Virus-induced expression of IFNβ and effect of IFNβ on bacterial phagocytosis.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with <b>(A)</b> H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) or <b>(C)</b> M37 RSV or a UV-irradiated aliquot of virus (UV-RSV) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for IFNβ gene expression by RT-PCR (X31 n = 9, RSV n = 7). MDM were differentiated for 12 d as above before treatment without (NT) or with 50 IU/ml IFNβ for 24 h and <b>(B)</b> cells harvested for CD36 gene expression by RT-PCR (n = 5) or <b>(D)</b> phagocytosis of <i>S</i>. <i>pneumonia</i> was detected after a further 2 h incubation with live bacteria by culture (n = 5). PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM. Data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05, ** p<0.01.</p

Role of CD36 in bacterial phagocytosis by MDM.

<p><b>(A)-(E)</b> MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d before transfection with non-specific siRNA or siRNA specific for CD36 for 24 h before cells were harvested for analysis of (<b>A)</b> CD36 mRNA expression by RT-PCR (n = 4) and <b>(B) & (C)</b> CD36 surface expression by flow cytometry (n = 6) or <b>(D)</b> phagocytosis of <i>S</i>. <i>pneumonia</i> was detected after a further 2 h incubation with live bacteria by culture (n = 4). <b>(E)</b> Total cellular expression of CD36 was assessed by western blot 24 h after transfection. β-actin is used as a loading control. Blot representative of 3 independent experiments. <b>(F)</b> MDM were differentiated for 12 d as above before phagocytosis of <i>S</i>. <i>pneumonia</i> was detected in the presence or absence of 10 μg/ml αCD36 blocking antibody or isotype control (ISO) after a further 2 h incubation with live bacteria by culture (n = 5). PCR data were normalised to β2MG and are expressed as mean fold induction over the not treated (NT) sample ± SEM of n independent experiments. <b>B)</b> Representative flow cytometry histograms of CD36 surface expression in response to transfection are shown. Grey = NT, Blue = HiPerfect (HF), Red = CD36 siRNA. Flow cytometry and phagocytosis data are expressed as means ±SE of n independent experiments. <b>(A) & (C)</b> Data analysed using a paired t-test ** p<0.01. <b>(B) & (D)</b> Data analysed using a Wilcoxon-signed rank test * p<0.05.</p

Effect of influenza infection on bacterial phagocytosis by MDM.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for <b>(A) & (B)</b> influenza NP1 expression (% cells, n = 7) by flow cytometry. Phagocytosis of <i>S</i>. <i>pneumonia</i> was detected in <b>(C)</b> X31-infected (n = 5) MDM after a further 2 h incubation with live bacteria by culture. <b>(A)</b> Representative flow cytometry plot of MDM expressing influenza NP1. Data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05, ** p<0.01.</p

Effects of influenza infection on MDM cytokine expression.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with 500 pfu of H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h in the presence or absence of oseltamivir phosphate (Tam– 10 μM) or concanamycin A (ConA– 10 nM). After washing, media containing inhibitors was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for <b>(A)</b> Viral NP1 expression (% cells, n = 6) by flow cytometry, <b>(B)</b> release of hemagglutinin into supernatants (n = 6), <b>(C)</b> CD36 cell surface expression by flow cytometry (SMFI–n = 6) and <b>(D)</b> IFNβ release by MSD are expressed as percentage of X31-infection (n = 6). Data are expressed as means ±SE of n independent experiments. Data analysed using a Wilcoxon-signed rank test. * p<0.05.</p

Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36

<div><p>Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of <i>Streptococcus pneumoniae</i>, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM) were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV) or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of <i>S</i>. <i>pneumoniae</i> by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNβ gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031) and CD36 gene expression (p = 0.031) by MDM cultured for 24 h in 50IU/ml IFNβ. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNβ production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.</p></div

Effect of viral infection on CD36 expression by MDM.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with <b>(A) & (B)</b> H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) or <b>(C) & (D)</b> M37 RSV or a UV-irradiated aliquot of virus (UV-RSV) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for <b>(B) & (E)</b> CD36 cell surface expression by flow cytometry (specific mean fluorescence intensity–SMFI n = 6) by flow cytometry or <b>(C) & (F)</b> CD36 gene expression by RT-PCR (X31 n = 6, RSV n = 7). PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM. Representative flow cytometry histograms of cell surface CD36 expression in response to <b>(A)</b> influenza infection and <b>(D)</b> RSV infection are shown. Grey = NI, Blue = UV virus, Red = Live virus. Cell surface data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05, ** p<0.01.</p

Effect of influenza infection on scavenger receptor expression by MDM.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for cell surface <b>(A)</b> CD206 expression (n = 5), <b>(B)</b> CD163 expression (n = 5) or <b>(C)</b> CD204 expression by flow cytometry (n = 5). Data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05.</p

Effect of RSV infection on bacterial phagocytosis by MDM.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with M37 RSV or a UV-irradiated aliquot of virus (UV-RSV) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested for <b>(A) & (B)</b> RSV-F protein expression (% cells, n = 10) by flow cytometry or <b>(C)</b> RSV-N gene expression by RT-PCR (n = 7). Phagocytosis of <i>S</i>. <i>pneumonia</i> was detected in RSV-infected (n = 6) MDM after a further 2 h incubation with live bacteria by culture. <b>(A)</b> Representative flow cytometry plot of MDM expressing RSV F-protein. The units for RSV-N gene expression are 2^ΔCT. Data are expressed as means ±SE of n independent experiments and analysed using a Wilcoxon-signed rank test * p<0.05, ** p<0.01.</p