Correspondence: Deming Zhao, State Key Laboratories for Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Tel.: +86 1 062 732 980; fax: +86 1 062 732 975; e-mail: email@example.com
Bovine tuberculosis (BTB) is a chronic infectious disease caused by the pathogen Mycobacterium bovis and poses a long-standing threat to livestock worldwide. To further elucidate the poorly defined BTB immune response in cattle, we utilized monocyte-derived macrophages (MDMs) to assess the gene expression related to M. bovis Beijing strain stimulation. Here, we demonstrate the existence of distinctive gene expression patterns between macrophages of healthy cattle and those exposed to BTB. In comparing MDMs cells from healthy cattle (n=5) and cattle with tuberculosis (n=5) 3 h after M. bovis stimulation, the differential expressions of seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in M. bovis response were examined. The expressions of these seven genes were increased in both the tuberculosis-infected and the healthy cattle to M. bovis stimulation, and two of them (TLR2 and IL10) were significantly different in the tuberculosis and the healthy control groups (P≤0.05). The increase in the expression of the TLR2 gene is more significant in healthy cattle response to stimulation, and the change of IL10 gene expression is more significant in tuberculosis cattle. Additionally, we investigated the cytopathic effect caused by M. bovis stimulation and the relationship between M. bovis and MDMs cells to obtain a general profile of pathogen–host interaction.
Mycobacterium bovis is an important pathogenic bacterial species and the causative agent of most cases of tuberculosis in cattle. Bovine tuberculosis (BTB) continues to pose a threat to livestock worldwide and also has serious implications for human health (Tiruviluamala & Reichman, 2002). Macrophages are the major host cell of M. bovis infection and they mediate the host immune response to BTB through pathogen recognition and activation of an inflammatory response (Casadevall, 2008; Ahmad, 2011). Studies have shown differential gene expression between animals with tuberculosis and healthy animals (MacHugh et al., 2009; Moller & Hoal, 2010; Maertzdorf et al., 2011). These studies have implicated innate immune responses, TLR signaling and Th1 cytokines in the cellular response to M. bovis (Werling & Jungi, 2003; Netea et al., 2005). Innate immunity relies heavily on the behavior of inflammatory molecules. Proinflammatory cytokines TNF-α, IL1, and its receptor IL1R1, play an important role in the innate immune response, which is an essential mechanism for host defense against invading M. bovis (Salgame, 2005). TLR2 and TLR4 could recognize BTB products and rapidly generate a defensive response involving numerous proinflammatory cytokines and Th1 cytokines to restrict the growth of intracellular M. bovis. IL10 could downregulate the Th1 response and upregulate Th2 response in pathogen–host interaction, which may lead to the lack of protection of the host from M. bovis (Jacobs et al., 2000).
We hypothesized that macrophages from tuberculosis infection and healthy cattle may have distinctive immune regulation and gene expression in response to M. bovis stimulation. In this study, we use a monocyte-derived macrophages (MDMs) model to study the interaction of M. bovis and macrophages from tuberculosis cattle as well as healthy controls. Using real-time quantitative PCR, the expression of seven genes implicated in BTB (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) was examined in MDMs from tuberculosis and healthy control cattle stimulated with M. bovis, respectively.
We also observed the cytopathic effect (CPE) caused by M. bovis stimulation in MDMs cells by microscopy directly. Using Ziehl–Neelsen staining, bacteria entering into MDMs cells were detected to obtain a general impression of pathogen–host interaction. The growth and survival status of intracellular M. bovis may directly reflect the capability of cells in resisting and killing intracellular bacterium. We assessed the survival status of intracellular M. bovis by bacterial CFU in the MDMs to see the difference in the bacterial load between MDMs from tuberculosis and healthy control cattle.
Materials and methods
Bacterial strains and culture
Virulent M. bovis Beijing strain (bovis strain 93006) was received from the China Institute of Veterinary Drug Control (CVCC, China). This is a wild-type strain isolated from tuberculosis lesions of a tuberculin skin test-positive cow in Beijing in 1953. Bacteria were grown at 37 °C under a shaking condition for 8 weeks in Middlebrook 7H9 broth (Difco) supplemented with 10% v/v albumin–dextrose–catalase (ACD) and 0.05 v/v Tween 80. The CFU was determined by plating 100 μL of serial dilutions onto Petri dishes containing Middlebrook 7H10 agar, supplemented with Tween 80 and albumin–dextrose–catalase (ACD). These dilutions were stored at −80 °C and were subsequently used for virulent challenges.
Cattle and control subjects
Ten Holstein cows recruited from herds of a cattle farm in Shandong province, China, were used for this study. The five infected animals were selected on the basis of the skin-fold thickness response to bovine tuberculin in the single intradermal tuberculin test (SITT). The SITT reactor animals were selected where the skin-fold thickness response to bovine pure protein derivative (PPD) exceeded at least 4 mm. All of these animals were also tested positive in a whole-blood interferon-γ (IFN-γ) enzyme immunoassay (Bovigam, Prionics AG), which is based on the use of the Bovigam avian PPD- and Bovigam bovine PPD-stimulating antigens. None of the infected subjects had any symptom of active tuberculosis. The five noninfected control animals were selected from a herd without a recent history of tuberculosis and were PPD tested and IFN-γ EIA negative.
