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. 2015 Oct;96(10):2951-2960.
doi: 10.1099/jgv.0.000240. Epub 2015 Jul 9.

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype

Gillian M Campbell  1 , Marlynne Q Nicol  1 , Ian Dransfield  2 , Darren J Shaw  1 , Anthony A Nash  1 , Bernadette M Dutia  1
Affiliations

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype

Gillian M Campbell et al. J Gen Virol. 2015 Oct.

Abstract

The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection.

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Figures

Fig. 1.
Fig. 1.. Influenza virus infection of 129Sv/Ev BMDMs. BMDMs cultured from femurs of female 129Sv/Ev mice were mock-infected or infected with A/WSN/33 and stained with antiserum to viral antigens. (a) Mock-infected cells, (b) 1 h p.i., m.o.i. 10, (c) 6 h p.i., m.o.i. 10 and (d) 48 h p.i., m.o.i. 10.
Fig. 2.
Fig. 2.. Treatment of 129Sv/Ev BMDMs with IFN-γ or IL-4 leads to polarization of the macrophages to classical or alternative phenotypes. BMDMs derived from 129Sv/Ev and IFN-γR− / −  mice were treated for 16 h with 1 ng IFN-γ ml− 1 or 4 ng IL-4 ml− 1. (a–c) Expression of Arg-1 (a) or iNOS (c) mRNA was measured by qRT-PCR, or by biochemical assay for Arg-1 activity (b) or iNOS activity (d).
Fig. 3.
Fig. 3.. Alternatively activated macrophages are more readily infected with A/WSN/33 than classically activated macrophages. BMDMs derived from (a–e) 129Sv/Ev and (f–j) IFN-γ R− / −  mice were cultured in medium containing M-CSF alone (a, f) or treated with IFN-γ (b, g) or IL-4 (c, h) for 16 h and infected with 10 p.f.u. A/WSN/33 per cell followed by staining with antiserum to virus antigens. The percentage of cells positive for antigen was quantified at 6 (d, i) and 48 h (e, j) *P < 0.05; ***P < 0.001.
Fig. 4.
Fig. 4.. Alternatively activated BMDMs produce more M1 mRNA than classically activated macrophages. BMDMs were activated with IFN-γ (classically activated) or IL-4 (alternatively active) and infected with 10 p.f.u. A/WSN/33 per cell (WSN). At 48 h p.i., M1 mRNA levels were measured by qRT-PCR. ***P < 0.001.
Fig. 5.
Fig. 5.. Alternatively activated BMDMs are more susceptible to cell death following infection with A/WSN/33, but do not support higher levels of virus replication. BMDMs from 129Sv/Ev and IFN-γR− / −  mice were untreated or activated with IFN-γ or IL-4 and infected with 10 p.f.u. A/WSN/33 per cell (WSN). Cell viability was measured by a CellTiter-Blue Viability Assay at various times after infection. (a) 129Sv/Ev BMDMs and (b) IFN-γR− / −  BMDMs. ***P < 0.001. (c) Virus titres in cell supernatants at 1 and 48 h p.i. were measured by titration on MDCK cells. Results are representative of three independent experiments.
Fig. 6.
Fig. 6.. Expression of pro- and anti-inflammatory markers in classically and alternatively activated macrophages infected with influenza virus A/WSN/33. BMDMs were treated with 1 ng IFN-γ ml− 1 or 4 ng IL-4 ml− 1 for 16 h and then infected with 10 p.f.u. A/WSN/33 per cell (WSN). Cells were harvested at 48 h p.i. and expression of cellular markers was monitored by qRT-PCR. Results are representative of three independent experiments. (a, b) iNOS, (c, d) Arg-1, (e, f) TNF-α and (g, h) CD206. *P < 0.05; **P < 0.005; ***P < 0.001.
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