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Innate Immunity Porcine Reproductive and Respiratory Syndrome Virus Induced Immunosuppression Exacerbates the Inflammatory Response to Porcine Respiratory Coronavirus in Pigs Gourapura J Renukaradhya 1 Konstantin Alekseev 1 Kwonil Jung 1 Ying Fang 2 and Linda J Saif 1 Abstract We performed a comprehensive analysis of innate and adaptive immune responses in dual virus infected pigs to understand whether a pre existing immunomodulatory respiratory viral infection affects the overall immunity to a subsequent porcine respiratory coronavirus PRCV infection in pigs Pigs were either mock infected or infected with porcine reproductive and respiratory syndrome virus PRRSV a virus known to cause immu nosuppressive respiratory disease and then pigs were co infected with PRCV which normally causes subclinical respiratory infection We collected samples for six independent experiments from 178 pigs that were also used for pathological studies We detected a significant reduction in innate NK cell mediated cytotoxic function in PRRSV infected pigs which was synergistically further decreased in pigs co infected with PRCV Subsequently in association with clinical signs we observed elevated levels of proinflammatory IL 6 Th 1 IL 12 and regulatory IL 10 and TGF b cytokines Increased frequencies of CD4CD8 double positive T lymphocytes and myeloid cells in addition to the elevated Th 1 and proinflammatory cytokines in dual infected pigs contributed to the severity of lung disease in pigs The results of our study clarify how each virus modulates the host innate and adaptive immune responses leading to inflammatory reactions and lung pathology Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of respiratory viral co infections and provide more definitive approaches for treatment Introduction C ytokines are key regulators in governing the host defense against pathogens and are produced following microbial infections They are potent immunomodulatory molecules that act as mediators of inflammation and the im mune response Proinflammatory cytokines TNF a IL 1 IL 6 and IL 8 are produced early in viral infection triggering theproductionoftheTh 1cytokines IFN gandIL 2 involved in cellular immune responses Both IL 10and TGF b suppress the host s cell mediated immune response by reducing cell recruitment and downregulation of cytokine production by innate immune cells 14 Natural killer NK cells are popu lations of lymphocytes recognized for their ability to provide a first line of innate defense against viral pathogens 8 Pigs possess relatively more NK cells up to 10 of lymphocytes than other species of animals and humans 16 We used a porcine reproductive and respiratory syndrome virus PRRSV infection to understand how immune modu lation induced by a prior respiratory viral pathogen influ ences a subsequent porcine respiratory coronavirus PRCV respiratory infection Coronaviruses CoVs are members of the family Coronaviridae and the order Nidovirales 34 38 They are enveloped viruses with a single stranded positive sense RNA The coronaviruses infect a broad range of vertebrates and cause a variety of disorders including gas troenteritis and respiratory tract disease In this study we used PRCV a deletion mutant of transmissible gastroenter itis virus TGEV of pigs 44 PRCV alone causes mostly subclinical respiratory tract infection in pigs but in con junction with other viral or bacterial infections it causes severe respiratory disease in swine 44 PRCV replicates in epithelial cells of the nasal mucosa and lung 13 15 32 PRRSV is an enveloped RNA virus and a member of the 1 Food Animal Health Research Program Ohio Agricultural Research and Development Center Department of Veterinary Preventive Medicine The Ohio State University Wooster Ohio 2 Veterinary Science Department South Dakota State University Brookings South Dakota Contributed equally to this research VIRAL IMMUNOLOGY Volume 23 Number 5 2010 Mary Ann Liebert Inc Pp 457 466 DOI 10 1089 vim 2010 0051 457 family Arteriviridae and the order Nidovirales 28 PRRSV has a specific tropism for macrophages in the lung and other tissues 7 17 35 and infected pigs have weak and delayed adaptive immune responses as suggested by low levels and deferred generation of IFN g secreting cells 27 PRRSV is a strong inducer of the immunoregulatory cytokine IL 10 in the lungs 40 The IL 10 is produced by antigen presenting cells APCs and lymphocytes which are also important targets of IL 10 23 Overall the immune responses against PRRSV are ineffective in resolving the infection completely and they induce immune modulation resulting in prolonged viremia and persistent infection in lung and lymphoid tis sues potentiating the effects of other swine pathogens 31 To determine if dual virus infections compared to single virus infections result in enhanced clinical manifestations in pigs Van Reeth s group carried out experiments and de tected more persistent fever