Jemma R. Mayall

Interferon-ε Protects the Female Reproductive Tract from Viral and Bacterial Infection

A Role for IFN-ɛ Type I interferons (IFNs) are critical cytokines involved in host defense against pathogens, particularly viruses. IFN-ɛ is an IFN-like gene encoded within the type I IFN locus in mice and humans whose function has not been characterized. Fung et al. (p. 1088) created mice with a genetic deletion in Ifn-ɛ and found that, like other type I IFNs, IFN-ɛ signals through the IFN-α receptors 1 and 2. However, unlike these other cytokines, which are primarily expressed by immune cells and are induced upon immune cell triggering, IFN-ɛ was expressed exclusively by epithelial cells of the female reproductive tract in both mice and humans and its expression was hormonally regulated. IFN-ɛ–deficient mice were more susceptible to infection with herpes simplex virus 2 and Chlamydia muridarum, two common sexually transmitted pathogens. The cytokine interferon-ε is expressed in the female reproductive tract and protects against sexually transmitted diseases. The innate immune system senses pathogens through pattern-recognition receptors (PRRs) that signal to induce effector cytokines, such as type I interferons (IFNs). We characterized IFN-ε as a type I IFN because it signaled via the Ifnar1 and Ifnar2 receptors to induce IFN-regulated genes. In contrast to other type I IFNs, IFN-ε was not induced by known PRR pathways; instead, IFN-ε was constitutively expressed by epithelial cells of the female reproductive tract (FRT) and was hormonally regulated. Ifn-ε–deficient mice had increased susceptibility to infection of the FRT by the common sexually transmitted infections (STIs) herpes simplex virus 2 and Chlamydia muridarum. Thus, IFN-ε is a potent antipathogen and immunoregulatory cytokine that may be important in combating STIs that represent a major global health and socioeconomic burden.

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.

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.

Mechanisms and treatments for severe, steroid-resistant allergic airway disease and asthma

Severe, steroid‐resistant asthma is clinically and economically important since affected individuals do not respond to mainstay corticosteroid treatments for asthma. Patients with this disease experience more frequent exacerbations of asthma, are more likely to be hospitalized, and have a poorer quality of life. Effective therapies are urgently required, however, their development has been hampered by a lack of understanding of the pathological processes that underpin disease. A major obstacle to understanding the processes that drive severe, steroid‐resistant asthma is that the several endotypes of the disease have been described that are characterized by different inflammatory and immunological phenotypes. This heterogeneity makes pinpointing processes that drive disease difficult in humans. Clinical studies strongly associate specific respiratory infections with severe, steroid‐resistant asthma. In this review, we discuss key findings from our studies where we describe the development of representative experimental models to improve our understanding of the links between infection and severe, steroid‐resistant forms of this disease. We also discuss their use in elucidating the mechanisms, and their potential for developing effective therapeutic strategies, for severe, steroid‐resistant asthma. Finally, we highlight how the immune mechanisms and therapeutic targets we have identified may be applicable to obesity‐or pollution‐associated asthma.

Role of iron in the pathogenesis of respiratory disease

Iron is essential for many biological processes, however, too much or too little iron can result in a wide variety of pathological consequences, depending on the organ system, tissue or cell type affected. In order to reduce pathogenesis, iron levels are tightly controlled in throughout the body by regulatory systems that control iron absorption, systemic transport and cellular uptake and storage. Altered iron levels and/or dysregulated homeostasis have been associated with several lung diseases, including chronic obstructive pulmonary disease, lung cancer, cystic fibrosis, idiopathic pulmonary fibrosis and asthma. However, the mechanisms that underpin these associations and whether iron plays a key role in the pathogenesis of lung disease are yet to be fully elucidated. Furthermore, in order to survive and replicate, pathogenic micro-organisms have evolved strategies to source host iron, including freeing iron from cells and proteins that store and transport iron. To counter these microbial strategies, mammals have evolved immune-mediated defence mechanisms that reduce iron availability to pathogens. This interplay between iron, infection and immunity has important ramifications for the pathogenesis and management of human respiratory infections and diseases. An increased understanding of the role that iron plays in the pathogenesis of lung disease and respiratory infections may help inform novel therapeutic strategies. Here we review the clinical and experimental evidence that highlights the potential importance of iron in respiratory diseases and infections.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/β receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK–STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100–1000-fold reduced potency compared with rmIFNα1 and rmIFNβ. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

