IFN-β induces IL-15 in macrophages and rhinovirus induction is via IFN-αβ receptor signalling.

<p>(A) THP-1-derived macrophages (THP-1) or monocyte-derived macrophages were stimulated with diluent control (0), or recombinant IFN-β at concentrations of 10, 100 and 1000 IU/mL, supernatants harvested at 72 hours and IL-15 quantified by ELISA. Means and SEM from at least three independent experiments (performed in triplicate) are shown. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001, compared with diluent control. (B) THP-1-derived macrophages and (C) monocyte-derived macrophages were pre-treated for 1 hour with IFN-αβ receptor blocking antibody (anti-IFNAR) or isotype control (both at 5 µg/mL), the same concentration of antibody was added to the medium after infection with RV16, supernatants were harvested at 24 hours (C) and 72 hours (B) and IL-15 quantified by ELISA. Means and SEM from at least four independent experiments (performed in triplicate) are shown. ** <i>P</i><0.01 and *** <i>P</i><0.001 compared with isotype control. (D) Lysates of THP-1-derived macrophages incubated with medium alone (medium), or stimulated with IFN-β (IFN-β) at 1000 IU/mL or RV16 (RV16) were analysed for the presence of IRF-1 by Western blot at 4, 8 and 24 hours. A representative image of three independent experiments with similar results is shown. (E) Monocyte-derived macrophages treated as in (C) were lysed after 24 hours and analysed for IRF-1 by Western blot. RV16 induction of IRF-1 protein over medium control (medium) was clearly inhibited by prevention of αβ IFN signalling with an IFN-αβ receptor blocking antibody (anti-IFNAR), but not by isotype control (IgG control), or diluent alone (control). A representative image of three independent experiments with similar results is shown.</p

Rhinovirus induction of IL-15 release from alveolar macrophages; induction is inversely related to severity of lower respiratory symptoms following rhinovirus infection <i>in vivo.</i>

<p>(A) BAL cells were collected at bronchoscopy from normal (circles, <i>n</i> = 10) or asthmatic (squares, <i>n</i> = 7) subjects. Cells were incubated for 48 hours with medium alone (open symbol, medium) or live rhinovirus (closed symbol, RV16) and IL-15 release into supernatants assessed by ELISA. Bars are median values, * <i>P</i><0.05, RV16 vs medium and ** <i>P</i><0.01 RV16 asthmatic vs. RV16 normal subjects. (B) Levels of IL-15 released <i>ex vivo</i> in RV16 infected cultures from normal (closed circles, <i>n</i> = 8) and asthmatic (closed squares, <i>n</i> = 6) subjects were significantly related to severity of total lower respiratory symptoms during the 2 weeks following experimental infection with RV16 <i>in vivo</i>.</p

IFN-β and IFN-α are induced by rhinovirus infection of macrophages via NF-<i>κ</i>B-dependent mechanisms.

<p>(A–B) Monocyte-derived macrophages were infected with RV16 (closed squares) or incubated with medium alone (open squares) at time 0, supernatants were harvested at 4, 8, 24 and 48 hours and levels of IFN-β (A) and IFN-α (B) quantified by ELISA. Means and SEM from at least four independent experiments (performed in triplicate) are shown. (C–D) Monocyte-derived macrophages were exposed to medium alone, UV-inactivated RV16 (UV RV16) or infected with RV16, RV9 or RV1B, cultured for 24 hours, supernatants harvested and IFN-β (C) and IFN-α (D) quantified by ELISA. Means and SEM from at least five independent experiments (performed in triplicate) are shown. <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 for live virus compared to medium, and ## <i>P</i><0.01 for live RV16 compared to UV-inactivated RV16. (E–F) Monocyte-derived macrophages were pre-treated for 1 hour with an inhibitor of NF-<i>κ</i>B activation (the NF-<i>κ</i>B inhibitor AS602868 5 µM) or diluent control, before infection with RV16. The same concentration of drug/diluent was added to the medium after infection. Supernatants were harvested at 24 hours and IFN-β (E) and IFN-α (F) quantified by ELISA. Means and SEM from at least five independent experiments (performed in triplicate) are shown. ** <i>P</i><0.01 for live virus infected cells, NF-<i>κ</i>B inhibitor compared to diluent control. G. BAL cells from the baseline bronchoscopy of normal (circles, <i>n</i> = 9) or asthmatic (squares, <i>n</i> = 7) subjects were incubated for 48 hours with medium alone (open symbol, medium) or live rhinovirus (closed symbol, RV16) and IFN-α and IFN-β release into supernatants assessed by ELISA. Bars are median values, ** <i>P</i><0.01, RV16 vs medium.</p

Rhinovirus infection induces IL-15 protein and mRNA production in human monocyte-derived macrophages.

