Quantification of infection of human lung explants.

<p>Histograms showing flow cytometry data of viral NP1 (% cells) and PDL1 expression (specific mean fluorescence intensity—SMFI) in epithelial cells <b>(A & C)</b> and macrophages <b>(B & D).</b> Data are expressed as means ±SE of 9 independent experiments. Data analysed using a paired t-test ** p<0.01. **** p<0.0001.</p

Infection of human lung macrophages by X31.

<p>Human lung macrophages were isolated by plate adherence prior to infection with 4000 pfu/ml of 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. Cells were analysed for intracellular viral NP1 expression or cell surface expression of HLA-DR and PD-L1 using flow cytometry. Histograms showing flow cytometry data of <b>A)</b> Viral NP1 expression (% cells), <b>B)</b> HLA-DR expression (specific mean fluorescence intensity—SMFI) and <b>C)</b> PDL1 expression (SMFI) from isolated human lung macrophages are expressed as means ±SE of 4 independent experiments. <b>D)</b> Representative histograms demonstrating increase in PDL1 expression in response to influenza infection are shown. Data analysed using a paired t-test * p<0.05, ** p<0.01.</p

Functional effect of PDL1 on T cell activation.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with 500 pfu/ml of H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. Infected MDM were transferred to a coated ELISpot plate and autologous lymphocytes added and incubated for a further 22 h before IFNγ release was measured using ELISpot. <b>A)</b> 2.5 x 10⁵ monocyte-depleted PBMC co-cultured with 5 x 10⁴ autologous MDM not infected (NI) or treated with X31 or UVX31(n = 5). <b>B)</b> 1 x 10⁵ CD8+ T cells co-cultured with 5 x 10⁴ autologous MDM NI or infected with X31 (n = 6) <b>C)</b> 1 x 10⁵ CD8+ T cells co-cultured with 5 x 10⁴ autologous MDM infected with X31 in the presence of 10 μg/ml anti-PDL1 antibody or isotype control (n = 5). Data are expressed as means ±SE of n independent experiments and analysed using a paired t-test * p<0.05, ** p<0.01.</p

Infection of MDMs by X31.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with 500 pfu/ml of 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. Cells were analysed using flow cytometry and RT-PCR, supernatant was analysed using dot blot. Histograms showing infected MDM expression of <b>A)</b> Viral NP1 expression (% cells n = 7), <b>B)</b> release of hemagglutinin into supernatants (n = 10) <b>C)</b> cell surface HLA-DR expression (specific mean fluorescence intensity—SMFI n = 7) <b>D)</b>, PDL1 expression (SMFI n = 7) <b>E)</b> PDL1 gene expression (n = 6). Data are expressed as means ± SE of n independent experiments and analysed using a paired t-test * p<0.05, ** p<0.01. *** p<0.001.</p

Ex vivo infection of human lung explants.

<p>After resting explanted lung tissue overnight, 1 x 10⁶ pfu/ml H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) was added for 2 h. After washing, media was replaced and the tissue was incubated for a further 22 h followed by collagenase digestion and flow cytometry analysis <b>A)</b> Gating strategy for identification of CD45+HLADR+ macrophages and CD45-EpCAM+ epithelial cells expressing viral NP1 and PDL1 from lung tissue. <b>B)</b> FACS plots demonstrating increases in influenza infection (NP1) with corresponding increases in PDL1 expression. Plots are representative of nine independent experiments.</p

Regulation of PDL1 mRNA expression by influenza infection and IFNβ.

<p><b>A)</b> PDL1 gene expression was assessed in MDM differentiated in the presence of 2 ng/ml GM-CSF for 12 d before incubation with the indicated doses of rhIFNβ for 24 h using RT-PCR (n = 5). <b>B)</b> MDM were differentiated in the presence of 2 ng/ml GM-CSF for 11 d before transfection with non-specific siRNA or siRNA specific for IFNβ in the presence of GM-CSF for 24 h. MDM were then infected with 500 pfu/ml of 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 cells were harvested for RT-PCR analysis of IFNβ and PDL1 mRNA expression (n = 6). PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM of n independent experiments. Data analysed using a paired t-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/ml of 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. Inflammatory cytokine expression was measured by <b>A)</b> real time PCR (IFNα, IFNβ n = 6) and <b>B)</b> ELISA (IFNβ, TNFα, IL-6, IL-8 n = 6) N.D. = not detected. PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM of 6 independent experiments. ELISA data are expressed as means ±SE of 6 independent experiments. Data analysed using a paired t-test. * p<0.05, ** p<0.01.</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 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

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