H9N2 Avian Influenza Virus Downregulates FcRY Expression in Chicken Macrophage Cell Line HD11 by Activating the JNK MAPK Pathway

Abstract

The H9N2 avian influenza virus causes reduced production performance and immunosuppression in chickens. The chicken yolk sac immunoglobulins (IgY) receptor (FcRY) transports from the yolk into the embryo, providing offspring with passive immunity to infection against common poultry pathogens. FcRY is expressed in many tissues/organs of the chicken; however, there are no reports investigating FcRY expression in chicken macrophage cells, and how H9N2-infected HD11 cells (a chicken macrophage-like cell line) regulate FcRY expression remains uninvestigated. This study used the H9N2 virus as a model pathogen to explore the regulation of FcRY expression in avian macrophages. FcRY was highly expressed in HD11 cells, as shown by reverse transcription polymerase chain reactions, and indirect immunofluorescence indicated that FcRY was widely expressed in HD11 cells. HD11 cells infected with live H9N2 virus exhibited downregulated FcRY expression. Transfection of eukaryotic expression plasmids encoding each viral protein of H9N2 into HD11 cells revealed that nonstructural protein (NS1) and matrix protein (M1) downregulated FcRY expression. In addition, the use of a c-jun N-terminal kinase (JNK) activator inhibited the expression of FcRY, while a JNK inhibitor antagonized the downregulation of FcRY expression by live H9N2 virus, NS1 and M1 proteins. Finally, a dual luciferase reporter system showed that both the M1 protein and the transcription factor c-jun inhibited FcRY expression at the transcriptional level. Taken together, the transcription factor c-jun was a negative regulator of FcRY, while the live H9N2 virus, NS1, and M1 proteins downregulated the FcRY expression through activating the JNK signaling pathway. This provides an experimental basis for a novel mechanism of immunosuppression in the H9N2 avian influenza virus.

Keywords: FcRY; H9N2 avian influenza virus; HD11 cells; IgY; c-jun N-terminal kinase; chicken macrophage cell line.

Figure 1

Susceptibility of HD11 cells to H9N2 virus infection and identification of FcRY expression. (A) Expression of FcRY in different cells was assessed by reverse transcription polymerase chain reaction (RT-PCR). Lane 1: HD11. Lane N: Negative, Lane 2: DEF, Lane 3: DF-1, Lane 4: LMH. Cells were harvested at the 24 h post inoculation. (B) Western blot analysis of FcRY expression in different cells. Relative FcRY protein levels were calculated by Image J 1.53t software and normalized to GAPDH. (C) Expression of the HA gene of the H9N2 virus at different times as assessed by RT-PCR. Lane M: Marker. Lanes 1–4: Control (6, 12, 18, and 24 h post-infection) uninfected HD11 cells. Lanes 5–8: H9N2 (6, 12, 18, and 24 h post-infection, respectively) infected HD11 cells. Lane P: Positive. Lane N: Negative. (D) Indirect immunofluorescence detection of FcRY expression in HD11 cells. Scale bar: 20 µm. HD11 cells were fixed, permeabilized, and blocked at 16 h post inoculation. (E) Control group. (F) The H9N2 virus infection group. Infection with the H9N2 virus at a multiplicity of infection (MOI) = 1 at 18 h. Scale bar: 50 µm.

Figure 2

The effect of H9N2 virus infection on FcRY expression in HD11 cells. (A) RT-qPCR analysis of FcRY mRNA in HD11 cells by live H9N2 virus infection (MOI = 0.01, 0.1, 1) and UV-inactivation at 12 h. (B) RT-qPCR analysis of FcRY H9N2 virus mRNA in HD11 cells by infection (MOI = 0.1) for the indicated times. (C) HD11 cells were stimulated with the H9N2 virus (UV, MOI = 0.01, 0.1, and 1) for 18 h. Lysates of cells were used to test the FcRY protein expression level. (D) HD11 cells were stimulated with the H9N2 virus (MOI = 0.1) for the indicated times. The mRNA level of FcRY was standardized to β-actin. Relative FcRY protein levels were calculated by Image J 1.53t software and normalized to β-actin or GAPDH. All results were repeated at least three times, and data shown are means ± SEM (n = 3). * p < 0.05, ** p < 0.01 vs. the control group.

Figure 3

The effect of the MAPK signaling pathway on FcRY expression. (A–C) HD11 cells were continuously treated with the indicated doses of sp600125, U0126, and SB202190 for 12 h. (D–F) HD11 cells were pre-treated with sp600125 (10 µg/mL), U0126 (20 µg/mL), and SB202190 (20 µM) for 1 h, followed by incubation with the H9N2 virus (MOI = 0.1) for 12 h, respectively. FcRY mRNA levels were analyzed by RT-qPCR. The relative mRNA levels were normalized to the corresponding β-actin mRNA levels. * p < 0.05, ** p < 0.01. (G–I) The expression of FcRY and the ratio of p-JNK/JNK, p-ERK/ERK, p-p38/p38 in HD11 cells using the following inhibitors: sp600125 (10 µg/mL), U0126 (20 µg/mL), and SB202190 (20 µM), respectively. GAPDH was used as a loading control. The right panel represents protein band quantification determined by densitometry and normalized to GAPDH. All results were repeated at least three times, and data shown are means ± SEM (n = 3). * p < 0.05, ** p < 0.01, ns: not significant.

Figure 4

The effect of eukaryotic expression plasmids for each gene segment of the H9N2 virus on FcRY. (A,B) HD11 cells were transiently transfected with the constructed eukaryotic expression plasmids of H9N2 viral genes. Cells were harvested 24 and 36 h after transfection, and protein extracts were prepared for western blotting. Protein bands determined by densitometry were quantified and normalized to β-actin. (C,D) The same transfection operation as the previous step was followed, and the cell samples were harvested 24 and 36 h after transfection to extract RNA, as well as to measure the mRNA expression. FcRY mRNA levels were analyzed by RT-qPCR, and the relative mRNA levels were normalized to the corresponding β-actin mRNA levels. * p < 0.05, ** p < 0.01. Error bars represent the mean ± SEM (n = 3).

Figure 5

The JNK MAPK pathway regulates FcRY expression. (A) HD11 cells were used to inhibit pretreatment for 1 h, while HD11 cells were transfected by the indicated plasmids for 24 h, followed by examining the protein levels by western blotting. The right panel represents protein band quantification determined by densitometry and normalized to GAPDH, * p < 0.05. (B) HD11 cells were treated with Anisomycin at the indicated concentrations for 24 h. HD11 cells were harvested for protein analysis. The right panel represents protein band quantification determined by densitometry and normalized to GAPDH, * p < 0.05, ** p < 0.01.

Figure 6

Construction of FcRY promoter luciferase report plasmids. (A) Basal promoter activity of the FcRY-luc-F (1–4) in HD11 cells. (B) Predicted transcription factor binding site of c-jun in the primary active region (−1847/−1235). (C) HD11 cells were co-transfected with FcRY promoter luciferase report plasmids (−1847/+96, F1) and a Renilla luciferase vector (pRL-TK-luc). Cells were subsequently treated with the H9N2 virus (MOI = 0.1) for 12 h and harvested for luciferase assays. *** p < 0.001. (D,E) NS1, M1, and c-jun eukaryotic expression plasmids were co-transfected in HD11 cells with FcRY promoter constructs (−1847/+96, F1) and a luciferase reporter vector (pRL-TK-luc), respectively. Luciferase activity was measured 36 h post-transfection. ** p < 0.01, ns: not significant.