Role Of Neutrophils In Lung Injury Of PRRSV Infected Pigs And Construction Of A Model Of PRRSV Infection In Transplanted Pig Lung

Porcine reproductive and respiratory syndrome(PRRS)is the most important infectious viral disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which causes huge economic losses to China's animal husbandry every year.Diffuse interstitial pneumonia and a large number of bleeding spots in the lungs have been reported in the infected pigs.However,the pathological mechanism of how PRRSV causes pig lungs injury is still not very clear.Recent studies suggest that the number of neutrophils in the bronchoalveolar lavage fluid(BALF)of PRRSV infected pigs were remarkably increasing in inflammatory phase at 7-day post infection.Neutrophils are innate immune cells and play a crucial role in the first line of host defense.However,it is also known that excessive recruitment of neutrophils in the lungs may cause lung injury.To understand the mechanisms of neutrophil recruitment and lung infiltration in the PRRSV infected pig lung and to identify the biomarkers for lung injury,in the first part of our work,we examined the expression of several molecules involved in neutrophil infiltration and MPO activity in the swine lungs from experimental PRRSV infection and clinical lung samples with natural PRRSV infection.This part of the work is important for exploring how PRRSV causes pig lungs injury.However,because large animals such as pigs are not easy to knock out individual gene like mice,nor are they suitable for interference applications of small batches of biological agents,such as using antibodies or siRNA to neutralize or deplete a cytokine or protein,it is hard to further study the molecular mechanism of PRRSV infection.Therefore,the second part of our work is to establish a transplanted porcine lung PRRSV infection model on immunodeficient mice.This part of the work will provide an important experimental basis for further research on the molecular mechanism of lung injury caused by PRRSV infection.The main results are as follows: 1.Recruitment of neutrophils in the lungs of PRRSV-infected pigsFirstly,the mRNA expression of inflammatory factors were significantly activated after PRRSV infection;Hematoxylin-eosin(HE)staining showed significant interstitial pneumonia and thickening of alveolar septum accompanied by infiltration of a large number of inflammatory cells in both experimental and natural PRRSV infected samples;the diff-quik staining and immunohistochemical methods confirmed the presence of large number of neutrophils in these inflammatory cells,and by fluorescent quantitative PCR(qPCR)and enzyme-linked immunosorbent assay(ELISA)or Western blot showed that the mRNA and protein expression of the marker enzyme MPO released by neutrophils and its chemokine IL-8 were significantly up-regulated;In the adhesion factors associated with neutrophil transendothelial cell migration,the protein expression of VCAM-1 was significantly up-regulated,while the mRNA and protein expression of ICAM-1 was a slight decreased in the experimentally infected group and remarkablely increased in the naturally infected group.These results suggest that neutrophils are recruited in the lungs of PRRSV infected pigs.On the other hand,PRRSV infection also resulted in a significant up-regulation of mRNA expression of IL-23?IL-17A?G-CSF axis,which regulates the differentiation of neutrophil,indicating that neutrophils continue to differentiate and mature into the bloodstream in the lungs of PRRSV-infected pigs,promoting the occurrence of inflammatory reactions.2.Construction of a model of PRRSV infection in transplanted pig lungUsing immunodeficient mice,we transplanted pig lungs from newly born piglets under the kidney capsule of mice.The area and volume of the graft increased significantly 7 days and 14 days after the transplant operation,and HE staining showed that the transplanted lung tissue retained the morphology and structure of normal pig lung tissue,indicating the normal growth of the graft under the kidney capsule of the mouse.Next,we chose three ways to perform PRRSV challenge on transplanted pig lung tissue.Two weeks after infection,PRRSV was detected in the intraperitoneal injection group,the surgical injection group and the tail vein injection group by qPCR.HE staining showed histopathological changes in the lung tissue of pigs in all three ways,and Immunohistochemistry and Immunofluorescence detection also showed significant PRRSV positive signals.Furthermore,the mRNA expression of the inflammation-related factors IL-6 and CXCR3 was significantly up-regulated.These results indicate that the model of PRRSV infection in transplanted pig lungs was successfully constructed.Finally,we use this infection model to inject CXCR3 antagonist AMG487 in vivo.The results showed that PRRSV infection was significantly inhibited after drug administration.HE staining showed that the morphology and structure of lung tissue were significantly improved after administration,and immunohistochemistry and immunofluorescence showed that there was almost no or only a small number of positive signals after administration,indicating that AMG487 can significantly inhibit PRRSV infection and alleviate pig lung damage,which proved that the model can be used to screen and verify the drugs for the treatment of PRRSV infection.Taken together,our results suggest that inflammatory factors was significantly activated after PRRSV infection,histological tests and MPO activity tests confirmed the recruitment of neutrophils in the lungs of PRRSV-infected pigs,combined expression of VCAM-1 and IL-8 may serve as the candidate biomarkers for lung injury induced by virus infection,and the inflammatory response caused by excessive recruitment of neutrophils is an important factor of lung injury in pigs infected with PRRSV.Here we innovatively constructed a porcine lung transplantation model under the renal capsule of immunodeficient mice,and successfully infected PRRSV with this model.Furthermore,based on this,in vivo injection of drug AMG487 using this infection model successfully inhibited the infection of PRRSV in transplanted pig lung tissue,which proves that the model is feasible for the study of some small molecule compounds in porcine respiratory diseases,thus opens a new avenue to better understand the molecular pathogenesis of porcine respiratory diseases.