Malcolm R. Starkey

Chlamydial respiratory infection during allergen sensitization drives neutrophilic allergic airways disease.

Neutrophilic asthma is a prevalent, yet recently described phenotype of asthma. It is characterized by neutrophilic rather than eosinophilic airway inflammation and airways hyperresponsiveness (AHR) and may have an infectious origin. Chlamydial respiratory infections are associated with asthma, but how these Th1-inducing bacteria influence Th2-mediated asthma remains unknown. The effects of chlamydial infection on the development of asthma were investigated using a BALB/c mouse model of OVA-induced allergic airways disease (AAD). The effects of current and resolved Chlamydia muridarum infection during OVA sensitization on AAD were assessed and compared with uninfected and nonsensitized controls. Current, but not resolved, infection attenuated hallmark features of AAD: pulmonary eosinophil influx, T cell production of IL-5, mucus-secreting cell hyperplasia, and AHR. Current infection also induced robust OVA-driven neutrophilic inflammation and IFN-γ release from T cells. The phenotype of suppressed but persistent Th2 responses in association with enhanced neutrophilia is reminiscent of neutrophilic asthma. This phenotype was also characterized by increased pulmonary IL-12 and IL-17 expression and activation of APCs, as well as by reduced thymus- and activation-regulated chemokine. Inhibition of pulmonary neutrophil influx during infection blocked OVA-induced neutrophilic inflammation and T cell IFN-γ production and reversed the suppressive effects on mucus-secreting cell hyperplasia and AHR during AAD. These changes correlated with decreased IL-12 and IL-17 expression, increased thymus- and activation-regulated chemokine and altered APC activation. Blocking IFN-γ and IL-17 during OVA challenge had no effect. Thus, active chlamydial respiratory infection during sensitization enhances subsequent neutrophilic inflammation and Th1/Th17 responses during allergen exposure and may have a role in the pathogenesis of neutrophilic asthma.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T-cells

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Macrolide therapy suppresses key features of experimental steroid-sensitive and steroid-insensitive asthma

Background Steroid-insensitive endotypes of asthma are an important clinical problem and effective therapies are required. They are associated with bacterial infection and non-eosinophilic inflammatory responses in the asthmatic lung. Macrolide therapy is effective in steroid-insensitive endotypes, such as non-eosinophilic asthma. However, whether the effects of macrolides are due to antimicrobial or anti-inflammatory mechanisms is not known. Objective To determine and assess the efficacy of macrolide (ie, clarithromycin) and non-macrolide (ie, amoxicillin) antibiotic treatments in experimental models of infection-induced, severe, steroid-insensitive neutrophilic allergic airways disease (SSIAAD), compared with steroid-sensitive AAD and to delineate the antimicrobial and anti-inflammatory effects of macrolide therapy. Methods We developed and used novel mouse models of Chlamydia and Haemophilus lung infection-induced SSIAAD. We used these models to investigate the effects of clarithromycin and amoxicillin treatment on immune responses and airways hyper-responsiveness (AHR) in Ova-induced, T helper lymphocyte (Th) 2 -associated steroid-sensitive AAD and infection-induced Th1/Th17-associated SSIAAD compared with dexamethasone treatment. Results Clarithromycin and amoxicillin had similar antimicrobial effects on infection. Amoxicillin did attenuate some features, but did not broadly suppress either form of AAD. It did restore steroid sensitivity in SSIAAD by reducing infection. In contrast, clarithromycin alone widely suppressed inflammation and AHR in both steroid-sensitive AAD and SSIAAD. This occurred through reductions in Th2 responses that drive steroid-sensitive eosinophilic AAD and tumour necrosis factor α and interleukin 17 responses that induce SSIAAD. Conclusions Macrolides have broad anti-inflammatory effects in AAD that are likely independent of their antimicrobial effects. The specific responses that are suppressed are dependent upon the responses that dominate during AAD.

