|Screen||Ex Vivo Macrophage Screen for Control of Viral Infection|
|Common Name||Peritoneal Macrophage|
|Posted On||02/18/2010 12:24 PM|
|Author||Celine Eidenschenk, Sungyong Won|
|Science Writer||Eva Marie Y. Moresco|
In this screen, thioglycolate-elicited peritoneal macrophages from ENU-mutagenized G3 mice are used to discover components important for the early control of mouse cytomegalovirus (MCMV), adenovirus, influenza virus, and Rift Valley Fever virus (RVFV) infections. Each virus used in this screen is titered so that only a small percentage of target cells is infected. After an incubation period during which the virus is replicated and released to infect new cells (except here in the case of adenovirus), the total numbers of virus-infected cells are compared between C57BL/6J control and G3 strains (Figure 1). Strains for which a significantly higher number of cells are infected relative to wild type are identified as potential mutants. In parallel with this screen, the supernatants from virus-infected macrophages are analyzed for tumor necrosis factor (TNF) and type I interferon (IFN) content (Figure 2). These cytokines are induced by most viral infections, and provide another indication of viral control. Compared to in vivo screens in which susceptible animals may die from viral infection (see MCMV Susceptibility and Resistance Screen and In Vivo RVFV Susceptibility Screen), the use of macrophages facilitates more rapid recovery of the mutant strain, since the animal is kept alive and only macrophages are taken for testing. However, because the full complement of cell types found in the intact organism is lacking, the macrophage screen cannot reveal all genes required for viral control.
Macrophages are critical in the defense against infection, functioning as phagocytes as well as interacting with and stimulating other components of the immune system. They are important factors in the pathogenesis and outcome of viral infections, and are restrictive for some viruses, such as herpes simplex virus and vesicular stomatitis virus (1). In the case of MCMV, peritoneal macrophages are permissive for its infection, support MCMV replication, and release plaque-forming virus into the culture medium to infect other cells (2;3). Latently infected mice harbor MCMV in peritoneal macrophages for extended periods of time, from which virus can be recovered upon macrophage activation (2;4). It has been proposed that exacerbation of latent MCMV infections in vivo may be triggered by the activation of macrophages (2).
Adenoviruses are a frequent cause of acute upper respiratory tract infections, as well as eye, gastrointestinal tract and bladder infections. In the respiratory tract, adenoviruses infect epithelial cells, causing an early, rapid recruitment of neutrophils, macrophages and monocytes which initiate the inflammatory response. Macrophages do not express the 46-kDa cell surface receptor CAR (coxsackie/adenovirus receptor) for the adenovirus fiber knob, which mediates high-affinity binding of the virion to the host cell, and thus internalize virus 100- to 1000-fold less efficiently than lung epithelial cells (5). Instead, adenoviruses utilize CD46 as their primary attachment receptor on macrophages (6). Adenoviruses have been intensively studied as a tool for gene transfer to a variety of cell types. This screen utilizes the human adenovirus 5 serotype expressing the fiber protein from serotype 16 (Ad5-F16), which is able to infect mouse macrophages. As this adenoviral vector is non-replicating, an abnormally elevated fraction of cells expressing the GFP tag must reflect enhanced infectivity or greater permissiveness of the cells in expressing the transfected marker rather than propagation of the vector.
Peritoneal macrophages may also be infected by influenza, although with lower efficiency compared to alveolar macrophages that normally encounter the virus in vivo (7). Once infected, at least one round of viral replication occurs followed by apoptosis of the infected macrophages, and their phagocytosis by surrounding uninfected macrophages and dendritic cells (8-10). Phagocytosis of infected cells by alveolar macrophages is an important mechanism limiting the propagation of influenza in vivo (9). For this screen, a mouse-adapted human influenza A virus (PR8 strain) is used (see Influenza Resistance Screen).