and growth retardation in PRRSV PRCV and PRRSV SIV dual virus infected pigs than in pigs with single virus infections 45 In another study the cytokine analysis of PRRSV PRCV and SIV single virus in fected pigs revealed that changes in proinflammatory cyto kine levels are associative and do not demonstrably cause viral respiratory disease in pigs 43 44 Suppressed innate immune responses to TGEV infection in pigs were associated with a reduced NK cell response 35 However compre hensive immunological responses to cytokines in systemic and local mucosal sites of lungs and lymphoid and myeloid cell populations in the dual respiratory virus infected pigs were unexplored The aim of our study was to understand how viral co infections modulate innate and adaptive im muneresponses andhowtheseresponsesrelatetotheclinical outcome in pigs Materials and Methods Virus inoculation and management of pigs Conventional Large White Duroc crossbred specific pathogen free piglets n 178 were weaned at 16 20 d of age and transported to animal facilities at the Ohio Agri cultural Research and Development Center The Ohio State University Wooster Ohio The swine herd was seronegative for antibodies to PRRSV PRCV TGEV and porcine circo virus type 2 PCV2 The piglets were bled on arrival and pre bleed sera were tested to confirm the absence of neu tralizing antibody to PRCV using a fluorescent focus neu tralization test 48 and for PRRSV antibody screening serum samples were sent to the Animal Disease Research and Diagnostic Laboratory South Dakota State University for serological study and confirmed as negative The pigs were allowed to acclimate for an additional week before the initiation of the experiments Six sequential batches of pigs were obtained for six individual trials The piglets were randomly assigned to one of four treatment groups mock infected n 43 PRRSV infected n 39 PRCV infected n 48 and PRCV PRRSV infected n 48 The ISU 1 strain of PRCV PRCV ISU 1 18 and the PRRSV strain SD23983 were used to infect pigs in this study To establish PRCV infection after progressive PRRSV disease subsequent to PRRSV viremia the pigs were inoculated in tranasally IN with 3C210 4 TCID 50 and intramuscularly IM with2C210 4 TCID 50 ofthePRRSVSD23983strain At10dafter PRRSV infection Telazol C210 anesthetized pigs were inoculated IN with 4C210 6 plaque forming units PFU and intra tracheally with 6C210 6 PFU of the PRCV ISU 1 strain Mock infected control pigs received 5mL of DMEM by similar routestothosedescribedfortherespectiveviruses Aftervirus inoculation we assessed clinical signs body weight gains breathing rates and rectal temperatures every other day as previously described 19 Nasal swabs and blood were col lectedeveryotherdaythroughouttheexperimentalperiodfor detection of virus shedding and serum cytokine andantibody analysis The pigs were maintained in accordance with the standards of the Institutional Laboratory Animal Care and Use Committee The Ohio State University Determination of cytokine concentrations in serum and lung by ELISA The lung tissue from all of the euthanized pigs was col lected and lung lysates were prepared in DMEM without serum Approximately 2 5g of lung tissue of individual pigs was minced into tiny pieces To make homogenates the tissues were blended for 2min in a Stomacher 400 laboratory blender Seward Long Island NY and clarified by centri fugation Then the supernatant collected was aliquoted and frozen at C0208C until subjected to cytokine analysis Pigs were bled on PRCV PRRSV post infection days PID C010 0 C06 4 C02 8 0 10 2 12 4 14 6 16 8 18 10 20 12 22 14 24 16 26 18 28 and 21 31 and serum samples were aliquoted and frozen at C0208C until used for cytokine anal ysis Representative proinflammatory IL 6 Th 1 IL 12p35 and p40 and anti inflammatory T regulatory IL 10 and TGF b cytokine levels in serum and lung were determined by ELISA as previously described 4 20 49 Isolation of PBMCs and flow cytometric analysis of lymphoid and myeloid cell populations For the isolation of peripheral blood mononuclear cells PBMCs bloodwascollectedinacid citratedextrosesolution from the euthanized pigs and processed as previously de scribed 47 The PBMCs 1C210 6 cells well were incubated with optimal dilutions of fluorophore conjugated anti bodies in staining buffer 0 02 PBS 0 5 sodium azide and bovine serum albumin for 30min at 48C For CD4 and CD8 cell staining the cells were incubated with fluorescein isothiocyanate conjugated mouse anti pig CD4a or CD8a mAb BD Biosciences San Jose CA For CD3 staining biotin conjugated mouse anti pig CD3e mAb Southern Biotechnology Associates Birmingham AL was used fol lowed by incubation with streptavidin PerCP peridinin chlorophyll protein complex conjugate BD Biosciences The CD172 cells were stained with mouse anti pig CD172 R phycoerythrin R PE Southern Biotechnology Fifty thou sand PBMCs were analyzed out of the 1C210 6 cells stained for each sample by flow cytometry Lymphocytes were defined