Targeting PI3K-p110α Suppresses Influenza Virus Infection in Chronic Obstructive Pulmonary Disease

RATIONALE Chronic obstructive pulmonary disease (COPD) and influenza virus infections are major global health issues. Patients with COPD are more susceptible to infection, which exacerbates their condition and increases morbidity and mortality. The mechanisms of increased susceptibility remain poorly understood, and current preventions and treatments have substantial limitations. OBJECTIVES To characterize the mechanisms of increased susceptibility to influenza virus infection in COPD and the potential for therapeutic targeting. METHODS We used a combination of primary bronchial epithelial cells (pBECs) from COPD and healthy control subjects, a mouse model of cigarette smoke-induced experimental COPD, and influenza infection. The role of the phosphoinositide-3-kinase (PI3K) pathway was characterized using molecular methods, and its potential for targeting assessed using inhibitors. MEASUREMENTS AND MAIN RESULTS COPD pBECs were susceptible to increased viral entry and replication. Infected mice with experimental COPD also had more severe infection (increased viral titer and pulmonary inflammation, and compromised lung function). These processes were associated with impaired antiviral immunity, reduced retinoic acid-inducible gene-I, and IFN/cytokine and chemokine responses. Increased PI3K-p110α levels and activity in COPD pBECs and/or mice were responsible for increased infection and reduced antiviral responses. Global PI3K, specific therapeutic p110α inhibitors, or exogenous IFN-β restored protective antiviral responses, suppressed infection, and improved lung function. CONCLUSIONS The increased susceptibility of individuals with COPD to influenza likely results from impaired antiviral responses, which are mediated by increased PI3K-p110α activity. This pathway may be targeted therapeutically in COPD, or in healthy individuals, during seasonal or pandemic outbreaks to prevent and/or treat influenza.

TLR2, but Not TLR4, Is Required for Effective Host Defence against Chlamydia Respiratory Tract Infection in Early Life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient (−/−), 4−/− or 2/4−/− BALB/c mice. Wt mice had moderate disease and infection. TLR2−/− mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4−/− mice were asymptomatic. TLR2/4−/− mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4−/− mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4+ and CD8+ T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2−/− mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4−/− mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.

Th2 cytokine antagonists: potential treatments for severe asthma

Introduction: Asthma is a major disease burden worldwide. Treatment with steroids and long acting β-agonists effectively manage symptoms in many patients but do not treat the underlying cause of disease and have serious side effects when used long term and in children. Therapies targeting the underlying causes of asthma are urgently needed. T helper type 2 (Th2) cells and the cytokines they release are clinically linked to the presentation of all forms of asthma. They are the primary drivers of mild to moderate and allergic asthma. They also play a pathogenetic role in exacerbations and more severe asthma though other factors are also involved. Much effort using animal models and human studies has been dedicated to the identification of the pathogenetic roles of these cells and cytokines and whether inhibition of their activity has therapeutic benefit in asthma. Areas covered: We discuss the current status of Th2 cytokine antagonists for the treatment of asthma. We also discuss the potential for targeting Th2-inducing cytokines, Th2 cell receptors and signaling as well as the use of Th2 cell antagonists, small interfering oligonucleotides, microRNAs, and combination therapies. Expert opinion: Th2 antagonists may be most effective in particular asthma subtypes/endotypes where specific cytokines are known to be active through the analysis of biomarkers. Targeting common receptors and pathways used by these cytokines may have additional benefit. Animal models have been valuable in identifying therapeutic targets in asthma, however the results from such studies need to be carefully interpreted and applied to appropriately stratified patient cohorts in well-designed clinical studies and trials.

Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease.

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13−/− mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13−/− mice induced persistent AHR. Infection and associated pathology were restored in infected Il13−/− mice by reconstitution with IL-13. Stat6−/− mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.

