAD was resistant to dexamethasone treatment. All features of infection-induced neutrophilic AAD, including eosinophil and neutrophil influx, antigen-specific IL-5, IL-13 and Interferon (IFN)-γ, NTHi-specific IL-17, and AHR were unchanged with steroid treatment. This study also demonstrated that neutrophil and macrophage activation and function was inhibited in neutrophilic AAD. This lack of innate immune response may enable chronic bacterial infection. The final study investigated clarithromycin, a macrolide, and combination therapy with dexamethasone, as possible treatment strategies for neutrophilic asthmatics. This study demonstrated that clarithromycin alone significantly reduced neutrophil influx and IL-17 responses, but increased Th2-mediated eosinophilic inflammation. However, the combination of clarithromycin and dexamethasone suppressed all key features of AAD, including eosinophilic and neutrophilic inflammation, ovalbumin (OVA)-specific IL-5, IL-13, and IFN-γ, NTHi-induced IL-17, and AHR. These novel findings further the understanding of the potential role of NTHi in the development of neutrophilic asthma. We have identified some mechanisms of how infection may lead to features observed in neutrophilic asthma, and importantly, possible treatment strategies for neutrophilic asthmatics, and perhaps, other neutrophilic airway diseases with evidence of infection

Altered lung function at mid-adulthood in mice following neonatal exposure to hyperoxia

Infants born very preterm are usually exposed to high oxygen concentrations but this may impair lung function in survivors in later life. However, the precise changes involved are poorly understood. We determined how neonatal hyperoxia alters lung function at mid-adulthood in mice. Neonatal C57BL/6J mice inhaled 65% oxygen (HE group) from birth for 7 days. They then breathed room air until 11 months of age (P11mo); these mice experienced growth restriction. Controls breathed only room air. To exclude the effects of growth restriction, a group of dams was rotated between hyperoxia and normoxia during the exposure period (HE+DR group). Lung function was measured at P11mo. HE mice had increased inspiratory capacity, work of breathing and tissue damping. HE+DR mice had further increases in inspiratory capacity and work of breathing, and reduced FEV₁₀₀/FVC. Total lung capacity was increased in HE+DR males. HE males had elevated responses to methacholine. Neonatal hyperoxia alters lung function at mid-adulthood, especially in males

PD-L1 promotes early-life chlamydia respiratory infection-induced severe allergic airway disease

However, the immune responses that protect against early-life infection and the mechanisms that lead to chronic lung disease are incompletely understood. In the current study we investigated the role of programmed death (PD)-1 and its ligands PD-L1 and PD-L2 in promoting early-life Chlamydia respiratory infection, and infection-induced airway hyperresponsiveness (AHR) and severe allergic airway disease (AAD) in later life. Infection increased PD-1 and PD-L1, but not PD-L2, mRNA expression in the lung. Flow cytometric analysis of whole lung homogenates identified monocytes, dendritic cells, CD4+ and CD8+ T cells as major sources of PD-1 and PD-L1. Inhibition of PD-1 and PD-L1, but not PD-L2, during infection ablated infection-induced AHR in later life. Given that PD-L1 was the most highly up-regulated and its targeting prevented infection-induced AHR, subsequent analyses focused on this ligand. Inhibition of PD-L1 had no effect on Chlamydia load, but suppressed infection-induced pulmonary inflammation. Infection decreased the levels of the IL-13 decoy receptor in the lung, which were restored to baseline levels by inhibition of PD-L1. Finally, inhibition of PD-L1 during infection prevented subsequent infection-induced severe allergic airways disease in later-life, by decreasing IL-13 levels, Gob-5 expression, mucus production and AHR. Thus, early-life Chlamydia respiratory infection-induced PD-L1 promotes severe inflammation during infection, permanent reductions in lung function and the development of more severe AAD in later life

