Grouping Pig-Specific Responses to Mitogen with Similar Responder Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model

Abstract

Pig peripheral blood-derived mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) stimulated with mitogens ex vivo can show significant animal-to-animal variation lead to difficulty in interpreting responses in an out-bred animal species. Mixed-cell populations were stimulated ex vivo with 2.5 μg/ml Con A or 2.5 ng/ml PMA plus 250 ng/ml ionomycin (PMAi; (LPCMs only)) or media alone for 72 hours. Supernatants were then tested for cytokine production using a Bioplex assay for porcine IFNα, IFNγ, IL-10, and IL-12. Unstimulated PBMCs had significant levels of IL-10 and the median value for this group decreased in the presence of Con A. Con A did, however, induce production of IFNα and IFNγ, but not IL-12 in this cell population. In contrast, unstimulated and Con A-stimulated LPMCs produced negligible IL-10, IFNα, IFNγ, and the majority of animals' LPMCs showed negligible IL-12 production in response to Con A. In contrast, LPMCs stimulated with PMAi produced IFNγ suggesting cytokine production is mitogen-specific response. When we tracked animal-specific responses, we observed that discrete subsets of animal's PBMCs responded to Con A with significantly increased or decreased IL-10 production relative to unstimulated cells. Further, in the LPMCs, some cells produced no IL-12 in response to Con A but showed augmented production in response to PMAi, while others showed production of IL-12 in response to Con A but no response to PMAi. Flow cytometric analysis showed that the PBMCs were a mixture of CD3+ T cells>CD21+ B cells>CD172+ myeloid cells whereas the LPMCs consisted of mainly Cytotoxic T cells and Natural Killer cells. The percentage of CD8α+CD4+ antigen-experienced T cells was greater in the LPMCs relative to the PBMCs. As expected in an out-bred species, animal-specific differences in cytokine production in response to stimulants exist and may confound interpretation of results unless tracked individually.

Dynamics of cytokine gene expression in peripheral blood mononuclear cells of indigenous and exotic breeds of pigs in India

“…On the other hand, the comparatively low expression levels of IL-10 in PBMCs of both the breeds depict an overall control of the T H 1 and T H 2 response preventing the possibility of aberrant adaptive immune response. Earlier studies indicated that the cytokine production and T H 1/T H 2 ratios are highly variable between and within pig breeds (Pasternak et al 2014). Importantly, this study revealed that the T H 1 response of Zovawk pigs in terms of mRNA expression of IL-2 and IFN-c was much higher than the mRNA expression of those of LWY pigs.…”

“…Earlier studies indicated that the cytokine production and T H 1/T H 2 ratios are highly variable between and within pig breeds (Pasternak et al . ). Importantly, this study revealed that the T H 1 response of Zovawk pigs in terms of mRNA expression of IL‐2 and IFN‐γ was much higher than the mRNA expression of those of LWY pigs.…”

“…Earlier studies indicated that the cytokine production and T H 1/T H 2 ratios are highly variable between and within pig breeds (Pasternak et al 2014). However, the dynamics of IFN-c, the signature cytokine for a T H 1 response, in case of Zovawk peaked early (3 h) compared to a slow (peaked after 24 h) and lower expression level in LWY.…”

“…Lymphocyte proliferation induced by PHA or other mitogens in vitro has been preferentially used for assessing the cell-mediated immune responses (Nowell 1960;Schlottfeldt et al 1995). Apart from lymphocyte proliferation, PHA also possesses the ability to stimulate lymphocytes to secrete cytokines as immune regulators (Reddy & Wilkie 2000;Abdalla et al 2003;Ai et al 2013). Accordingly, we observed the expression of all the signature cytokine transcripts (IL-2, IFN-c, IL-4 and IL-10) in the PBMCs of both the pig breeds (Zovawk and LWY) after mitogenic stimulation.…”

“…The early responsiveness of adaptive immune mechanisms that can be assessed by the quantity of different signature cytokine genes expressed for regulation of immune response in a time scale is a major determinant for evaluating the immune competence of a breed of animals. Although cytokine gene expression studies on PBMCs from some exotic breeds have been reported (Rottman et al 1996;Reddy & Wilkie 2000;Duvigneau et al 2007), no scientific report in this regard could be traced in indigenous pigs in India. Therefore, in the present study, the comparative dynamics of different immune-regulating key cytokine genes' expression in the mitogen-stimulated PBMCs of indigenous (Zovawk) and exotic (Large White Yorkshire: LWY) pigs were carried out.…”