Chlamydia muridarum infection differentially changes smooth muscle contractility and responses to prostaglandins in uterus and cervix

Chlamydia trachomatis infection is a primary cause of reproductive tract diseases including chronic pelvic pain and infertility. Previous studies showed that this infection alters physiological activities in mouse oviducts. Whether this occurs in the uterus and cervix has never been investigated. This study characterized the physiological activity of the uterus and the cervix in a Chlamydia muridarum (Cmu) mouse model of reproductive tract infection. Uterine or cervix smooth muscle contractility, responses to oxytocin or prostaglandins (PGF2α and PGE2) and mRNA expression of oxytocin and PG receptors were assessed 14 days post infection. Cmu infection did not affect the contractions of the uterine horn but significantly decreased the contraction amplitude of the cervix. Cmu infection did not alter the responses of uterine horn or cervix to oxytocin, however PGF2α induced contractions of the uterine horn, but not the cervix, were significantly increased following Cmu infection. PGE2 contraction amplitude in both the uterine horn and cervix was unaffected by Cmu infection. An upregulation of Ptgfr and a down-regulation of Ptegr4 mRNA expression was observed in the uterine horn following Cmu infection. These results indicate that Cmu infection alters contractility and prostaglandin signalling in the female reproductive tract but the effects are localised to specific regions.

NLRP3 inflammasome-mediated, IL-1 beta-dependent inflammatory responses drive severe, steroid-insensitive asthma

Introduction: Studies suggest that 10% of children with an ARI have persistent cough at day 21. There are no studies in Indigenous children who have a high risk of chronic lung disease. We aimed to identify the incidence and outcomes of ARI with cough as a symptom in urban Indigenous children. Methods:This is a prospective study of Indigenous children aged<5 years registered with a primary health service. Children are followed for a period of 12 months via monthly contacts. Children who develop cough as a symptom at any time are followed weekly for 4 weeks to ascertain cough outcomes. Results: To date, 162 children are enrolled, totalling 1065 child-months of observation. Two-hundred ARI episodes with cough have been reported (29.6 episodes/100 child-months at risk). Thirty-four ARIs (17%) have progressed to persistent cough at day 28 in 24 children. Of these, 15 children had 1 episode, 5 had 2, 4 had 3 and 1 had 4 during the follow-up period. The majority of children with persistent cough were diagnosed (by a respiratory physician) with protracted bacterial bronchitis and/or bronchiectasis. Conclusions:The proportion of children developing persistent cough postARI is higher than that currently reported (10%) with the majority suggesting protracted bacterial infection. GrantSupport:AQUTAPAaward, aQCMRIProgramGrant, UQFoundation Research Excellence Award, a QUT Indigenous Health Research StartUpGrant and the NHMRCCRE for LungHealth in Aboriginal and TorresStrait Islander Children. Declaration of Interest: None to Declare

A Sheep Model to Examine the Effects of Maternal Asthma On Fetal Outcomes

Introduction: Studies suggest that 10% of children with an ARI have persistent cough at day 21. There are no studies in Indigenous children who have a high risk of chronic lung disease. We aimed to identify the incidence and outcomes of ARI with cough as a symptom in urban Indigenous children. Methods:This is a prospective study of Indigenous children aged<5 years registered with a primary health service. Children are followed for a period of 12 months via monthly contacts. Children who develop cough as a symptom at any time are followed weekly for 4 weeks to ascertain cough outcomes. Results: To date, 162 children are enrolled, totalling 1065 child-months of observation. Two-hundred ARI episodes with cough have been reported (29.6 episodes/100 child-months at risk). Thirty-four ARIs (17%) have progressed to persistent cough at day 28 in 24 children. Of these, 15 children had 1 episode, 5 had 2, 4 had 3 and 1 had 4 during the follow-up period. The majority of children with persistent cough were diagnosed (by a respiratory physician) with protracted bacterial bronchitis and/or bronchiectasis. Conclusions:The proportion of children developing persistent cough postARI is higher than that currently reported (10%) with the majority suggesting protracted bacterial infection. GrantSupport:AQUTAPAaward, aQCMRIProgramGrant, UQFoundation Research Excellence Award, a QUT Indigenous Health Research StartUpGrant and the NHMRCCRE for LungHealth in Aboriginal and TorresStrait Islander Children. Declaration of Interest: None to Declare