Mechanisms underlying differential infection by pandemic H1N1 influenza A virus of human classically activated and alternativelyactivated macrophages

Abstract

Macrophages have well-established roles in the primary response to pathogens and hold essential functions during innate and adaptive immunity. Under activation by different growth factors and cytokines, human monocytes have been shown to differentiate and polarize into two main types of macrophage, classically-activated macrophages (caMφ) and alternatively-activated macrophages (aaMφ), displaying distinct properties and phenotypes. For instance, caMφ secrete pro-inflammatory cytokines, whereas aaM secrete anti-inflammatory cytokines. Additionally, aaMφ displays stronger phagocytic ability and are equipped with different endosomal proteases.

While it has been established that monocyte-derived macrophages can be infected by Influenza A virus, most studies utilized a macrophage population obtained by differentiation in the presence of autologous plasma. My research project aimed at systematically comparing susceptibility of the infection by Influenza A virus to the recently described caMφ and aaMφ.

Here I show that monocytes cultured in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ or in presence of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-4 or IL-10 can be differentiated into distinct populations. According to immunophenotyping results, a distinct expression profile was observed for Cluster of Differentiation (CD) 36, CD86, Mannose Receptor (MR or CD206), and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209) among differentiated macrophages. Except for CD86 expression, my results were in accordance with previous reports and thus allowed me to classify all populations into caMφ (M1 macrophages), and aaMφ (M2a and M2c macrophages).

I then assessed the susceptibility of the above mentioned macrophages to pandemic Influenza A/California/04/2009 H1N1 virus (CA04) infection. My results demonstrate a marked difference, caMφ showing low to moderate permissivity, whereas aaMφ – and in particular M2a macrophages – were consistently highly infected. In contrast, no difference was observed with Influenza A/WSN/1933 H1N1 virus (WSN/33) infection.

Because sialic acids are regarded as the primary receptor for influenza virus, I investigated the cell surface distribution of sialic acids with α2-3 linkage (SAα-2,3) or α2-6 linkage (SAα-2,6) among the population of human macrophages. By using lectin staining with Maackia amurensis lectin (MAL) II and Sambucus nigra lectin (SNA), which bind sialic acids with α2-3 linkage (SAα-2,3) and α2-6 linkage (SAα-2,6) respectively, I found all the monocyte-derived macrophages exhibited a comparable expression of SAα-2,3 and SAα-2,6, which unlikely explain the differential susceptibility to infection by CA04.

In addition to sialic acids, C-type lectins were also proposed to mediate entry of influenza viruses into macrophages. All macrophages expressed CD206 but only M2a expressed CD209. However assay aiming at interfering with CD209 binding (MAb blocking assay or EGTA treatment) did not inhibit pdmH1N1 infection. Surprisingly, infection in presence of EGTA, which is believed to reduce the functional ability of C-type lectins, exacerbated susceptibility of the macrophages.

Altogether my results show that susceptibility to Influenza A virus infection of in vitro differentiated primary human macrophages is unlikely to rely on the sialic acid expression profile and is dependent on viral strain. Further studies are needed to understand what difference from caMφ and aaMφ – either phenotypic and/or biochemical – confer them distinct susceptibilities to some viral subtype/strain of Influenza A.