Human C1q Regulates Influenza A Virus Infection and Inflammatory Response via Its Globular Domain

Abstract

The Influenza A virus (IAV) is a severe respiratory pathogen. C1q is the first subcomponent of the complement system's classical pathway. C1q is composed of 18 polypeptide chains. Each of these chains contains a collagen-like region located at the N terminus, and a C-terminal globular head region organized as a heterotrimeric structure (ghA, ghB and ghC). This study was aimed at investigating the complement activation-independent modulation by C1q and its individual recombinant globular heads against IAV infection. The interaction of C1q and its recombinant globular heads with IAV and its purified glycoproteins was examined using direct ELISA and far-Western blotting analysis. The effect of the complement proteins on IAV replication kinetics and immune modulation was assessed by qPCR. The IAV entry inhibitory properties of C1q and its recombinant globular heads were confirmed using cell binding and luciferase reporter assays. C1q bound IAV virions via HA, NA and M1 IAV proteins, and suppressed replication in H1N1, while promoting replication in H3N2-infected A549 cells. C1q treatment further triggered an anti-inflammatory response in H1N1 and pro-inflammatory response in H3N2-infected cells as evident from differential expression of TNF-α, NF-κB, IFN-α, IFN-β, IL-6, IL-12 and RANTES. Furthermore, C1q treatment was found to reduce luciferase reporter activity of MDCK cells transfected with H1N1 pseudotyped lentiviral particles, indicative of an entry inhibitory role of C1q against infectivity of IAV. These data appear to demonstrate the complement-independent subtype specific modulation of IAV infection by locally produced C1q.

Keywords: RNA viruses; classical pathway; complement; cytokine storm; human C1q; immune evasion; influenza A virus; innate immunity.

Figure 1

Interaction of H1N1 and H3N2 subtypes of influenza A virus (IAV) with C1q (A), ghA (B), ghB (C) and ghC (D). Decreasing concentrations (5, 2.5, and 1.25 μg) of human C1q and its recombinant globular head modules were coated overnight in a 96-microtiter well plate in carbonate/bicarbonate (CBC) buffer, pH 9.6 at 4 °C. Next, the wells were washed three times with PBS. Then, 20 µL of H1N1 or H3N2 virus (1.36 × 10⁶ pfu/mL) was added to corresponding wells and incubated at 37 °C for 2 h. After removing and washing off the unbound viruses, the wells were probed with primary antibodies (100 μL/well): monoclonal anti-influenza virus H1 or anti-influenza virus H3 (1:5000) antibodies, respectively. VSV-G pseudo-typed lentivirus was used as a negative control. The data were expressed as the mean of three independent experiments carried out in triplicate ± SD. The background was subtracted from all samples. In addition, the absorbance of Maltose Binding Protein (MBP) (5, 2.5, and 1.25 μg) was subtracted from the respective absorbance of the recombinant MBP tagged globular head modules (B–D).

Figure 2

Far-Western blotting analysis to assess C1q binding to individual IAV proteins in the virus lysate, or purified H1N1 (A) and H3N2 (B). H1N1 and H3N2 virus lysates, or recombinant IAV glycoproteins (5 μg/mL) were separated using SDS-PAGE (12% w/v) under reducing conditions, and then transferred onto an activated PVDF membrane. Following blocking with PBS + 5% w/v BSA, the membrane was incubated with 20 μg/mL of C1q. After PBS washes, the membrane was probed with rabbit anti-human C1q antibody (1:1000). C1q bound M1 (~25 kDa), HA (~70 kDa) and NA (~55 kDa) of both IAV subtypes. C1q was also found to bind to the M2 protein of H1N1 alone.

Figure 3

IAV pre–treatment with human C1q (A), ghA (B), ghB (C) or ghC (D) suppresses replication of H1N1-infected A549 cells while upregulating replication in H3N2. mRNA transcript levels of M1 expression of H1N1 and H3N2 IAV subtypes (IAV) (MOI 1) 6 h post-infection in A549 cells were measured. A549 cells were incubated with H1N1 or H3N2, pre–treated with or without human C1q or its recombinant globular head modules (20 μg/mL). Following cell lysis, RNA was extracted and converted into cDNA. M1 expression levels were measured via qRT–PCR using M1 primers to assess IAV replication; 18S was used as an endogenous control. Data are shown as the normalized mean of three independent experiments performed in triplicate ± SEM. C1q (A) was normalized to M1 levels of its control (cells + virus only), and the globular heads (B–D) were normalized to M1 levels of their control (Cells+ virus pre–treated with 20 μg/mL of MBP). Significance was determined using the two–way ANOVA test (* p < 0.05, ** p < 0.01, *** p < 0.001, (n = 3)).

Figure 4

C1q, ghA, ghB, or ghC treatment modulates IAV entry in MDCK cells in a subtype–dependent manner. Matched H1N1 or unmatched H3N2 pseudo–typed lentiviral particles were pre–treated with C1q or its recombinant globular head modules (20 μg/mL). Luciferase reporter activity of MDCK cells transduced with either treated or untreated pseudo–typed lentiviral particles was measured to assess if the protein treatment affected the ability of the virus to enter the cells. The pre––treatment of the viral particle with immune proteins was found to inhibit viral entry in the case of H1N1 particles but to promote viral entry in H3N2 particles. C1q (A) was normalized to relative luminescence unit of its control (cells + virus only), and the globular head modules (B–D) were normalized to relative luminescence unit levels of their control (Cells+ virus pre–treated with 20 μg/mL of MBP). Data are shown as the normalized mean of three independent experiments performed in triplicate ± SEM. Significance was determined using the two–way ANOVA test (* p < 0.05, ** p < 0.01, *** p < 0.001, (n = 3).

Figure 5

Pre-treatment of IAV with human C1q restricts H1N1 viral particles binding to the cell surface. A549 cells (1 × 10⁵ cells/mL) were infected with H1N1 or H3N2 viral particles pre-incubated with or without human C1q (A), ghA (B), ghB (C) or ghC (D) (20 µg/mL). Then, 2 h post-infection, unbound protein and viral particles were removed, and the wells were fixed with 1% v/v paraformaldehyde for 1 min. The wells were probed with the corresponding primary antibodies: monoclonal anti-influenza virus H1 or polyclonal anti-influenza virus H3 antibodies (1:5000). Data are shown as the normalized mean of three independent experiments performed in triplicate ± SEM. Data were normalized to the absorbance of its untreated control (cells + virus only). Significance was determined using the two-way ANOVA test (* p < 0.05, *** p < 0.001, (n = 3)).

Figure 6

C1q treatment triggers NF-κB activation during H1N1 infection. NF–κB activation was measured via luciferase reporter assay in A549–NF–κB–Luc cells following challenge with C1q (A), globular-head- or MBP- (B) treated H1N1 or H3N2 subtypes. The relative NF-kB activity of C1q treated IAV challenged A549–NF–κB–Luc was calculated by using untreated sample (cells + virus only) as the baseline. The NF–κB activity of ghA-, ghB- or ghC-treated IAV-challenged A549–NF–κB–Luc cells only was calculated by using MBP-treated IAV-challenged A549–NF–κB–Luc the baseline. Cells treated with TNF-α and IL-β were used as a positive control for NF–κB activation (C). Data shown as the relative mean of three independent experiments ± SEM. Significance was determined using the two-way ANOVA test (* p < 0.05, ** p < 0.01, *** p < 0.001, (n = 3)).