<p>(A) THP-1-derived macrophages and (B) peripheral blood monocyte-derived macrophages were infected with RV16 (closed squares) or incubated with medium alone (open squares) at time 0. Supernatants were harvested at 4, 8, 24, 48 and 72 hours and levels of IL-15 released determined by ELISA. (C) THP-1-derived macrophages were exposed to major group rhinovirus (RV16 and RV9), minor group rhinovirus (RV1B), medium alone and UV-inactivated RV16 (UV RV16) at time 0 and supernatants harvested at 72 hours. (D) THP-1-derived macrophages were infected as for (A). Total RNA was extracted from cell lysates at 4, 8, 24, 48 and 72 hours post-infection. IL-15 mRNA was quantified by PCR and results normalised to constitutive 18S ribosomal RNA and expressed as fold induction over medium alone. Mean and SEM from at least four independent experiments (performed in triplicate) are shown. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 for live virus compared to medium, and # <i>P</i><0.05 for live RV16 compared to UV-inactivated.</p

Microbiome balance in sputum determined by PCR stratifies COPD exacerbations and shows potential for selective use of antibiotics

BACKGROUND: While a subgroup of patients with exacerbations of chronic obstructive pulmonary disease (COPD) clearly benefit from antibiotics, their identification remains challenging. We hypothesised that selective assessment of the balance between the two dominant bacterial groups (Gammaproteobacteria (G) and Firmicutes (F)) in COPD sputum samples might reveal a subgroup with a bacterial community structure change at exacerbation that was restored to baseline on recovery and potentially reflects effective antibiotic treatment. METHODS: Phylogenetically specific 16S rRNA genes were determined by quantitative real time PCR to derive a G:F ratio in serial sputum samples from 66 extensively-phenotyped COPD exacerbation episodes. RESULTS: Cluster analysis based on Euclidean distance measures, generated across the 4 visit times (stable and exacerbation day: 0,14 and 42) for the 66 exacerbation episodes, revealed three subgroups designated HG, HF, and GF reflecting predominance or equivalence of the two target bacterial groups. While the other subgroups showed no change at exacerbation, the HG cluster (n = 20) was characterized by G:F ratios that increased significantly at exacerbation and returned to baseline on recovery (p<0.00001); ratios in the HG group also correlated positively with inflammatory markers and negatively with FEV1. At exacerbation G:F showed a significant receiver-operator-characteristic curve to identify the HG subgroup (AUC 0.90, p<0.0001). CONCLUSIONS: The G:F ratio at exacerbation can be determined on a timescale compatible with decisions regarding clinical management. We propose that the G:F ratio has potential for use as a biomarker enabling selective use of antibiotics in COPD exacerbations and hence warrants further clinical evaluation

Cluster analysis of qPCR-determined G:F ratios reveals three subgroups with different patterns of change through exacerbation.

<p><b>(A)</b> Heatmap representing the clustering of exacerbation episodes based on G:F ratio pattern across the four visit times. Blue shows Firmicute dominance and yellow Gammaproteobacterial dominance. <b>(B)</b> Changes in G:F across visit times. Mean ±SD. Points represent the individual sample G:F ratios.(****) p<0.0001. HF = High Firmicutes, HG = High Gammaproteobacteria and GF = Balanced G:F.</p

A single G:F ratio assay at exacerbation identifies membership of the HG cluster.

<p>Abbreviations G—Gammaproteobacteria; <i>M</i>. <i>cat–M</i>. <i>catarrhalis</i>; <i>H</i>. <i>inf–H</i>. <i>influenzae</i>; F–Firmicutes; <i>S</i>. <i>pneum–S</i>. <i>pneumoniae</i>; nph—neutrophils; eos–eosinophils.</p

Comparison of patient characteristics between G:F clusters.

<p>Comparison of patient characteristics between G:F clusters.</p

Comparison of inflammatory markers between the three clusters.

<p>Comparison of inflammatory markers between the three clusters.</p

Patient characteristics.

<p>Patient characteristics.</p