Modulation in host innate immunity
Our rationale for conducting this study was to investigate how PRRSV rapidly modulates host innate immune mediators at an early time point post-infection, because the virus-induced adaptive immune response is always weak and delayed [34, 35]. It is known that younger pigs suffer severely from PRRS than adult animals , likely resulting from poorly developed innate immune system or an efficient immune evasion strategy adapted by the virus. Innate NK cells are the lymphocyte subpopulation known for their ability to provide the first line of defense against viral infections . Younger pigs have underdeveloped NK cell-mediated cytotoxic function [7, 9], and in our study, approximately 50% of pigs at day 0 (preinfection) did not have detectable NK cell cytotoxicity. Moreover, in pigs with satisfactory NK cell cytotoxicity, PRRSV significantly suppresses the NK function by 50-80% from post-infection day 7-24 . In the current study, as early as day 2 post-infection, a significant reduction in NK cell-cytolytic activity was observed. In addition, an increased frequency in NK cells rich fraction in virus-infected pigs did not result in rescued NK cytotoxicity, suggesting PRRSV-induced modulation in NK cell function. This could be one of the innate immune evasion mechanisms of PRRSV. All pigs with reduced NK cell cytotoxicity were viremic with titers greater than 2 logs, suggesting replicating PRRSV mediated suppression of NK cell function. In pigs, a precise selection marker for phenotypic analysis of NK cells is not available, however, CD56 is used in some studies [36, 37]. Recently, we identified comparable frequency of CD56+ and CD3-CD4-CD8α+ cells in the pig PBMC (data not shown).
PRRSV is a poor inducer of IFN-α and its level remains low throughout the course of infection [12, 13, 38]. Suppression of IFN-α production in PRRSV infected pigs is mediated by viral nsp1β . In vitro stimulation of porcine monocytes and macrophages with low levels of IFN-α (10 units/ml) stimulates increased expression of sialoadhesin, the receptor required for PRRSV internalization in macrophages. Interestingly, such a stimulation of macrophages for the initial 2-3 days is sufficient to enhance the efficiency of PRRSV infection by nearly 20-fold . Consistent with that, our results identified a slight increase in IFN-α production early post-infection in greater than 50% of PRRSV-infected pigs which maybe sufficient to augment the yield of replicating PRRSV.
Critical and coordinated functions of IFN-α/β, IL-12, and IL-15 in regulating NK cell responses during viral infections have been reported . There have been many studies demonstrating the important role played by IFN-α in modulating NK cell cytotoxicity [41–46]. Moreover, reduction in PRRSV-induced pig NK cell cytotoxicity is independent of NK cell population observed in this and earlier reports [10, 30]. Impaired basal NK cell cytolytic activity, despite the presence of normal NK cell numbers, was observed due to impairment in the STAT1 pathway . Recently, more than 1000-fold increase in IFN-α production in mice by NK cells mediated through MIP-1β and LFA was reported . Thus, PRRSV-mediated NK cell modulation in pigs would have affected both its cytotoxic function as well as IFN-α production.
PRRSV rapidly alters the cytokine response
Cytokine IL-4 mediates reduction in IL-2-stimulated pig NK cell function . Enhanced production of IL-4 in greater than 90% of PRRSV-infected pigs detected in our study appears to be having an important immune modulatory role on NK cell function. In both mice and humans, IL-4 is essential for antibody production and is a soluble diagnostic marker of Th2 immune response. In contrast, IL-4 is not a stimulatory factor for porcine B cells, and in fact in pigs, IL-4 blocks antibody and IL-6 secretion and also suppresses antigen-stimulated proliferation of B cells . IL-4 suppresses the transcriptional activity of all the inflammatory cytokines and plays an important role in regulation of inflammatory activity of pig alveolar macrophages in respiratory disease conditions . Therefore, the role of IL-4 in pigs is different compared to mice and humans. Overall, our study has demonstrated the correlative evidences on PRRSV-mediated suppression of NK cell function by coordinated modulation of multiple cytokines.
