The effect of immunological status, in-vitro treatment and culture time on expression of eleven candidate reference genes in bovine blood mononuclear cells

Quantification of mRNA expression to investigate the cellular mechanism in both physiological and pathological state of cells or tissues has been a method of choice in many research projects [1‐3]. Various techniques including northern blotting, microarray, and real-time RT-PCR have been developed to help researchers to quantify very small amounts of mRNA, accurately [4]. Nowadays, the analysis of mRNA expression by real-time RT-PCR is the most feasible method due to the low cost, high sensitivity, and possibility of in-house species-specific designing of primers/probes. Despite of the advantages of this technique, the results, and procedure need to be verified due to the several pitfalls which may cause unreliable results [5]. A normalization step that removes variation in the quantity of starting material and efficiency of amplification between samples and runs, can greatly affect the reliability of quantitative RT-PCR results. Several methods have been proposed to normalize data such as normalization according to weight/volume of sample, quantity of RNA and/or using internal control genes [1, 3].

Housekeeping genes (HKGs) are considered to be expressed constantly and have been widely used as an internal control or reference gene. However, if the expression of the internal control genes are affected by the experimental procedure (i.e. treatments) then the results would not be valid [1]. Recently, expression stability of many HKGs have been investigated in different cells and tissues, including animals, and plants tissues, under treatment and/or untreated condition [1, 6‐10]. The common conclusion from those studies emphasized that there is no general housekeeping gene to be used as a reference gene among all cells and tissue types, as well as under different treatments. On the other hand, in any experiment that aims to study gene expression, it is necessary that a gene or a set of genes that are not affected by the treatment or cell/tissue type are identified to be used as an experimental control [1].

The relative quantification of mRNA by real-time RT-PCR is a preferred method, particularly when species specific monoclonal antibody are lacking to analyze cellular phenomenon [11]. Nevertheless, quantification of mRNA in cultured cells, in particular those of the adaptive immune system, is complicated by several factors including immunological memory, in-vitro cell culture, and treatments by mitogens and/or antigens. Thus, many genes, including housekeeping genes, which may be constitutively expressed in non-immune system cells are likely differentially expressed in cells of the immune system. In the current study, the stability of the expression of eleven housekeeping genes (18s, 24s, ACTB, B2M, CTBP, GAPDH, MDM4, PPIA RAD50, SDHA, and YWHAZ) was analyzed in cultured bovine blood mononuclear cells (BMCs). BMCs were isolated from individuals with different in-vivo exposure to test antigens (immunological status) and treated in-vitro with a mitogen or a type 1 or type 2 test antigen. The hypothesis being that the history of host exposure to test antigens and culturing time affect the expression of housekeeping genes. The objective of this study was to identify potential instability of HKG expression in BMCs in immunological research due to immunological memory and experimental treatment when samples are collected from an outbred species in uncontrolled environmental settings.

Normalization of real time RT-PCR data to a constitutively expressed gene is the ideal method to eliminate technical variations in relative quantification of mRNA. However, considerable variation has been recently reported in expression of housekeeping genes in different cells and tissue [3, 13, 14]. The expression of housekeeping genes in cultured primary cells is likely more prone to variation due to several steps of physical and/or chemical treatments to isolate the cells. Additionally, for the cells of adaptive immune system, immunological memory, the unique feature of adaptive immune system, can potentially affect the expression of housekeeping genes in these cells. The current study, for the first time, evaluated potential sources of inconsistency and variation in housekeeping gene expression including the effect of immunological memory in cultured bovine BMCs. Results of the study showed cell culture alone, and in-vitro treatment of the cells with a mitogen or test antigens significantly affects the expression of HKGs commonly used as reference genes. Notably, the effect of in-vitro antigenic stimulation of BMCs on some housekeeping genes depends on if and when the host has been previously exposed to the antigen.

Restricted maximum likelihood method in a linear mixed effects model was chosen to analyze the results in this study, despite of availability of numerous databases and algorithms such as Genevestigator, Bestkeeper, geNorm and NormFinder [15]. Bestkeeper and geNorm utilize similar pair-wise analysis without including any further classification of samples while NormFinder use a model based approach [16‐18]. Regardless of similarity or differences between these programs, they all return the most stable gene(s) based on variation between samples or groups. However, the source of inconsistency remains undescribed in the results of these programs. The PROC MIXED procedure in SAS software, which was used in this study, provides the user options to include sources of variation in a randomized block or repeated measure design. In addition, post-hoc analysis revealed differences between the levels of each effect.