by their light scatter characteristics 36 For discrimination of positive and negative populations quadrant markers were set and these were controlled by non stained samples and samples incubated only with isotype control antibodies Flow cytometry data were analyzed using FlowJo software Tree StarInc Ashland OR Thefrequencyofeachindividualtype of lymphocyte or CD172 cell was expressed as the frequency percentage of these cells within the 50 000 PBMCs counted 458 RENUKARADHYA ET AL Lymphocyte subpopulations were separated initially by CD3 and CD3 C0 gates Based on CD4 and CD8 staining characteristics each population was then further grouped as CD3 C0 CD4 C0 CD8 as NK cells 16 CD3 CD4 CD8 C0 as T helper cells CD3 CD4 C0 CD8 as CTL and CD3 CD4 CD8 double positive T cells All of the CD172 cells were grouped as total myeloid cell population Natural killer cell cytotoxicity assay The basic assay for colorimetric determination of NK cell cytotoxicity has been previously described by others 24 25 and by us 20 In brief the target cell line K 562 human myeloblastoid leukemia cell line was maintained in RPMI 1640 medium supplemented with FBS The PBMCs isolated from mock infected and virus infected pigs were used as effectors source of NK cells The cells were washed three times using medium 199 containing HEPES buffer gentami cin and bovine serum albumin assay medium to remove free lactate dehydrogenase LDH and then the cells were resuspended in assay medium The effectors were transferred to a 96 well round bottom plate and twofold dilutions were performed Fixednumbersoftargetcellswere addedtoattain different effector target cell ratios Appropriate controls were included in each plate such as only targets lysed only tar gets only effectors and medium control The plates were incubated at 378CinaCO 2 incubator overnight Control tar get cells were lysed with 2 Triton X 100 for 15min All the plates were centrifuged briefly and 100mL of the superna tant was transferred to a fresh 96 well flat bottom plate and then equal amounts of LDH substrate 5 4C210 C02 ML lactic acid 6 6C210 C04 M 2 p idophenyl 3 p nitrophenyl tetrazolium chloride 2 8C210 C04 Mphenazine methosifate and1 3C210 C03 M NAD Sigma Aldrich St Louis MO in 0 2M Tris buffer pH 8 2 was added to all of the wells and incubated at room temperature TomeasuretheamountofLDHreleasedintothe supernatant the plates were read in a microtiter plate reader at 490nm after 5 10min The quantity of LDH released into the supernatant is directly proportional to NK specific lysis of targets in experimental wells 24 The percentage of NK cell specific lysis was calculated as follows OD E T C0OD E C0OD T spon OD T total C0OD T AM C2100 where E effectors T targets AM assay medium T total targets with 0 5 NP40 T AM targets with assay medium and T spon T AM minus the OD of AM control wells Data analysis Statistical analyses were performed for each PID among the four experimental groups using the nonparametric Kruskal Wallis test and in addition for serum samples a repeated measures ANOVA was used due to repeated ana lyses of sera from the same animals Statistical significance was set at p39 58C were considered to be febrile responses A significantly higher incidence of fever p 0 05 was evident in more than 70 of dual virus infected pigs compared to pigs infected with PRRSV alone 52 Fever appeared in PRCV PRRSV infected pigs on PID 2 12 and persisted until PID 21 31 Fig 1A However the pigs in fected only with PRRSV had sporadic fever beginning at PID 4untilPID 31 and incontrast to thisfinding pigsinfected with PRCV alone had no fever The PRRSV only infected pigs had less body weight gain than PRCV infected and mock infected pigs However the dual virus infected pigs had 100 125 150 175 200 225 250 275 300 325 350 10 0 4 6 0 10 2 12 4 14 6 16 8 18 10 20 12 22 14 24 16 26 18 28 21 31 38 8 39 3 39 8 10 0 4 6 0 10 2 12 4 14 6 16 8 18 10 20 12 22 14 24 16 26 18 28 21 31 PRRSV PRCV PRRSV PRCV Mock Fever Rectal Temperature Rectal Temperature C PRCV PRRSV PID Body Weight Gain Body Weight Gain 39 5 fever A B FIG 1 Clinical responses in pigs infected with PRRSV and then infected with PRCV 10 d later Clinical signs of pigs mock infected n 43 PRRSV infected n 39 PRCV infected n 48 or infected with both PRRSV and PRCV n 48 were recorded on the indicated post inoculation days PID A Rectal temperature B Body weight gain Each data point represents the meanC6SEM of 5 48 pigs A sig nificantly higher incidence of fever was observed in PRCV PRRSVco infectedpigsthaninPRRSV alonepigs p 0 027 and a significantly higher proportion had less body weight gain in the PRCV PRRSV co infected pigs than in those infected with PRRSV alone p 0 005 as indicated by the asterisks Statistical analysis was performed using Fischer s exact test to evaluate the proportions of pigs with fever and decreased body weight gain and p 0 05 was considered statistically significant PRRSV AND PRCV CO INFECTION AND HOST RESPONSES 459 significantly