Murine models of infectious exacerbations of airway inflammation

Airway inflammation underpins the pathogenesis of the major human chronic respiratory diseases. It is now well recognized that respiratory infections with bacteria and viruses are important in the induction, progression and exacerbation of these diseases. There are no effective therapies that prevent or reverse these events. The development and use of mouse models are proving valuable in understanding the role of infection in disease pathogenesis. They have recently been used to show that infections in early life alter immune responses and lung structure to increase asthma severity, and alter immune responses in later life to induce steroid resistance. Infection following smoke exposure or in experimental chronic obstructive pulmonary disease exacerbates inflammation and remodeling, and worsens cystic fibrosis. Further exploration of these models will facilitate the identification of new therapeutic approaches and the testing of new preventions and treatments.

Role for NLRP3 Inflammasome-mediated, IL-1β-dependent Responses in Severe, Steroid-resistant Asthma.

&NA; Rationale: Severe, steroid‐resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing 3 (NLRP3) inflammasome and concomitant IL‐1&bgr; responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid‐resistant asthma. Objectives: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL‐1&bgr; in severe, steroid‐resistant asthma. Methods: We developed mouse models of Chlamydia and Haemophilus respiratory infection‐mediated, ovalbumin‐induced severe, steroid‐resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL‐1&bgr; responses. The roles and potential for targeting of NLRP3 inflammasome, caspase‐1, and IL‐1&bgr; responses in experimental severe, steroid‐resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase‐1 inhibitor Ac‐YVAD‐cho; and neutralizing anti‐IL‐1&bgr; antibody. Roles for IL‐1&bgr;‐induced neutrophilic inflammation were examined using IL‐1&bgr; and anti‐Ly6G. Measurements and Main Results: Chlamydia and Haemophilus infections increase NLRP3, caspase‐1, IL‐1&bgr; responses that drive steroid‐resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL‐1&bgr; expression. Treatment with anti‐IL‐1&bgr;, Ac‐YVAD‐cho, and MCC950 suppressed IL‐1&bgr; responses and the important steroid‐resistant features of disease in mice, whereas IL‐1&bgr; administration recapitulated these features. Neutrophil depletion suppressed IL‐1&bgr;‐induced steroid‐resistant airway hyperresponsiveness. Conclusions: NLRP3 inflammasome responses drive experimental severe, steroid‐resistant asthma and are potential therapeutic targets in this disease.

MicroRNA-21 drives severe, steroid-insensitive experimental asthma by amplifying phosphoinositide 3-kinase-mediated suppression of histone deacetylase 2.

Background: Severe steroid‐insensitive asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms of disease pathogenesis. Steroid‐insensitive asthma is associated with respiratory tract infections and noneosinophilic endotypes, including neutrophilic forms of disease. However, steroid‐insensitive patients with eosinophil‐enriched inflammation have also been described. The mechanisms that underpin infection‐induced, severe steroid‐insensitive asthma can be elucidated by using mouse models of disease. Objective: We sought to develop representative mouse models of severe, steroid‐insensitive asthma and to use them to identify pathogenic mechanisms and investigate new treatment approaches. Methods: Novel mouse models of Chlamydia, Haemophilus influenzae, influenza, and respiratory syncytial virus respiratory tract infections and ovalbumin‐induced, severe, steroid‐insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed and interrogated. Results: Infection induced increases in the levels of microRNA (miRNA)‐21 (miR‐21) expression in the lung during SSIAAD, whereas expression of the miR‐21 target phosphatase and tensin homolog was reduced. This was associated with an increase in levels of phosphorylated Akt, an indicator of phosphoinositide 3‐kinase (PI3K) activity, and decreased nuclear histone deacetylase (HDAC)2 levels. Treatment with an miR‐21–specific antagomir (Ant‐21) increased phosphatase and tensin homolog levels. Treatment with Ant‐21, or the pan‐PI3K inhibitor LY294002, reduced PI3K activity and restored HDAC2 levels. This led to suppression of airway hyperresponsiveness and restored steroid sensitivity to allergic airway disease. These observations were replicated with SSIAAD associated with 4 different pathogens. Conclusion: We identify a previously unrecognized role for an miR‐21/PI3K/HDAC2 axis in SSIAAD. Our data highlight miR‐21 as a novel therapeutic target for the treatment of this form of asthma. GRAPHICAL ABSTRACT Figure. No caption available.