The Differences In Inflammatory Responses To PRRSV Infection Of PAMs And PIMs And Its Proteomics Study

Porcine Reproductive and Respiratory Syndrome(PRRS)is an important viral infection caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),which has seriously jeopardized the development of the pig industry worldwide and caused huge economic losses.Inflammation plays an important role in the infection and pathogenesis of PRRSV,and interstitial pneumonia is one of the most important pathological features of PRRS.Porcine alveolar macrophages(PAMs)are the main target cells of PRRSV in vivo,and recent studies have confirmed that porcine pulmonary intravascular macrophages(PIMs)are also highly susceptible to PRRSV and are involved in inducing inflammatory responses.PRRSV infection can cause viremia,suggesting that PIMs colonizing the pulmonary vasculature may be involved in the establishment of PRRSV infection,but the role of PIMs in this process is unclear,and the low isolation efficiency has limited the application of PIMs in PRRSV infection studies.In this study,we first optimized the isolation method of PIMs,compared the differences in the inflammatory response and barrier damage induced by PRRSV infection of PAMs and PIMs,and further carried out a comparative proteomics study of PRRSV infection of both types of cells.This study elucidates the molecular mechanisms of PRRSV-induced inflammation from the perspective of PIMs and provides useful data to delineate the role of PIMs in PRRSV infection and pathogenesis.Details of the study are as follows:1.Optimization of isolation method and identification of PIMsTo improve the isolation rate and activity of PIMs,the isolation method was optimized in several aspects:(i)the In vitro isolation method was used to improve the stability and adjust-ability of exogenous dynamics during cell isolation;(ii)the location of PIMs collection was shifted from the aorta to the left ventricle to shorten the time to isolate cells;(iii)the digestive effect of collagenase was terminated by using serum to reduce the effect of collagenase on PIMs activity;(iv)isolating both PIMs and PAMs from the same lung lobe to improve the cell yield.PIMs and PAMs were isolated simultaneously using the optimized method and identified.Morphological observations revealed that both cells had homogeneous morphology,relatively smooth surface,and PIMs were significantly smaller than PAMs.expression levels of differentially expressed genes Ma FB,CD14 and CD16 in PIMs and PAMs were detected by Real-time quantitative PCR,and it was found that Ma FB and CD16 in PIMs were significantly higher than those in PAMs,while the m RNA expression level of CD14 in PIMs was significantly lower than that in PAMs.The results were consistent with those reported in the literature,indicating that PIMs and PAMs with higher purity were obtained.2.The differential study in inflammatory responses upon PRRSV infection in PIMs and PAMsFirstly,the results of real-time quantitative PCR showed the expression of TNF-αand IL-1β was upregulated by PRRSV infection of both PAMs and PIMs,but the expression of TNF-α and IL-1β were upregulated earlier and at a higher fold in PIMs,suggesting that there are differences in the expression of inflammation induced by PRRSV infection of PAMs and PIMs.A co-culture system of PAMs/PIMs and pulmonary microvascular endothelial cells(PMVECs)was further constructed,and the barrier permeability of PMVECs was enhanced by PRRSV infection of both types of cells as detected by fluorescein isothiocyanate-dextran(FD-4)permeability assay,and the barrier permeability enhancement caused by PRRSV infection of PIMs was earlier.Subsequently,the cells were treated with neutralizing antibodies to TNF-α(adalimumab)and antagonists to IL-1R(raleukin)alone or in combination with both before inoculation with PRRSV.The effect of combined treatment was stronger,suggesting that both TNF-α and IL-1 β were involved in the barrier permeability changes induced by PRRSV infection.It was also found that TNF-α and IL-1 βinduced by PRRSV infection of PAMs and PIMs down-regulated the expression of the tight junction proteins CLDN8 and OCLN and up-regulated the expression of CLDN1 in PMVECs,indicating that PRRSV infection induced cytokines to regulate the expression of tight junction proteins,thus causing barrier permeability