Combined haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma

Background: 20–30% of patients with asthma have neutrophilic airway inflammation and reduced responsiveness to steroid therapy. They often have chronic airway bacterial colonisation and <i>Haemophilus influenzae</i> is one of the most commonly isolated bacteria. The relationship between chronic airway colonisation and the development of steroid-resistant neutrophilic asthma is unclear. Objectives: To investigate the relationship between <i>H influenzae</i> respiratory infection and neutrophilic asthma using mouse models of infection and ovalbumin (OVA)-induced allergic airways disease. Methods: BALB/c mice were intratracheally infected with <i>H influenzae</i> (day 10), intraperitoneally sensitised (day 0) and intranasally challenged (day 12–15) with OVA. Treatment groups were administered dexamethasone intranasally during OVA challenge. Infection, allergic airways disease, steroid sensitivity and immune responses were assessed (days 11, 16 and 21). Results: The combination of <i>H influenzae</i> infection and allergic airways disease resulted in chronic lung infection that was detected on days 11, 16 and 21 (21, 26 and 31 days after infection). Neutrophilic allergic airways disease and T helper 17 cell development were induced, which did not require active infection. Importantly, all features of neutrophilic allergic airways disease were steroid resistant. Toll-like receptor 4 expression and activation of phagocytes was reduced, but most significantly the influx and/or development of phagocytosing neutrophils and macrophages into the airways was inhibited. Conclusions: The combination of infection and allergic airways disease promotes bacterial persistence, leading to the development of a phenotype similar to steroid-resistant neutrophilic asthma and which may result from dysfunction in innate immune cells. This indicates that targeting bacterial infection in steroid-resistant asthma may have therapeutic benefit

Data_Sheet_1_The DmsABC S-oxide reductase is an essential component of a novel, hypochlorite-inducible system of extracellular stress defense in Haemophilus influenzae.DOCX

Defenses against oxidative damage to cell components are essential for survival of bacterial pathogens during infection, and here we have uncovered that the DmsABC S-/N-oxide reductase is essential for virulence and in-host survival of the human-adapted pathogen, Haemophilus influenzae. In several different infection models, H. influenzae ΔdmsA strains showed reduced immunogenicity as well as lower levels of survival in contact with host cells. Expression of DmsABC was induced in the presence of hypochlorite and paraquat, closely linking this enzyme to defense against host-produced antimicrobials. In addition to methionine sulfoxide, DmsABC converted nicotinamide- and pyrimidine-N-oxide, precursors of NAD and pyrimidine for which H. influenzae is an auxotroph, at physiologically relevant concentrations, suggesting that these compounds could be natural substrates for DmsABC. Our data show that DmsABC forms part of a novel, periplasmic system for defense against host-induced S- and N-oxide stress that also comprises the functionally related MtsZ S-oxide reductase and the MsrAB peptide methionine sulfoxide reductase. All three enzymes are induced following exposure of the bacteria to hypochlorite. MsrAB is required for physical resistance to HOCl and protein repair. In contrast, DmsABC was required for intracellular colonization of host cells and, together with MtsZ, contributed to resistance to N-Chlorotaurine. Our work expands and redefines the physiological role of DmsABC and highlights the importance of different types of S-oxide reductases for bacterial virulence.</p

Peptide methionine sulfoxide reductase from Haemophilus influenzae is required for protection against HOCl and affects the host response to infection

Peptide methionine sulfoxide reductases (Msrs) are enzymes that repair ROS-damage to sulfur-containing amino acids such as methionine, ensuring functional integrity of cellular proteins. Here we have shown that unlike the majority of pro- and eukaryotic Msrs, the peptide methionine sulfoxide reductase (MsrAB) from the human pathobiont (Hi) is required for the repair of hypochlorite damage to cell envelope proteins, but more importantly, we were able to demonstrate that MsrAB plays a role in modulating the host immune response to Hi infection. Loss of MsrAB resulted in >1000-fold increase in sensitivity of Hi to HOCl-mediated killing, and also reduced biofilm formation and in-biofilm survival. Expression of was also induced by hydrogen peroxide and paraquat, but a Hi2019 strain was not susceptible to killing by these ROS in vitro. Hi2019 fitness in infection models was low, with a 3-fold reduction in intracellular survival in bronchial epithelial cells, increased susceptibility to neutrophil killing, and a 10-fold reduction in survival in a mouse model of lung infection. Interestingly, infection with Hi2019 led to specific changes in the antibacterial response of human host cells, with genes encoding antimicrobial peptides (BPI, CAMP) upregulated between 4 and 9 fold compared to infection with Hi2019, and reduction in expression of two proteins with antiapoptotic functions (BIRC3, XIAP). Modulation of host immune responses is a novel role for an enzyme of this type and provides first insights into mechanisms by which MsrAB supports Hi survival in vivo