Studies have demonstrated the adjuvant role of IL-12 to live PRRSV vaccine in pigs by induction of IFN-γ production resulting in a protective immune response . Although, increased secretion of IL-12 in PRRSV-infected pigs was detected early post-infection in our study, secretion of IFN-γ remained low. 2 days post-infection maybe too early to expect IFN-γ production, but PRRSV-induced upregulation of IL-12 did not increase the IFN-γ secretion even after 3 weeks post-infection . Generally, IL-12 is a heterodimer consisting of p40 and p35 subunits [50, 51], and it is a critical regulator of T and NK cell functions [52, 53]. But IL-12 can also exist as a p40 homodimer which is a potent antagonist of immune responses . The cytokine IL-12 mediates multiple roles in the immune system which include activation of macrophages during intracellular infection . At present, reagents to differentiate homo- and heterodimers of porcine IL-12 are not available. Interestingly, our data further confirmed that PRRSV-induced enhanced IL-12 is not helping to elicit protective immunity against PRRSV infection. PRRSV has restricted cell tropism, with differentiated macrophages expressing various receptor molecules such as heparin sulphate, sialoadhesin, and CD163, which all aid in PRRSV invasion [56–58]. Therefore, PRRSV-induced upregulation of IL-12 might be involved in differentiation of macrophages which aids viral infection. However, targeted approach to investigate such a mechanism of action of IL-12 in PRRSV-infected pigs is important.
PRRSV rapidly modulates the frequency of immune cells
Lymphocytes expressing the CD8 marker are important in viral clearance by secreting IFN-γ and mediating pathogen-specific cytotoxicity [21, 22]. Pigs have abundant CD4+CD8+ T cells and these cells possess memory, T-helper, and cytolytic properties, and are called as "Th/memory cells," and they too secrete IFN-γ [21, 23]. The CD4+CD8+ T cells were found to be associated with protection in pigs vaccinated against pseudorabies virus [21, 23]. Interestingly, we detected a rapid downregulation of both CD8+ and CD4+CD8+ T cells, suggesting one mechanism of the PRRSV-mediated delay in initiation of virus-specific adaptive immunity is by altering the function and frequency of important lymphocytes early post-infection. The reason behind an increased frequency of CD8+ T cells detected in contact pigs needs further investigation.
Swine contain a large population of γδ T cells in the periphery, capable of acting in both innate and adaptive immunity [59, 60]. In PRRSV-infected pigs, γδ T cells secrete IFN-γ, indicating their role in antiviral defense . In our study, the population of γδ T cells at 2 days post-infection was decreased in contact pigs and remained unaltered in infected pigs compared to this population at preinfection, suggesting that the data on PRRSV-mediated effect on γδ T cells is not conclusive at day 2 post-infection to make any meaningful conclusion. In pigs, the cell surface marker CD172 is expressed on myeloid cells and granulocytes . A rapid reduction in the total myeloid cell pool in PBMC of infected pigs indicates their migration to the lungs, a primary site of infection.
Like in mice and humans, Foxp3+CD4+CD25+ Tregs are present in pigs and have comparable immunosuppressive properties . PRRSV-mediated upregulation of Tregs bearing a natural Treg phenotype in infected pigs indicates the role of Tregs in viral pathogenesis [26–28]. Recently, a significant decrease in the frequency of Tregs in pigs vaccinated with a modified live PRRSV vaccine co-administered with a potent mucosal adjuvant and challenged with a virulent heterologous PRRSV MN184 has suggested the cross-protective immunity . Increased frequency of Tregs in unvaccinated PRRSV challenged pigs was associated with an increased secretion of immunosuppressive cytokines, IL-10 and TGF-β . Our study has demonstrated that the increased population of Tregs in PRRSV-infected pigs at day 2 post-infection is associated with an increased production of IL-10. Within the contact pig group, a significant modulation in the cytokine secretion profile and the frequency of immune cells was observed principally in pigs which were viremic.