The Inhibition Of JAK3 Kinase Activation Protects Against The Immune Injury Of Peripheral Immne Organ Mediated By The H5N1 Virus Hemagglutinin Protein

Objective:Human Avian Influenza caused by highly pathogenic Avian Influenza Virus A (H5N1) is a human acute respiratory infectious disease leading a high mortality rate. H5N1 avian influenza virus not only leads to acute lung injury(ALI),but also to immune system damage.A large number of studies have shown that the pathological immune response of host can be promoted by virus,which is the main mechanism underlying acute respiratory failure and immune system dysfunction.Recent studies also prompted that a combined anti-viral and immunodulatory agent maybe a new strategy for prevention of ALI and of effective anti-viral infection.In previous studies,we found that SARS-CoV Spike protein can activate the JAK-STAT signaling pathway,through which a pathological immune reaction can be promoted,and that JAK3 is a key target signal molecular.JAK3 selective inhibitor has the capability to protect against the immune injury mediated by SARS-CoV and to improve the survival rate of mice infected with SARS-CoV.The JAK3 selective inhibitor plays a protective role of immune cells in SARS-CoV-infected mice as well.Based on the evidence that the pathophysiological process caused by H5N1 virus is similar to that by SARS-CoV,we speculated that H5N1 might activate a common key signal target causing acute immune injury.To address this issue,we investigated whether inhibition of JAK3 kinase could protect against immune injury mediated by HA protein by using JAK3 gene knockout mice,aim to find a anti-inflammatory agent with specificly targeting to immune injury mediatd by virus,thereby protecting the immune system from injury by pathological immune reaction. Methods:PartⅠ:Comparison of the spleen injury induced by intratracheal injection of H5N1 HA in the mice between JAK3 wild-type(JAK3 +/+) and JAK3 knockout (JAK3-/-)1.JAK3-/- and JAK3 +/+ mice,aged six to eight weeks,were randomly divided into PBS control and HA (+) group.The mice in the groups were intratracheally administrated with PBS or HA(recombinant protein)(90ug).2.Seventy-two hrs after HA instillation,the lungs and spleens were isolated from the mice.Histopathological sections of the isolated lung and spleen were prepared for pathological examination and immuno-staining for IP-10. PartⅡ:Comparison of the levels of chemokines/cytokines released from the spleen cells of the mice challenged by H5N1 HA intratracheal administration between JAK3 wild-type and JAK3 knockout. As described above,the mice of JAK3 knockout (JAK3-/-) and JAK3 wild type (JAK3 +/+), aged six to eight weeks ,were randomly divided into PBS control group and HA (+) group, and challenged as mentioned above. The spleen cells of those mice were isolated at 72hrs after challenge.1.The isolated spleen cells were treated with LPS or PBS for 12~24 hrs, and then those treated cells were subjected to evaluation of injury index by using MTT assay.2.The supernatants released from the treated spleen cells were collected and subjected to Liquid-Chip detection of the levels of chemokines/cytokines, including eotaxin,IFN-γ,IL-2,IL-4,IL10,IL-13,IP-10,MIP-1a,MCP-1,Rantes. Result:PartⅠ:1.In response to HA challenge with intratracheal instillation,JAK3+/+ mice were observed with significant shortness of breath,decreased activity,the symptoms lasted for a long time whereas JAK3-/ - mice were less serious.2.Pathological examination revealed spleen swelling,destruction of local structure of germinal center,vacuolar changes and a decrease in lymphocyte in JAK3+/+ mice challenged by HA,whereas there were no significant difference of the histological morphology in JAK3-/- mice treated with HA or PBS. 3.Lung pathological examination showed diffuse alveolar damage,inflammatory cell infiltration in JAK3+/+ mice challenged by HA whereas a decreased inflammatory reaction was observed in JAK3-/- mice.PartⅡ:1.The injury index of spleen cells after LPS stimulation in JAK3+/+ and JAK3-/ - mice of HA pretreatment was higher than that in those of PBS pretreatment.The injured index of LPS stimulation in JAK3-/- mice of HA pretreatment was significantly lower than that in JAK3 +/+ of HA pretreatment (P﹤0.05).2.Under basal condition, the levels of IP-10,MCP-1a and RANTES released from the spleen cells of JAK3 +/+ mice in HA pretreatment were significantly higher than than those from the JAK3 +/+ mice of PBS pretreatment group (P <0.05). Twenty-four hours after stimulation of LPS,the levels of IFN-γ,IP-10,MCP-1a and RANTES released from the spleen cells of HA pre-treated mice was significantly higher than those from PBS pre-treated mice(P﹤0.05).3.Under basal condition, the levels of IP-10,MCP-1a and RANTES released from the spleen cells of JAK3-/- mice in HA pretreatment group were significantly lower than those of JAK3+/+ mice in HA pretreatment group(P﹤0.05).After stimulation of LPS,the levels of IFN-γ,IP-10,MCP-1a and RANTES released from the spleen cells of JAK3-/- mice in HA or pretreatment group were increased,but significantly lower than those of the JAK3 +/+ mice in HA pretreatment group (P﹤0.05).Conclusion:1.The H5N1 virus HA membrane protein by intratracheal instillation elicits a strong pathological immune response response in the host,leading to immune injury of peripheral lymphoid organs and immune cells and acute lung injury.JAK3-/- mice are able to tolerate immune injury mediated by H5N1 virus HA as well as attenuate the induction of IP-10,an important chemokine,in the spleen. 2.JAK3-/- mice have a protective effect on HA-mediated immune injury,significantly reducing the damage of the spleen cells after the LPS stimulation,which is associated with the decrease in release of chemokines/cytokines from the challenged cells.3.It is suggested that inhibition of activated JAK3 kinase by virus could reduce immune injury mediated by the H5N1 virus,decreasing the injury in the immune system.