Multiple inflammasomes may regulate the interleukin-1-driven inflammation in protracted bacterial bronchitis

Protracted bacterial bronchitis (PBB) in young children is characterised by prolonged wet cough, prominent airway interleukin (IL)-1β expression and infection, often with nontypeable Haemophilus influenzae (NTHi). The mechanisms responsible for IL-1-driven inflammation in PBB are poorly understood. We hypothesised that the inflammation in PBB involves the NLRP3 and/or AIM2 inflammasome/IL-1β axis. Lung macrophages obtained from bronchoalveolar lavage (BAL), peripheral blood mononuclear cells (PBMCs), blood monocytes and monocyte-derived macrophages from patients with PBB and age-matched healthy controls were cultured in control medium or exposed to live NTHi. In healthy adult PBMCs, CD14+ monocytes contributed to 95% of total IL-1β-producing cells upon NTHi stimulation. Stimulation of PBB PBMCs with NTHi significantly increased IL-1β expression (p<0.001), but decreased NLRC4 expression (p<0.01). NTHi induced IL-1β secretion in PBMCs from both healthy controls and patients with recurrent PBB. This was inhibited by Z-YVAD-FMK (a caspase-1 selective inhibitor) and by MCC950 (a NLRP3 selective inhibitor). In PBB BAL macrophages inflammasome complexes were visualised as fluorescence specks of NLRP3 or AIM2 colocalised with cleaved caspase-1 and cleaved IL-1β. NTHi stimulation induced formation of specks of cleaved IL-1β, NLRP3 and AIM2 in PBMCs, blood monocytes and monocyte-derived macrophages. We conclude that both the NLRP3 and AIM2 inflammasomes probably drive the IL-1β-dominated inflammation in PBB.Alice C-H. Chen, Hai B. Tran, Yang Xi, Stephanie T. Yerkovich, Katherine J. Baines, Susan J. Pizzutto, Melanie Carroll, Avril A.B. Robertson, Matthew A. Cooper, Kate Schroder, Jodie L. Simpson, Peter G. Gibson, Greg Hodge, Ian B. Masters, Helen M. Buntain, Helen L. Petsky, Samantha J. Prime, Anne B. Chang, Sandra Hodge, and John W. Upha

Study protocol for preventing early-onset pneumonia in young children through maternal immunisation: a multi-centre randomised controlled trial (PneuMatters)

Background: Preventing and/or reducing acute lower respiratory infections (ALRIs) in young children will lead to substantial short and long-term clinical benefits. While immunisation with pneumococcal conjugate vaccines (PCV) reduces paediatric ALRIs, its efficacy for reducing infant ALRIs following maternal immunisation has not been studied. Compared to other PCVs, the 10-valent pneumococcal-Haemophilus influenzae Protein D conjugate vaccine (PHiD-CV) is unique as it includes target antigens from two common lower airway pathogens, pneumococcal capsular polysaccharides and protein D, which is a conserved H. influenzae outer membrane lipoprotein. Aims: The primary aim of this randomised controlled trial (RCT) is to determine whether vaccinating pregnant women with PHiD-CV (compared to controls) reduces ALRIs in their infants' first year of life. Our secondary aims are to evaluate the impact of maternal PHiD-CV vaccination on different ALRI definitions and, in a subgroup, the infants' nasopharyngeal carriage of pneumococci and H. influenzae, and their immune responses to pneumococcal vaccine type serotypes and protein D. Methods: We are undertaking a parallel, multicentre, superiority RCT (1:1 allocation) at four sites across two countries (Australia, Malaysia). Healthy pregnant Australian First Nation or Malaysian women aged 17-40 years with singleton pregnancies between 27+6 and 34+6 weeks gestation are randomly assigned to receive either a single dose of PHiD-CV or usual care. Treatment allocation is concealed. Study outcome assessors are blinded to treatment arms. Our primary outcome is the rate of medically attended ALRIs by 12-months of age. Blood and nasopharyngeal swabs are collected from infants at birth, and at ages 6- and 12-months (in a subset). Our planned sample size (n = 292) provides 88% power (includes 10% anticipated loss to follow-up). Discussion: Results from this RCT potentially leads to prevention of early and recurrent ALRIs and thus preservation of lung health during the infant's vulnerable period when lung growth is maximum. The multicentre nature of our study increases the generalisability of its future findings and is complemented by assessing the microbiological and immunological outcomes in a subset of infants. Clinical Trial Registration: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=374381, identifier: ACTRN12618000150246.Anne B. Chang, Maree Toombs, Mark D. Chatfield, Remai Mitchell, Siew M. Fong, Michael J. Binks ... at al

Suppressor of cytokine signalling-3 at pathological levels does not regulate lipopolysaccharide or interleukin-10 control of tumour necrosis factor-α production by human monocytes

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that suppresses the production of tumour necrosis factor-α (TNF-α) by monocytes and macrophages. Suppressor of cytokine signalling-3 (SOCS3), a negative regulator of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, is induced following IL-10 exposure but recent studies in mice suggest that SOCS3 only targets gp-130-dependent signal transduction pathways. Understanding the signalling pathways responsible for IL-10-mediated effects in primary human monocytes is relevant to human inflammatory disease and necessary for the identification of potential therapeutic targets. An adenoviral transfection system was used to express different levels of SOCS3 (quantified experimentally with its tag green fluorescent protein (GFP)) with the aim of investigating the role of SOCS3 in LPS-induced and IL-10-mediated suppression of TNF-α production by non-transformed human monocytes. SOCS3 over-expression had no effect on TNF-α mRNA levels induced by LPS or LPS plus IL-10, or on IL-10 phosphorylation of STAT3, STAT1 and ERK1/2. When data from all donors were combined, adenoviral overexpression of SOCS3 significantly reversed the suppressive effects of IL-10 on LPS-induced TNF-α production after 2 hr. However, there was a direct correlation between mean GFP intensity (extent of viral infection) and extent of reversal of IL-10's inhibitory effects. Physiological levels of SOCS3 detected in IL-10-exposed human monocytes had no effect on LPS-induced TNF-α production. Although overexpression of SOCS3 to supraphysiological levels transiently antagonized the regulatory properties of IL-10 by a post-transcriptional mechanism, these findings suggest that under pathological conditions SOCS3 does not control LPS-activation or the anti-inflammatory properties of IL-10 in primary human monocytes