HIV co-infection negatively impacts MTB-specific CD4+ functional cell subsets secreting IFN-γ and IL-2.

<p>Graphs show frequency and median of CD4+ cell functional subsets secreting IFN-γ and IL-2 (top row) and IL-2 only (middle row) and IFN-γ, IL-2 and TNF-α (bottom row) in response to overnight stimulation with PPD (left column) and MTB-peptides (right column). p values on graphs show Mann-Whitney U tests for HIV-infected vs. HIV-uninfected (n = 34). Closed circles represent those with TB, and open circles those with LTBI with or without HIV co-infection.</p

Association of PD-1 expression on CD4+ MTB-specific T-cells secreting IFN-γ with HIV VL.

<p>Graphs show Spearman’s rank correlation of HIV VL with the percentage of cells expressing PD-1, for PPD-specific CD4+ cells secreting IFN-γ-only (top left), IFN- γ and TNF-α (top right) and IFN-γ, TNF-α and IL-2 (bottom left). Those without a positive response in the relevant functional subset were excluded. Those with TB/HIV (closed circles) and LTBI/HIV (open circles) are shown.</p

HIV VL is negatively correlated with the proportion of CD4+ MTB-specific T-cells secreting IFN-γ and IL-2.

<p>Graph shows Spearman’s rank correlation of proportion of PPD-specific CD4+ IFN-γ and IL-2-dual-secreting cells with HIV VL. Participants with both active TB/HIV (closed circles) and LTBI/HIV (open circles) and a positive response to PPD are shown (n = 16).</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 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 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

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

Example of a 2D-DIGE image (pI 6–9) depicting differences in protein abundance between sarcoidosis and healthy control spleen tissue.

<p>Example of a 2D-DIGE image (pI 6–9) depicting differences in protein abundance between sarcoidosis and healthy control spleen tissue.</p

Change in levels of fractalkine in PBMCs following <i>in vitro</i> infection with RV16 and RV1B.

<p>Soluble fractalkine protein was measured in cell supernatants from PBMCs obtained from (A) non-asthmatic (n = 15) and (B) asthmatic (n = 15) subjects and compared between subject groups at 8hrs post infection (C). Fractalkine mRNA expression was measured in PBMC cell lysate cDNA obtained from (D) non-asthmatic and (E) asthmatic subjects. The results are expressed as mean ± SEM. Protein data were analysed by one-way ANOVA with Bonferroni post-test and mRNA by Kruskal Wallis with Dunn’s post test (*<i>P</i><0.05, ***<i>P</i><0.001).</p

Change in soluble fractalkine in BAL fluid during experimental <i>in vivo</i> RV16 infection, related with upper respiratory symptom scores.

<p>Soluble fractalkine protein was measured in filtered BAL fluid collected at baseline and day 4 post RV16 infection from non-asthmatic (n = 10), mild-asthmatic (n = 11) and moderate-asthmatic (n = 14) subjects. (A) Data is presented as soluble fractalkine (pg/mL) per subject and horizontal bars for median levels for each group in BAL fluid obtained at baseline and day 4. Data were analysed within groups by Wilcoxon-matched pairs signed rank tests and between groups by Mann Whitney U test, *P<0.05. (B) Levels of fractalkine in BAL fluid on Day 4 were correlated with peak upper respiratory symptom scores for each subject infected using Pearson’s correlation (r = 0.289, <i>P</i> = 0.098).</p