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. 2020 Mar 25;11:355.
doi: 10.3389/fimmu.2020.00355. eCollection 2020.

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

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Free PMC article

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

Valarmathy Murugaiah et al. Front Immunol. .
Free PMC article

Abstract

The complement system is an ancient innate immune defense mechanism that can recognize molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesized factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV entry, as evident from down-regulation of matrix protein 1 (M1) in H1N1 subtype-infected cells and up-regulation of M1 expression in H3N2-infected A549 cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ~70 kDa), neuraminidase (NA, ~60 kDa), and M1 (~25 kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, and RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. A recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%), and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory responses, independent of its complement-related functions.

Keywords: complement; cytokine storm; factor H; influenza A virus; innate immunity; pseudotyped lentiviral particles; vaccinia virus complement control protein.

Figures

Figure 1
Figure 1
12% (w/v) SDS-PAGE analysis of complement factor H purified from human plasma (A), and a recombinant form of vaccinia virus complement control protein (VCP) expressed in HEK 293FT cells (B). The purified protein was run under reducing conditions: Factor H appears as ~155 kDa band, and VCP was observed at ~35 kDa.
Figure 2
Figure 2
Hemagglutination assay. Inhibition of hemagglutination by purified human factor H was tested on human IAV subtypes. Guinea pig red blood cells were incubated with either PBS or IAV subtypes with and without various concentrations of factor H (20, 10, 5, and 2.5 μg).
Figure 3
Figure 3
Binding between factor H or VCP with H1N1 (A), and H3N2 (B) IAV subtypes were determined via ELISA. Microtiter wells were coated with various concentrations of factor H, VCP or VSV-G (0.625, 1.25, 2.5, and 5 μg/well) in carbonate bicarbonate buffer, pH 9.6 overnight at 4°C. H1N1 or H3N2 virus (1.36 × 106 pfu/ml) was added to each well in the presence of 5 mM CaCl2 and probed with either monoclonal anti-influenza virus H1 or polyclonal anti-influenza virus H3 antibody. VSV-G pseudotyped particles were used as a negative RNA virus control. The data were expressed as mean of three independent experiments done in triplicates ± SEM.
Figure 4
Figure 4
Cell-binding assay to show binding of factor H (A) and VCP (B) to A549 cells challenged with H1N1 and H3N2. A549 cells (1 × 105 cells/ml) were seeded in a 96-well microtiter plate and incubated at 37°C overnight. Decreasing concentrations of factor H and VCP (10, 5, 2.5, and 1.25 μg), pre-incubated with IAV subtypes, were added to the corresponding wells, and incubated at room temperature for 1 h. After removing unbound protein and viral particles, the wells were fixed with 4% v/v paraformaldehyde, and probed with monoclonal anti-influenza virus H1 or polyclonal anti-influenza virus H3 antibodies. BSA was used as a negative control protein. Three independent experiments were carried out in triplicates and error bars expressed as ± SEM.
Figure 5
Figure 5
Far western blot analysis to show factor H (A) and VCP (B) binding with purified H1N1 and H3N2. IAV subtypes (1.36 × 106pfu/ ml) were first run on the SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane. The transferred membrane was then incubated with 10 μg/ml of factor H or VCP. The membrane was probed with either monoclonal mouse anti-human factor H or polyclonal rabbit anti-VCP antibody. Factor H and VCP bound to HA (~70 kDa), NA (~55 kDa), and M1 (~25 kDa) in the case of both IAV subtypes. The identities of factor H and VCP bound IAV glycoproteins were validated using a separate blot that was directly probed with monoclonal anti-HA (C), anti-NA (D), and anti-M1 (E) antibodies.
Figure 6
Figure 6
Replication of IAV subtypes and inflammatory responses of A549 cells. (A) Following entry inhibition, replication of IAV subtypes (H1N1 and H3N2) is affected by factor H and VCP in human A549 cells. M1 expression was monitored in A549 cells challenged with either H1N1 or H3N2 IAV subtypes (MOI 1) at 6 h. A549 cells were challenged with H1N1 and H3N2 pre-incubated with or without factor H and VCP (40 μg/ml). Cell pellets were harvested at 6 h post-infection. 18S rRNA was used as an endogenous control. Significance was determined using the unpaired one-way ANOVA test (**p < 0.01, and ***p < 0.001) (n = 3). (B) Transcription expression profile of cytokines and chemokines produced by A549 in response to H1N1 and H3N2 challenge with and without factor H. 18S rRNA expression was used as an endogenous control. Cells only was used as a calibrator sample to calculate relative quantitation (RQ); RQ = 2−ΔΔCt. Experiments were conducted in triplicates, and error bars represents ± SEM. Unpaired one-way ANOVA test was used calculate the significance (*p < 0.05, **p < 0.01, and ***p < 0.001) (n = 3). (UT, untreated sample; T, treated sample). (C) Expression levels of type I interferon alpha (IFN-α) following 6 h treatments with factor H and VCP. 18S rRNA was used as an endogenous control. RQ = 2−ΔΔCt was used to calculate the RQ value. Significance was determined using the unpaired one-way ANOVA test (**p < 0.01) (n = 3).
Figure 7
Figure 7
Factor H and VCP inhibit transduction of MDCK cells by H1N1-pseudotyped lentiviral particles. Western blotting to show expression of hemagglutinin (HA) in purified H1N1 and H3N2 pseudotyped lentiviral particles (A). HA expression was evident at ~70 kDa. Luciferase reporter activity of H1N1 and H3N2 pseudotyped lentiviral particles-transduced cells with factor H and VCP (B). Significance was determined using the unpaired one-way ANOVA test (**p < 0.01 and ***p < 0.001) (n = 3).

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