Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages

Abstract

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors.IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

Keywords: IL-1α; MARCO; MPI cells; adenovirus; cGAS; cytokines; innate immunity; macrophages; scavenger receptor.

FIG 1

Efficiency of Ad infection in different cell types. (A) GFP expression was analyzed in different cell types 16 h p.i. with AdGFP by fluorescence (top) and light (phase-contrast; bottom) microscopy. Bars, 50 µm. (B) FACS analysis of GFP expression in infected cells at the time points indicated. The value at the top of each graph is the frequency of GFP⁺ cells at 16 h p.i., and the gate indicates GFP⁺ cells.

FIG 2

Early immune activation of MPI cells and BMMs p.i. with AdGFP. (A) Western blot analysis of the cytoplasmic (p-p38, p-IRF3) and nuclear (NF-κB p65) cell fractions at the time points indicated. (B) Cytokine production was measured by ELISA in cell-free supernatants at the time points indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 3

Ad-induced cytokine production in MPI cells is independent of TLR2 and TLR4 but dependent on cGAS. (A) IL-6 induction in WT and TLR2/TLR4^−/− MPI cells p.i. with WT Ad2 or mutant Ad2 ts1. (B) Cytokine induction in cGAS knockdown MPI cells inoculated with AdGFP or mock infected for 16 h.

FIG 4

Ad-induced IL-1 production in MPI cells and BMMs. (A) Induction of IL-1α and IL-1β in cell-free supernatants of MPI cells and BMMs after Ad infection at the time points indicated. (B) Western blot analysis of MPI and BMM cell lysates at the time points indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 5

Ad transduction is strongly reduced by blocking MARCO but independent of serum factors. (A) GFP expression of cells infected with AdGFP in the presence of a MARCO-blocking (right) or isotype antibody (left). Scale bars, 100 µm. Top, fluorescence microscopy; bottom, phase-contrast microscopy. (B) Cytokine production of AdGFP-infected cells without antibody (control) and with a MARCO-blocking antibody or an isotype control. (C) GPF expression (top, fluorescence microscopy and corresponding phase-contrast microscopy) and IL-6 production (bottom) of AdGFP-infected MPI cells in the presence or absence of FCS. Scale bars, 100 µm. n.s., no statistically significant difference.

FIG 6

Effect of MARCO and SR-A deficiency on GFP expression and IL-6 production in macrophages infected for 18 h with AdGFP. (A) Fluorescence microscopy (top) and corresponding phase-contrast microscopy (bottom) of infected MARCO^−/− and WT MPI cells. Scale bars, 50 µm. (B) IL-6 concentrations in supernatants of mock-infected and infected MARCO^−/− and WT MPI cells. (C) FACS comparison of MARCO^−/− (gray-filled histogram) and WT (open histogram) MPI cells and BMMs at 16 h p.i. with AdGFP. (D) Fluorescence microscopy (top) and corresponding phase-contrast microscopy (bottom) of mock-infected and infected AMs from different knockout mice. Scale bars, 100 µm. (E) IL-6 concentrations in supernatants from mock-infected and infected AMs obtained from different knockout mice.

FIG 7

GFP expression and IL-6 response of peritoneal cells from naive WT and MARCO^−/− mice infected with AdGFP in vitro at 5 (top) and 16 (bottom) h p.i. Left, GFP expression in F4/80⁺ cells (histograms). Lines: black continuous, MARCO^−/− infected; black dotted, MARCO^−/− mock infected; red continuous, WT infected; red dotted, WT mock infected. Right, IL-6 levels in supernatants of peritoneal cells.

FIG 8

Expression of MARCO in RAW 264.7 macrophages increases the infection efficiency of and cytokine response to Ad. (A) Cells infected with AdGFP 16 h after transfection with murine MARCO or control plasmid DNA. Tomato was used as a reporter for successfully transfected cells. Cells were analyzed by FACS at 8 h p.i. (B) IL-6 was analyzed in cell-free supernatants 8 h after AdGFP infection.