PBMC isolation is time consuming and requires a relatively large volume of blood. The isolation procedure removes polymorphonuclear cells that may play a role in intracellular communication for the synthesis and release of cytokines. In contrast to PBMCs, WB contains granulocytes which can be subdivided into neutrophils, eosinophils, and basophils. Moreover, the membrane properties and activation state of PBMCs may be influenced by centrifugation (Damsgaard et al. 2009). Often, PBMCs require the addition of fetal calf serum, which contains various nutrients and hormones that may alter the immunological responsiveness of the cells (Albers et al. 2005). Furthermore, cells in isolated PBMCs undergo a significantly high level of apoptosis when compared with WB cultures (Hodge et al. 2000), and stained cell populations clearly separate in WB with better clarity (Appay and Rowland-Jones 2002). Therefore, the current study aimed to develop a time-saving method to assess porcine immune responses using a smaller volume of WB.
Undiluted WB stimulated for 6 h with PMA/ION displayed a rapid increase of TNF-α and IFN-γ (Ai et al. 2013). However, on stimulation of WB with PMA/ION for 24-h we were unable to detect any significant stimulation of T-cell, as evaluated by flow cytometry and ELISA. This may be due to the poor stability of undiluted WB when incubated for a longer time. Further, there are certain unknown factors in WB (May et al. 2013) that might interfere with the stimulation process, and the presence of RBCs in close proximity of lymphocytes may reduce the space available for stimulants to interact with immune cell receptors. Diluting WB with PMA/ION using the TM resulted in an increase in stimulation with increasing dilution as evaluated by flow cytometry and TNF-α ELISA. We observed better stimulation of IFN-γ secreting T-cells in 1:8 and 1:16 diluted WB. In contrast to IFN-γ secreting CD3+ T-cells, the TNF-α response started to increase from 1:2 dilution of WB, which might be due to the fact that TNF-α is one of the most abundant cytokines (Mascher et al. 1999) and is produced by a variety of cells, including monocytes (Olsnes et al. 2007), activated lymphocytes (Baran et al. 2001) and granulocytes (Dubravec et al. 1990). Our results are consistent with previous studies; 1:5 or 1:10 diluted WB is optimal to obtain measurable and accurate IFN-γ response (Godoy-Ramirez et al. 2004) and cell-mediated immune responses (Leroux et al. 1985; Weir et al. 1994; Gaines et al. 1996) in a long term culture.
The TM is a time-consuming process because all samples need to be treated separately. One alternative to this was to use a 96-well plate instead of microcentrifuge tubes. The 96-well plate cannot accommodate a large volume of diluted WB (100 µl WB + 800 µl RPMI = 900 µl); therefore, we sought to reduce the volume of WB for stimulation. To test our hypothesis, we diluted (1:8) 30 µl and 100 µl of WB and stimulated by the TM and PM and compared their stimulation efficiency. The PM revealed a higher mean with reduced standard deviation when compared with the TM for IFN-γ producing CD3+ cells and TNF-α response. This is consistent with previous reports of reduced intra-subject precision values using the TM when compared with PM (Russell et al. 2018). Using a smaller volume in a 96-well plate enables the simultaneous processing of a high number of samples with a reduced amount of WB. Additionally, reagent use is reduced, and the harvesting of samples is simple and time saving. For further experiments in this study, PM was used because it was easier and produced lower inter-individual variation.
Depending on the method employed, human PBMCs and/or WBA techniques provide fine conditions for the aim proposed and WB cultures are well-suited proxy-measures (Silva et al. 2013). On stimulating 1:8 diluted WB and 106 PBMCs, the results were at least comparable with no significant difference in the frequency of IFN-γ secreting CD3+ T-cells.