The expression of many housekeeping genes in different bovine cells or tissues, including cells [19], adipose tissue [9], pre-implantation embryos [10], cell lines [1] and primary cells of immune system have been extensively studied [6, 13]. Spalenza et al. have reported that YWHAZ, S24, and PPIA are the most stably expressed genes in bovine BMCs when the cells are freshly isolated and have not been cultured nor have undergone any in-vitro treatment [6]. In the current study, similar conditions resulted in similar finding, except for the expression of YWHAZ which was increased significantly after 24 hours culture. In another study by Goossens et al. YWHAZ along with SDHA and GAPDH were reported to be stably expressed in in-vitro fertilized bovine embryos up to seven days [10]. In contrast, none of these genes were stably expressed in cultured bovine BMCs, showing that the stability of HKGs expression depends on both the tissue and also the in-vitro environment. On the contrary, ACTB was found to be among the least consistently expressed genes in several studies, including the current study, despite the common use of this gene as a reference gene in relative expression studies [6, 9, 10]. These similarities and differences may be explained by cell adaptation to the in-vitro environment. Further, it is very likely that major physical and chemical differences between the in-vivo and in-vitro environment as well as cell injuries that may happen during the isolation procedure would alter the expression of genes that form the structure of cells, mediate cellular response to injury and or hyperoxia such as ACTB and SDHA [20‐22].

Treatment of either cells or the host, whether in-vitro, or in-vivo, is another potential source of variation in the expression of housekeeping genes. Ostrowska et al. demonstrated that oral glucogenic feed supplementation caused variation in housekeeping gene expression in bovine liver. Notably, ACTB was the least stably expressed gene in their study [23]. In alignment with these results, but in an in-vitro study, it has been shown that normalization against different reference genes will significantly affect the result of IL10 and TNF-α expression in lipopolysaccharide treated monocytes [13]. Likewise in the current study, significant changes were observed in gene expression after in-vitro treatment, which could potentially change the results of quantification if they were used for normalization. Only the expression of CTBP1 remained unchanged after in-vitro treatment of BMCs with Con A, C. albicans extract, or HEWL (Figs. 2, 3, and 4). In addition, previous exposure of the host to the antigen used for stimulation and culturing time significantly affected the expression of tested housekeeping genes (Table 2). A notable contrast was observed on the effect of the host by comparing mitogenic versus antigenic stimulation. The immunological status of the host did not have a significant effect when BMCs were treated with the mitogen Con A, however immunological status was significant on the expression of some HKGs under antigenic treatment (Table 2). Con A is a mitogen that nonspecifically stimulates T lymphocytes whereas antigens stimulate specific lymphocyte clones via T cell receptors (TCR) [24]. Thereby, as expected immunological memory affects responses of lymphocyte to antigenic stimulation but not mitogen. Furthermore, the effect of the host became greater the longer the cells were in culture. For instance, the expression of PPIA under treatment with both test antigens were significantly higher than zero only in groups that had been previously exposed to the antigens. The same was the case for RAD50 and 24 s under treatment with C. albicans (Figs. 3 and 4). In contrast, no significant difference was observed in expression of the tested housekeeping genes between individuals recently exposed to the test antigens or individuals exposed 2 years before sampling, with the exception of MDM4 (Figs. 3 and 4). MDM4 expression was significantly increased 4 hours post antigenic treatment in BMCs when individuals had been boosted one week prior to sampling, compared to untreated control. This increase is likely due to the anti-apoptotic role of this gene in lymphocyte proliferation in response to antigens [25, 26].

The stability of reference gene(s) expression over time after in-vitro treatment is critical when quantification of expression is relative to time zero or use of the kinetics of expression are intended. Both designs are common in gene expression studies but the stability of expression in these designs are often overlooked. Anstaett et al. have shown that B2M and YWHAZ expression are most stable among 8 candidate genes using Bestkeeper and geNorm programs in BL3 cells up to 48 hours post infection with Bovine Viral Diarrhea Virus. But, the expression of ACTB, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were reported from the same study to be the most stable when NormFinder was used [1]. This inconsistency may be due to different approaches and lack of compatibility of these software with multi-dimensional experimental designs. However, the results of the current study from statistical modeling showed that CTBP1 is the only constantly expressed gene after treatment of BMCs with C. albicans and CTBP1 and RAD50 are constantly expressed after stimulation of BMCs with HEWL up to 24 hours post treatment (Figs. 3 and 4). In another example, the expression of PPIA, which was shown to be constant by Spalenza et al. as well as in the first part of the current study, was significantly up regulated at 24 hours post in-vitro treatment. Con A stimulation during culture time also caused significant up regulation of all tested genes but varied among different host group for 24 s and MDM4 (Fig. 2).

The current study for the first time evaluated the effect of previous in-vivo antigenic exposure of the host on expression of HKGs in cultured BMCs. In agreement with previously published papers, this study did not find any common HKGs expressed constitutively under all in-vitro treatments, as well as over time in bovine BMCs. However, 24 s, CTBP1, and RAD50 were constantly expressed independent from previous exposure of the host under in-vitro treatment with ConA, C. albicans, and HEWL, respectively. The current study revealed that the immunological status of the host can be an important potential source of inconsistency when primary cells of the adaptive immune system are studied in samples from un-controlled environments common to outbred species. This is particularly true when cells are also exposed to antigenic in- vitro treatment. The combination of previous environmental exposure, in-vitro treatment, and culturing time are sources of variation that can alter the expression of housekeeping genes which if used for the normalization of gene expression data can inadvertently generate misleading results.