Hemagglutinin from the H5N1 virus activates Janus kinase 3 to dysregulate innate immunity

Abstract

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in humans. There are no effective vaccines or antiviral therapies currently available to control fatal outbreaks due in part to the lack of understanding of virus-mediated immunopathology. In our study, we used hemagglutinin (HA) of H5N1 virus to investigate the related signaling pathways and their relationship to dysregulated innate immune reaction. We found the HA of H5N1 avian influenza triggered an abnormal innate immune signalling in the pulmonary epithelial cells, through an unusual process involving activation of Janus kinase 3 (JAK3) that is exclusively associated with γc chain and is essential for signaling via all γc cytokine receptors. By using a selective JAK3 inhibitor and JAK3 knockout mice, we have, for the first time, demonstrated the ability to target active JAK3 to counteract injury to the lungs and protect immunocytes from acute hypercytokinemia -induced destruction following the challenge of H5N1 HA in vitro and in vivo. On the basis of the present data, it appears that the efficacy of selective JAK3 inhibition is likely based on its ability to block multiple cytokines and protect against a superinflammatory response to pathogen-associated molecular patterns (PAMPs) attack. Our findings highlight the potential value of selective JAK3 inhibitor in treating the fatal immunopathology caused by H5N1 challenge.

Figure 1. Evaluation of the expression and function of recombinant hemagglutinin protein (HA) of AIV H5N1.

(A) Preparation of the recombinant HA protein. Identification of the Bacmid/HA recombinant (a) M, Marker, Lane 1, PCR product of Bacmid-HA. Recombinant HA purified from Bacmid/HA-transfected SF9 cells by Ni-NTA affinity chromatography (Coomassie Brilliant Blue staining) (b) M, prestained protein marker, Lane 1, Control (from SF9 cells transfected with blank bacmid), Lane 2, HA Purified from Bacmid/HA-transfected SF9 cells. Confirmation of HA recombinant by western blot analysis (c) M, Marker, Lane 1, HA Purified from Bacmid/HA-transfected SF9 cells, Lane 2, control. (B) Morphology changes in the recombinant HA-treated human pulmonary epithelial cells. A549 cells treated with 40 µg/ml HA (b) or the control (a) for 12 h (bar = 50 µm). The cells treated with HA become swollen, rounded and irregular in size and shape.

Figure 2. Impact of H5N1 HA on JAK/STAT and NF-κB signalling in the challenged pulmonary epithelial cells.

(A) Detection of phosphorylated/nonphosphorylated JAK2, JAK3, STAT1 and NF-κB. Using specific antibodies, western blotting was performed in the A549 cells treated with the HA (40 µg/ml) for the indicated time periods. Representative blots from 3 replicates are shown. (B) The mRNA expression of IP-10 and IRF-1 on the HA-treated A549 cells. A549 cells were treated with the HA (40 µg/ml) for 1–4 h (Lane 1, 0 h; Lane 2, 1 h; Lane 3, 2 h; Lane 4, 4 h) or with the HA for 1 h at the indicated doses (Lane 5, control; Lane 6, 20 µg/ml; Lane 7, 40 µg/ml; Lane 8, 80 µg/ml) and then subjected to RT-PCR analysis for IP-10 and IRF-1. Representative gels from 3 replicates are shown. (C) Levels of IL-6, IL-8, MCP-1, MIP-1α, MIP-1β and RANTES in the supernatant of A549 cells treated with the indicated doses of HA for 12 h. *P<0.05 vs. control group; ^# P<0.05 vs. 20 µg/ml HA group; & P<0.05 vs. 40 µg/ml HA group.

Figure 3. The role of JAK3 activation in JAK/STAT and NF-κB signalling upon challenge of HA.

(A, B) Modulation of the phosphorylation of JAK3 and NF-κB and of the expression of the IP-10 and IRF-1 genes in the HA-treated A549 cells in the presence and absence of the JAK3 inhibitor VI. Western blot analysis (A) and RT-PCR (B) were performed to assess the signal pathways and the gene expression in the A549 cells challenged with HA (40 µg/ml) for 1 h in the absence and presence of the JAK3 inhibitor VI (760 nM). The measurement of the expression of GAPDH was performed simultaneously. Representative gels or blots from 3 replicates are shown. (C) Effects of treatment with the JAK3 inhibitor VI on the release of cytokines/chemokines from the HA-challenged A549 cells. A Liquidchip assay was performed on the supernatants of the A549 cells incubated with HA (40 µg/ml) for 12 h in the absence and presence of the JAK3 inhibitor VI (760 nM). *P<0.05 vs. control group; ^# P<0.05 vs. HA group.

