The impact of respiratory infections and probiotic use on the nasal microbiota of frail residents in long-term care homes

Background Residents in long-term care (LTC) homes, who tend to be of advanced age and frail, are at increased risk of respiratory infections. The respiratory microbiota is known to change with age, but whether these changes contribute to the risk of infection is not known.Aim Our goal was to determine how the nasal microbiota of frail older adults changes during symptoms of influenza-like illness (ILI) and how this may be impacted by enrollment in a placebo-controlled trial testing the feasibility of administering a Lactobacillus rhamnosus GG probiotic to prevent respiratory infection (2014–2017).Methods The microbiome of the nasal (mid-turbinate) of 150 residents of LTC homes was interrogated using 16S rRNA gene sequencing.Results We identified a diverse and individualized microbiota which could be separated into 9 distinct clusters based on Bray Curtis distances. Samples collected during symptoms of influenza-like illness (ILI) differed statistically from those collected pre- and post-cold and influenza season, and we observed decreased temporal stability – as measured by movement between clusters – in individuals who experienced ILI compared to those who did not.Conclusions The use of probiotics decreased ILI-induced changes to the microbiota; however, it is not clear whether this decrease is sufficient to prevent respiratory illness

An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses

Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions

Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking.

The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease

Cigarette smoke attenuates the nasal host response to Streptococcus pneumoniae and predisposes to invasive pneumococcal disease in mice.

Streptococcus pneumoniae is a leading cause of invasive bacterial infections, with nasal colonization an important first step for disease. While cigarette smoking is a strong risk factor for invasive pneumococcal disease, underlying mechanisms remain unknown. This is partly due to a lack of clinically relevant animal models investigating nasal pneumococcal colonization in the context of cigarette smoke exposure. We present a model of nasal pneumococcal colonization in cigarette smoke-exposed mice and document, for the first time, that cigarette smoke predisposes to invasive pneumococcal infection and mortality in an animal model. Cigarette smoke increased the risk of bacteraemia and meningitis without prior lung infection. Mechanistically, deficiency in IL-1α or PAFR, an important host receptor thought to bind and facilitate pneumococcal invasiveness, did not rescue cigarette smoke-exposed mice from invasive pneumococcal disease. Importantly, we observed cigarette smoke to attenuate nasal inflammatory mediator expression, particularly that of neutrophil recruiting chemokines, normally elicited by pneumococcal colonization. Smoking cessation during nasal pneumococcal colonization rescued nasal neutrophil recruitment and prevented invasive disease in mice. We propose that cigarette smoke predisposes to invasive pneumococcal disease by suppressing inflammatory processes of the upper respiratory tract. Given that smoking prevalence remain high worldwide, these findings are relevant to the continued efforts to reduce invasive pneumococcal disease burden