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editorial
. 2012 Jul;92(1):1–4. doi: 10.1189/jlb.0312130

Editorial: Macrophage heterogeneity and responses to influenza virus infection

Suki M Y Lee *,, Isabelle Dutry , J S Malik Peiris *,†,1
PMCID: PMC3975085  PMID: 22745457

Abstract

Discussion on the impact of microenvironments on macrophage permissiveness to influenza virus infection and to the innate immune responses elicited by such infection.

Keywords: alveolar macrophage, monocyte, lung, pathogenesis, H5N1, cytokine


Macrophages play a central role in innate immune responses and are found in abundance in lungs of patients with fatal pneumonia caused by avian influenza H5N1. Respiratory epithelial cells, alveolar epithelial cells, and lung endothelial cells are targets for virus infection (reviewed in ref. [1]). It is therefore relevant to study influenza virus tropism, replication competence, and innate immune responses in physiologically relevant macrophages.

In this issue of JLB, Friesenhagen et al. [2] report a lack of productive virus replication (i.e., infectious virus released into cell culture supernatant) in macrophages infected in vitro with HPAI viruses H5N1 and H7N1 or seasonal influenza virus H1N1 (PR8). Furthermore, the HPAI H5N1 and H7N7 viruses elicited weaker innate immune responses compared with H1N1 (PR8). They suggest that the HPAI viruses evade innate immune responses, including type 1 IFN responses. These results differ in some respects with previous data.

Earlier studies comparing influenza A H1N1 and H5N1 virus infection in primary human MDMs showed that both viruses replicated productively in these cells and that HPAI H5N1 viruses induced a more potent proinflammatory response than seasonal H1N1 viruses [3, 4]. On the other hand, seasonal, pandemic 2009 and avian H5N1 influenza virus replication and proinflammatory cytokine responses were much poorer in AMs when compared with MDMs [5, 6]. AMs are resident in the lung and have a distinct phenotype induced by the lung microenvironment (interactions with epithelial cells, surfactant proteins, GM-CSF, etc.) [7].

What accounts for these differences? The conditions used to culture peripheral MDMs can polarize these cells into different phenotypes—the classically activated macrophages (M1) and the alternatively activated macrophages (M2). The concept of polarization and plasticity has been applied with more or less stringency to illustrate macrophage phenotypic variation in vitro and in vivo [7] and is still undergoing refinement. The classically activated macrophages are induced with GM-CSF and IFN-γ or LPS. They are proinflammatory and mediate a Th1 response. Alternatively activated macrophages mediate a Th2 response and are induced by differentiation in M-CSF in the presence of IL-4/IL-13 (M2a), immune complexes (M2b), or IL-10 (M2c) cells, each of them expressing different phenotypes in vitro. However, it is not clear if any of these macrophage subsets, differentiated from peripheral blood monocytes in vitro, accurately reflect the biological phenotype of AMs.

Studies on the interaction of influenza and macrophages in vitro [16] have used different culture conditions, which may shape their phenotype and polarization and thus, influence the ultimate outcome of the experiment (Table 1). Some of the factors that may affect the cell phenotype include the source and concentration of the serum used in the culture medium, the substrate used for culture (e.g., plastic, Teflon), and the addition of GM-CSF or IFN-γ to the culture medium. In some of these studies, macrophages were differentiated from peripheral blood-derived monocytes in culture medium with autologous human serum on a plastic substrate. Others have allowed the differentiation in the presence of GM-CSF. However, when such macrophages were compared directly with AMs derived from BAL, they differed in their permissiveness and response to influenza viruses [6].

Table 1. Comparison of Relevant Studies Where Influenza Virus Infection of Macrophages Has Been Researched.