Figure 4. Pathological examination of lung tissues in the Jak3-deficient and wild-type mice following exposure to HA.

Jak3^+/+ and Jak3^−/− mice were administered PBS (B, D) or the HA (90 µg per mouse) (A, C) by intratracheal instillation. Meanwhile, the JAK3 inhibitor VI was administered to the Jak3^+/+ mice prior to the HA instillation (E). Arrows show a marked thickening of the interalveolar septa with infiltration of lymphocytes (black arrow) and interstitial exudation (red arrow). The pathological examination (H&E) of lung tissues was performed at 72 h after HA administration (bar = 50 µm). The lung injury score was assessed in the lung tissues of the mice treated as above (F) (n = 5 per group, , Jak3^+/+ PBS, , Jak3^−/− PBS, , Jak3^+/+ HA, , Jak3^−/− HA, , Jak3^+/+ HA+JAK3Inh). *P<0.05 vs. Jak3^+/+ PBS group; ^# P<0.05 vs. Jak3^+/+ HA group.

Figure 5. Pathological examination of splenic tissues in the Jak3^−/− and Jak3^+/+ mice following exposure to HA.

(A) Haematoxylin/eosin (H&E) staining of paraffin sections of splenic tissues from the Jak3^+/+ and Jak3^−/− mice intratracheally administered with PBS or HA for 72 h. Arrows show the necrosis of lymphocytes. (B) The cytokines/chemokines (IFN-γ, IP-10, MCP-1α and RANTES) that were released from the splenocytes of either Jak3^+/+ or Jak3^−/− mice pretreated with HA or PBS as described above. The measurement of the concentration of the cytokines/chemokines by Liquidchip assay. *P<0.05 vs. Jak3^+/+ PBS group; ^# P<0.05 vs. Jak3^+/+ HA group.

Figure 6. Impact of HA intratracheal instillation on the inflammatory reaction of splenocytes from Jak3^−/− mice.

Liquidchip assays were performed to determine the cytokines/chemokines (IFN-γ, MCP-1α, IP-10 and RANTES) released from the splenocytes after LPS (20 µg/ml for 12 h) challenge in PBS-pretreated (A) and HA-pretreated (B) mice of either the Jak3^−/− or Jak3^+/+ background. (A) *P<0.05 vs. PBS group (Jak3^+/+ or Jak3^−/−) without LPS treatment; ^# P<0.05 vs. Jak3^+/+ PBS group with LPS treatment. (B) *P<0.05 vs. HA group (Jak3^+/+ or Jak3^−/−) without LPS treatment; ^# P<0.05 vs. Jak3^+/+ HA group with LPS treatment. A comparison of the levels of the cytokines/chemokines in the supernatants of the splenocytes exposed to LPS from Jak3^−/− and Jak3^+/+ mice with or without HA pretreatment (C). (C) *P<0.05 vs. PBS group (Jak3^+/+ or Jak3^−/−) with LPS treatment; ^# P<0.05 vs. Jak3^+/+ HA group with LPS treatment. A comparison of the fold increase of cytokines/chemokines released from the splenocytes following LPS stimulation from Jak3^−/− and Jak3^+/+ mice with HA pretreatment (D).

Figure 7. The effects of injury on the splenocytes challenged with LPS in HA-pretreated Jak3^−/− mice.

The splenocytes isolated from mice pretreated with HA or intratracheal instillation with PBS for 72 h were treated with LPS for 12 h (A) and 24 h (B), and then the cells were subjected to a CCK-8 assay for the evaluation of the injury index. The injury index = (OD value of control cells−OD of LPS-treated cells)/control cells OD. *P<0.05 vs. PBS group (Jak3^+/+ or Jak3^−/−); ^# P<0.05 vs. Jak3^+/+ HA group with LPS treatment.