WB was optimally stimulated with PMA/ION at 1:8 dilution; therefore, we next assessed our assay with a wide array of immune stimuli, including TLR agonists (LPS and poly I:C), bacteria (S. suis), and virus (PRRSV). LPS induces human PBMCs to synthesize pro-inflammatory cytokines, including TNF-α (Janský et al. 2003; Ngkelo et al. 2012; Ai et al. 2013; Irtegun et al. 2016) and anti-inflammatory cytokines, including IL-10 (Janský et al. 2003). Stimulating WB with LPS produces a higher concentration of cytokines when compared with PBMCs (Chen et al. 2010). WB contains the LPS binding protein (LBP), which plays a vital role in eliciting an immune response by enhancing the transfer of LPS aggregates from an aqueous solution to the CD14-TLR4 receptor complex (Tobias 1986; Wright et al. 1990). Therefore, WB provides a more natural environment for measuring the inflammatory response. Moreover, poly I:C induces the synthesis of pro-inflammatory cytokines, including TNF-α (Dorresteijn et al. 2010; Duffy et al. 2014; Pjanova et al. 2019), and anti-inflammatory cytokines, including IL-10 (Dorresteijn et al. 2010), in human PBMCs or WB. Porcine PBMCs stimulated with poly I:C elicit differential gene expression, similar to those induced by RNA viruses (Wang et al. 2014). Our study indicates that both LPS and poly I:C induced immune cells in porcine WB to produce TNF-α and IL-10.
S. suis stimulates the release of pro-inflammatory cytokines in mice (Segura et al. 1999) and human mononuclear cells (Vadeboncoeur et al. 2003) in vitro. Stimulation of 1:3 diluted porcine WB with HK S. suis increases cytokine mRNA expression; however, a low level of protein secretion is displayed (Segura et al. 2006). The present study provides evidence that HK S. suis stimulates WB to secrete pro- and anti-inflammatory cytokines (TNF-α and IL-10, respectively). In this study, HK RS induced TNF-α (non-significantly) and IL-10 in WB at 108 cfu/ml, while FI induced the secretion of IL-10 alone. The discrepancy with Segura and colleagues might be because of differences in the pathogenicity of S. suis strains used and WB dilution. Interestingly, lower cfu/ml (106) in both strains stimulated IL-10 but there was no significant difference in TNF-α concentration.
Next, we extended our assay to analyze the induction of CTLs response by PRRSV strains: NA42, NA45, and 10D415. We analyzed the response at 7 and 14 DPC. The CTLs response was higher in samples re-stimulated with homologous strains when compared with mock treated samples. The PRRSV strain, NA42, significantly induced a higher (p < 0.05) CTLs response at 14 DPC, while there was no significant response upon re-stimulation with NA45 and 10D415. Our study is consistent with a previous report showing that CD3+CD8high, designated as CTLs, are induced at 14 DPC (Costers et al. 2009). This might be due to strain differences; pigs respond differently to different PRRSV strains (Shabir et al. 2018b). Additionally, the cellular immune response depends on the viral isolate used in vitro stimulation, which can interact differently with different immune cells (Díaz et al. 2006; Correas et al. 2017; Madapong et al. 2020). More importantly, the role of CTLs in combating PRRSV infection in hosts requires further investigation, which is out of the scope of the present study. The kinetics of neutrophils in blood changes after infection with PRRSV (Liu et al. 2013). An advantage of WBA over PBMCs is that WB can be used to evaluate lymphocytes, monocytes, and granulocytes all at once with a small volume of blood. In conclusion, our study defines porcine WBA as a model to study the cellular responses that are induced by different PRRSV strains.
We conducted this study to standardize the dilution of porcine WB for optimal stimulation to evaluate immune responses with chemical and biological stimulants. We observed that the dilution of WB is necessary for stimulation. This assay can be used to study the immune responses that are induced by a wide variety of stimulants, including chemical and biological stimulants. Furthermore, the PRRSV strain, NA42, significantly induced CTLs at 14 DPC. Taken together, we have shown that this assay provides reproducible results and is better suited to study immune responses in swine because it maintains immune cells in more natural conditions.