Mice with mutations in known genes were used to test the requirement for interferon (IFN) and Toll-like receptor (TLR) signaling in the control of MCMV, influenza and adenovirus by macrophages (Figure1 and Table 1). Relative to C57BL/6J control macrophages, increased percentages of Ifnar-/- and Stat1domino/domino macrophages are infected by each of the viruses, demonstrating a requirement for type I IFN signaling in viral control, consistent with published data (11-14). In contrast to type I IFN signaling, IFN-γ signaling is dispensable for control of all three viruses by macrophages. For MCMV, this result is in contrast to studies indicating a role for IFN-γ in macrophage activation during MCMV infection in vivo (15). However, the situation is complex, as IFN-γ-mediated macrophage activation has been shown to be negatively regulated by type I IFN (16). IFN-γ signaling is important for adenovirus control in respiratory airway epithelial cells (17), but its role in macrophages has not been reported. IFN-γ is not required for the control of influenza A virus in vivo (18;19). Interestingly, signaling through TLRs is not required for control of MCMV, influenza, or adenovirus in macrophages, as demonstrated by the infection of similar percentages of cells from wild type mice or mutants deficient in TLR3, TLR7, TLR9, Unc93b1 (3d), or MyD88 (pococurante) (Table 1). These findings suggest redundant macrophage sensing mechanisms for these viruses, and/or differing requirements for IFN and TLR signaling in macrophages versus other cell types.
RVFV (genus Phlebovirus, family Bunyaviridae), a mosquito-borne, hepatotropic virus, causes epidemics in ruminants of sub-Saharan Africa but also infects humans, where it typically results in flu-like illness, but may cause myalgia, hemorrhagic fever, ocular disease, encephalitis, and death (20). RVFV has been shown to infect the human monocytic cell line U937, and the mouse macrophage cell line J774.1 (21;22). The NSs protein of RVFV blocks transcription by the transcription factor TFIIH, resulting in suppression of type I IFN production and consequently, innate immune responses (23;24). IFN-α and IFN-γ therapies prevent or greatly suppress RVFV disease in rhesus monkeys (25;26). Accordingly, peritoneal macrophages lacking type I IFN signaling as a result of mutations in either IFNAR1 (macro-1), STAT1 (domino), or IRF1 (Endeka), cannot control RVFV infections as efficiently as wild type cells (Figure 3 and Table 1). In addition, TLR signaling is dispensable for control of RVFV in macrophages (Table 1). Two recombinant viruses are used in screening: a mutagen-attenuated RVFV vaccine strain containing the complete genome (arMP-12) (27), and a mutant virus derived from arMP-12 in which the gene encoding the viral NSs protein has been replaced with the coding sequence for GFP (arMP-12-delNSs/GFP).
|Reagents and Solutions|
Brewer’s thioglycolate medium, 4%
4% (w/v) Brewer’s thioglycolate medium powder (BBL Microbiology Systems, Cockeysville, MD) is added to distilled water pre-warmed to 37°C. Solution is autoclaved to sterilize and stored away from light.
PEC recovery solution
Hepes-buffered saline solution (Gibco, Invitrogen, Carlsbad, CA )
5% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA)
200 IU/mL penicillin (Gibco)
200 mg/mL streptomycin (Gibco)
Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)
5% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin
Phosphate buffered saline (PBS)
5% (v/v) heat-inactivated fetal bovine serum
Perm/Wash Buffer (BD Biosciences, #554723)
Perm/Fix Solution (BD Biosciences, #554722)
α-hemagglutinin antibody and appropriate FITC-conjugated secondary antibody
α-RVFV nucleoprotein (N) rabbit polyclonal antibody (28), and α-rabbit FITC-conjugated secondary antibody
Peritoneal exudate cell (PEC) isolation
Viral infections and FACS analysis
The MCMV-GFP strain, generated by insertion of the GFP gene into the MCMV bacterial artificial chromosome plasmid pSM3fr (29), is from Dr. Chris Benedict (La Jolla Institute of Allergy and Immunology, La Jolla, CA). The virus is propagated on mouse embryonic fibroblasts and purified as described (30). Titres are determined by plaque assay on mouse embryonic fibroblasts.
Ad5-F16-GFP is from Dr. Glen Nemerow (The Scripps Research Institute, La Jolla, CA), and was constructed as described (31). The virus is grown in 293 cells, purified by CsCl gradient, and particle number is calculated as described (31;32).
Influenza virus is grown in chicken eggs and titred using a standard hemagglutination assay (Influenza Resistance Screen).
The RVFV strain arMP-12 was recovered from cDNA through a reverse genetics system (33). arMP-12-delNSs/GFP was generated from the parental arMP-12 strain. The viruses are propagated and titred by plaque assay in Vero E6 cells as described (22).
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