decreased body weight gain compared to pigs infected with PRCV p 0 05 or PRRSV alone Fig 1B Suppressed innate NK cell cytotoxicity occurred in the dual infected pigs Innate immunity is the first line of defense and is essential for effective adaptive immune responses and protection against pathogens To examine the systemic innate immune responses in the blood of PRCV PRRSV co infected pigs the NK cell population and its cytotoxic function were ana lyzed Only marginal changes were detected in the frequency of total NK cells CD3 C0 CD4 C0 CD8 in single virus and co infected pigs Fig 2A The NK cell specific lysis seen at different time points post infection indicated that PRCV in fection alone resulted in only a 10 30 reduction in NK cell lytic activity whereas PRRSV infection alone resulted in significant reductions 50 80 at different post infection days In dual virus infected pigs NK cell mediated cyto toxicity was synergistically reduced by 80 100 Fig 2B The percentage reduction in NK cell cytotoxicity was calcu lated in relation to mock infected pigs 100 at the re spective post infection days The reductions in NK cell cytotoxic function in dual infected pigs was statistically sig nificant on PRCV PRRSV PID 2 12 8 18 and 14 24 com pared to mock infected pigs and compared to pigs infected with PRCV alone at PID 14 24 Significant reductions in NK cell function were detected at a higher effector target cell ratio in PRCV only infected pigs at PRCV PRRSV PID 2 12 and 8 18 compared to mock infected pigs Thus prior in fection with PRRSV suppressed the innate NK cell cytotoxic function in pigs which resulted in a further synergistic re duction in NK cell mediated cytotoxicity following PRCV infection Interestingly the NK cell cytotoxicity observed was independent of their frequency in virus infected pigs Co infection of pigs with PRRSV and PRCV resulted in elevated Th 1 and proinflammatory cytokines in pigs Effective adaptive immune responses are important for protection against viral infections To measure Th 1 IL 12 and proinflammatory IL 6 cytokine responses in pigs in fected with PRCV and or PRRSV we collected serum at 14 different time points over a period of 30 d post infection To measure cytokine responses in the lung lung lysates were prepared from all of the euthanized pigs and used in the assay In both lung and serum levels of the Th 1 cytokine IL 12 in PRCV only infected pigs remained low compared to the other groups Fig 3A In PRRSV alone infected pigs IL 12 was detected at higher levels at both middle and later time FIG 2 Significant reductions occurred in NK cell cytotoxicity despite a lack of significant changes in NK cell populations following PRRSV and PRCV co infection in pigs A Percentages of CD3 C0 CD4 C0 CD8a lymphocytes NK cells in PBMCs of pigs were evaluated in mock infected PRRSV infected PRCV infected or dual infected PRCV PRRSV pigs on the indi cated post inoculation days PID by flow cytometric analysis Each bar represents the meanC6SEM of NK cells from 4 8 pigs and total numbers of pigs at each PRCV PRRSV PID C02 8 n 17 2 12 n 18 4 14 n 18 8 18 n 20 10 20 n 20 14 24 n 21 and 21 31 n 21 B Percentages of NK specific cytotoxicity were measured using pig PBMCs effectors harvested from mock infected or infected pigs as described above against target cells K 562 Effectors and targets at the different indicated ratios were cultured together and the supernatants harvested were analyzed for amounts of LDH released using its substrate at 490nm Each line is from one pig and each data point on the line is the mean of triplicate readingsC6SEM at the respective E T ratios tested Altogether there were 19 pigs from one independent experimental trial a total of four PIDs A similar trend in NK specific lysis was detected in another two independent experimental trials comprised of the same numbers of pigs Statistical analyses were performed from the pooled results of all three independent experimental trials and a denotes a statistically significant difference p 0 05 between the mock and dual infected groups b denotes a statistically significant difference between the mock and PRRSV infected groups c denotes a statistically significant difference between the mock and PRCV infected groups and d denotes a statistically significant difference between the PRCV and dual infected groups as analyzed by the nonparametric Kruskal Wallis test 460 RENUKARADHYA ET AL points post infection PID 8 24 in both lung and serum compared to the PRCV alone and mock infected groups and it was significantly higher at PID 14 20 Fig 3A In dual virus infected pigs serum IL 12 levels were significantly higher at both early and later time points post infection PRCV PRRSV PID 2 12 to 21 31 compared to the mock infected and PRCV alone infected groups Fig 3A and 4A In the lungs of dual virus infected pigs substantially higher levels of IL 12 were detected only during the middle period of infection PRCV PRRSV PID 8 18 and 10 20 compared