Reference Cheung et al. [3] Yu et al. [5] Perrone et al. [4] van Riel et al. [6] Sakabe et al. [8] Monteerarat et al. [10] Geiler et al. [9] Friesenhagen et al. [2]
Source of cells MDM from peripheral blood leukocytes AM from human lung and MDM from peripheral blood leukocytes MDM from peripheral blood leukocytes AM from BAL and MDM from peripheral blood leukocytes MDM from peripheral blood leukocytes MDM from peripheral blood leukocytes MDM from peripheral blood leukocytes MDM from peripheral blood leukocytes
Culture conditions for MDM Plastic dishes in RPMI medium with 5% autologous plasma differentiated for 14 days Plastic dishes in RPMI medium with 5% autologous plasma differentiated for 14 days Serum-free medium with 20% autologous plasma and GM-CSF differentiated for 7 days Teflon flasks in RPMI with 5% human AB serum or 5% FCS with GM-CSF differentiated for 7 days Medium with GM-CSF RPMI with normal human serum and GM-CSF differentiated for 10 days IMDM medium with 10% pooled human serum and GM-CSF differentiated for 14 days Teflon bags in RPMI with 10% human AB serum differentiated for 7 days
Virus strains used H5N1: A/HK/483/97
    A/HK/486/97
    A/Vietnam/3212/04
H5N1: A/HK/483/97 H5N1: A/Thailand/16/04
    A/Thailand/SP/83/04
H5N1: A/Vietnam/1194/04 H5N1: A/HK/483/97
    A/VN/UT31203A/2007
    A/VN/UT3028II/2003
    A/IDN/UT3006/2005
    A/Ck/Miyazaki/K11/2007
H5N1: A range of clade 0, clade 1, clade 2.1, and clade 2.3.4 viruses used H5N1: A/Thailand/Kan-1/04 H5N1: A/Thailand/Kan-1/04
H1N1: A/HK/54/98 H1N1: A/HK/54/98 H1N1: A/South Carolina/1/18
    A/Texas/36/91
H3N2: A/NL/213/03 H1N1: A/Kawasaki/UTK-4/2009 H1N1: Four viruses used H1N1: A/New Caledonia/20/99 H1N1: PR8
pH1N1: A/NL/602/09 H3N2: A/Kawasaki/UTK-20/2008 H3N2: Three viruses used (see paper for details) H3N2: A/California/7/04 H7N7: A/FPV/Bratislava/79
Productive virus replication detected Yes for all AM: Yes for H5N1; marginal for H1N1 Yes for all AM: No for all pH1N1: A/California/04/2009 pH1N1: A/HH/01/09
MDM: Yes for all MDM: Yes for H3N2 and H5N1; no for pH1N1 Yes for all; levels of replication differed among virus strains Yes for all; levels of replication differed among virus strains Yes for all No for all
Evidence for more potent cytokine/chemokine responses in H5N1 virus-infected cells MDM: Yes MDM: Yes MDM (GM-CSF): Yes MDM: Yes MDM (GM-CSF): Virus strain-dependent MDM (GM-CSF): Virus strain-dependent MDM (GM-CSF): Yes; H5N1 induced cytokines similar to H3N2 but much higher than H1N1 and pH1N1 MDM: No
AM: Equivocal Responses much less than seen in MDM AM: No Responses much less than seen in MDM H1N1 induced cytokines much higher than H5N1 and H7N7

pH1N1, Pandemic H1N1.

Although HPAI H5N1 viruses in general appear to be more potent inducers of proinflammatory cytokines compared with seasonal influenza viruses in MDMs, there is variation between different H5N1 virus strains, and some H5N1 strains induce cytokines to a similar extent of seasonal influenza viruses [8, 10]. Furthermore, there is variation between donors in the intensity of the cytokine/chemokine response.

Some of these experimental factors may explain the differences in the results observed by Friesenhagen et al. [2] with others (Table 1). The strain of H5N1 virus A/Thailand/KAN-1/2004, which they use, has also been found by others to be a poor cytokine inducer [10]. The cytokine induction phenotype of HPAI H7N7 has not been investigated in detail by others, but our own unpublished data suggest that H7N7 viruses are not potent inducers of proinflammatory cytokines, which is in agreement with the findings of Friesenhagen and colleagues[2]. Their finding that H5N1, H7N7, and H1N1 fail to replicate productively in MDM differs from that of others and is more akin to what has been observed for some viruses in AMs. Differences in experimental strategy (e.g., virus inoculum not removed after infection of cells) may also contribute to the different conclusions by making it more difficult to detect low-level virus replication.

This review of influenza virus–macrophage interactions highlights the importance of defining the physiological state and relevance of the macrophages used for experimental study. It is clear that AMs differ dramatically from MDMs in their response to viral infection; they are generally less permissive to virus and release less innate immune mediators in response to infection. However, lungs of patients with severe influenza caused by viruses, such as H5N1, have massive infiltration with macrophage-like (CD68-positive) cells. Some of these cells are likely derived from infiltration of peripheral blood monocytes [7]. If so, are these newly infiltrated MDMs as docile as resident AMs, or do they retain the MDM phenotype? Which (if any) of the “alternatively activated” macrophages generated in vitro (see above) physiologically and phenotypically resemble AMs, lung interstitial macrophages, or newly infiltrating macrophages in the infected lung? The influenza-infected lung will have an altered cytokine and chemokine milieu, and what effect does this have on the resident AM and on newly recruited monocytes? It has been reported previously that macrophages may switch from one subset to another depending on their environment [7]. It has also been reported that cross-talk between innate immune mediators released from virus-infected macrophages can amplify and broaden cytokine responses in alveolar epithelial cells, thereby amplifying mediator cascades [1].

As Friesenhagen and colleagues [2] point out, alveolar epithelial cells and endothelial cells may be at least as, if not more pathophysiologically, important in this context. HPAI H5N1 viruses and seasonal viruses efficiently replicate in type I and type II alveolar epithelial cells. In comparison with seasonal influenza viruses, HPAI H5N1 viruses are more potent inducers of IL-6, RANTES, MCP-1, and IFN-inducible protein 10 in these cells [1, 5]. HPAI H5N1, but not seasonal H1N1, productively replicated in polarized, differentiated lung microvascular endothelial cells [11, 12] and dysregulates innate immune responses [13]. There is a need for a systematic study of these complex interactions to understand their implications for disease pathogenesis.

ACKNOWLEDGMENTS

The authors' research has been supported by research grants from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project No. AoE/M-12/06), and the U.S. National Institute of Allergy and Infectious Diseases (Contract HSN266200700005C).

SEE CORRESPONDING ARTICLE ON PAGE 11

AM
alveolar macrophage
HPAI
highly pathogenic avian influenza
MDM
monocyte-derived macrophage
PR8
A/Puerto Rico/8/34 virus

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