changes in PMVECs.The above results suggest that PRRSV infection of PAMs and PIMs differ in the timing and intensity of induction of TNF-α and IL-1β production,but both TNF-α and IL-1β can cause altered barrier permeability in PMVECs by regulating the expression of tight junction proteins.3.Proteomics differences in PRRSV-infected PIMs and PAMsIn order to gain a better understanding of the differences in the inflammatory responses induced by PRRSV infection of PAMs and PIMs,the effects of different culture conditions and different inoculation doses on cell activity and cell membrane integrity were examined by CCK8 and Homogeneous Membrane Integrity Assay(HMIA).It was determined that PAMs and PIMs were inoculated with 1 MOI of PRRSV,respectively,and serum-free 1640 was used as the cell maintenance solution after inoculation,and cells(intracellular proteins)and supernatants(extracellular proteins)were collected 24 h after inoculation for comparative proteomic studies.Analysis of the proteomic data revealed that 36 proteins were changed in both PRRSV-infected PAMs and PIMs,9 were up-regulated and 14 were down-regulated;86 proteins were changed in supernatants,16 were up-regulated and 5 were down-regulated.The clustering analysis revealed that PRRSV infection of PAMs was mainly enriched in intracellular differentially expressed proteins in inflammation-related biological processes and the KEGG pathway,while PRRSV infection of PIMs was enriched in supernatants in differentially expressed proteins in inflammation-and tissue repair-related biological processes or the KEGG pathway.In addition,clustering analysis and comparison of intracellular differential proteins following PRRSV infection of both cells revealed that the biological processes and KEGG pathways upregulated in PIMs compared to PAMs were mainly associated with tissue repair,whereas clustering analysis and comparison of differential proteins in supernatants following PRRSV infection of both cells revealed that the biological processes and KEGG pathways upregulated in PIMs compared to PAMs were involved in inflammatory response.This suggests that PRRSV infection of PIMs induces an earlier inflammatory response than PAMs,which is consistent with the above findings.PRRSV infection of both PIMs and PAMs enriched the arachidonic acid metabolic pathway associated with inflammation,and arachidonic acid metabolism in which cyclooxygenase(COX-1/2)with its product prostaglandin E2(PGE2)is an important mediator of inflammation.Proteomics data analysis revealed that the expression level of COX-1 was higher in PIMs than in PAMs under both PRRSV-infected and uninfected conditions;while the expression level of COX-2 was slightly higher in PIMs than in PAMs under uninfected conditions,and there was no significant difference in the expression level of COX-2 between the two types of cells under infected conditions.Real-time quantitative PCR and Western blot assays revealed that the expression trends of COX-2 in the two cell types was essentially the same;while the expression of COX-1was more obviously up-regulated in the early stage of infection in PIMs and in the late stage of infection in PAMs,and PGE2 upregulated the expression of TNF-α,IL-1β,IL-6and IL-8,and the fold of upregulation was higher in PIMs,indicating that PRRSV infection of PIMs induced earlier and higher level of cytokines expression may be related to COX-1.Adhesion molecules participate in the inflammatory response by targeting inflammatory cells with inflammatory mediators.Proteomics data showed that the expression of adhesion molecules was upregulated by PRRSV infection in both PAMs and PIMs,and the expression of adhesion molecules(ICAM-1 and VCAM-1)was also upregulated by PRRSV infection in both cell types as confirmed by real-time quantitative PCR and Western blot assays,and PGE2 dose-dependently upregulated the expression of ICAM-1 and VCAM-1,and the upregulation was higher in PIMs,indicating that PGE2 could up-regulate the expression of adhesion molecules.Further detection by co-culture system revealed that PRRSV infection of PIMs promoted ICAM-1 and VCAM-1expression in PMVECs,PGE2 also dose-dependently upregulated ICAM-1 and VCAM-1 expression in PMVECs as well,suggesting that PRRSV may up-regulate the expression of adhesion molecules in PMVECs by promoting PGE2 secretion from PIMs.