IFN-γ ELISA assays
ELISA assays were performed according to the manufacturer's instructions (Bovigam, Prionics AG). Briefly, whole heparinized blood was mixed in a 24-well culture plate in a 1 : 1 ratio with RPMI 1640 medium (Invitrogen), and then blood was stimulated with avian PPD or bovine PPD (25 000 IU each tuberculin) in 100 μL in three replicates. Phosphate-buffered saline (PBS) was used as a negative control (nil antigen). The results are calculated as mean nil antigen, avian and bovine PPD absorbance values for each sample. Blood plasma collected from cattle, within 3–30 days postapplication of the skin test, having an OD value greater than that of avian PPD and nil (PBS) antigen by over 0.100 indicates the presence of M. bovis infection (Supporting Information, Table S1).
Peripheral blood mononuclear cell (PBMC) isolation and stimulation of MDMs
PBMCs were separated from acid citrate dextrose (ACD) anticoagulated blood of cattle (five infected and five noninfected) by OptiPrep (Asix-Shield, Norway) gradient centrifugation according to the manufacturer's protocol. From 10 mL of blood, we obtained approximately 2–5 × 106 PBMCs. To derive monocytes, PBMCs were plated in six-well plates (Costar, Corning), 5 × 106 cells per well, containing RPMI-1640 (Invitrogen) with 10% fetal calf serum (FCS; Hyclone), 2 mM l-glutamine, 10 mM HEPES and antibiotics (100 U mL−1 penicillin and 100 U mL−1 streptomycin) for 2 h at 37 °C, 5% CO2. Nonadherent cells were removed by washing with PBS. Then, adherent cells were incubated for 5 days at 37 °C, with 5% CO2 to obtain MDMs.
Cell infection with M. bovis
MDMs (2 × 105 cells per well) were washed with PBS three times to remove antibiotics before infection. Cells of treatment groups were challenged with M. bovis (MOI=10 : 1) for 4 h at 37 °C, with 5% CO2. Cells were then washed three times with PBS to remove extracellular bacteria and incubated again with fresh RPMI-1640 containing 10% FCS for 24 h. The challenge of M. bovis was substituted with PBS in the control groups.
RNA extraction and cDNA preparation
Cells were resuspended in Trizol (Invitrogen) after 3 h of stimulation and stored at −80 °C. RNA was isolated from MDMs from the treatment and the control groups, according to the manufacturer's protocol (Invitrogen), and then stored in RNase-free water at −80 °C. Total RNA was reverse transcribed to cDNA using the RevertAid first-strand cDNA synthesis Kit (Fermentas, Lithuania).
Real-time quantitative PCR
For animal samples, expression levels in eight genes (seven selected genes and one control) were examined with real-time PCR. The H3 histone family 3A gene (H3F3A) was used as a control gene for animal samples to normalize expression data for target genes (MacHugh et al., 2009). Gene expression levels were detected using the DNA Engine Opticon TM2 fluorescence detection system (MJ Research Inc.) and SYBR Green (RealMasterMix, Tiangen). The specific gene primer pairs are shown in Table 1. Real-time quantitative PCR data were analyzed using the method, and differences between groups were analyzed with a t-test by spss software.
Table 1. Real-time quantitative PCR primer pairs and amplicon size for all analyzed genes
Forward primer (5′–3′)
Reverse primer (5′–3′)
Amplicon size (bp)
Cell smear and acid-fast staining
Cells were collected at various time points (3, 12 and 24 h) to prepare a cell layer smear. The cell smear was stained using the acid-fast staining method according to the Ziehl–Neelsen stain protocol. The intracellular M. bovis number count was performed using CFU assessment. Cells from each timepoint (3, 12 and 24 h) were washed three times with PBS to remove the extracellular bacteria. Cells were then lysed with a 0.1% Triton X-100 solution, serially diluted in 7H9 medium with 0.05% Tween 80 and plated onto 7H10 agar plates containing 10% ADC. CFU were counted after incubation at 37 °C for 3–4 weeks.
Gene expression of MDMs
The gene expressions of IL1β, IL1A, IL1R1, TNF, TLR2, TLR4 and IL10 were examined by real-time PCR in MDMs in response to M. bovis stimulation from tuberculosis and healthy cattle (n=5 in each group).
Of the seven genes examined in MDMs from tuberculosis animals, six genes (except IL1A) showed significant differential expression after 3 h of stimulation with M. bovis as compared with nonstimulated controls at the P≤0.05 level (Fig. 1, Table S2). IL1, IL1R1 and TNF-α genes showed increased expression 3-h poststimulation in both groups, which shows that the proinflammatory cytokine TNF-α, IL1 and its receptor IL1R1 play a role in the early interaction of host cells and M. bovis. Increased expression of TLR2 and TLR4 genes (2.64-fold and 6.49-fold) was also noted. These genes regulate the innate immune system. Anti-inflammatory cytokine IL-10 showed increased expression by 8.74-fold over the nonstimulated control.
Healthy control animals
Of the seven genes from MDMs from healthy control animals, six genes (except IL1A) showed significant differential expression after 3 h of stimulation with M. bovis as compared with nonstimulated controls at the P≤0.05 level (Fig. 1, Table S3). Similar to that seen in the stimulated tuberculosis group, the expressions of proinflammatory cytokine TNF-α, IL1 and its receptor IL1R1 were significantly increased 3-h poststimulation. TLR2 and TLR4 genes increased 6.54-fold and 5.28-fold, respectively, in the healthy control group. However, the expression of IL10 (2.90-fold) 3-h poststimulation was less compared than that seen in the stimulated tuberculosis group (8.74-fold).
Difference of two groups
We compared the gene expression levels in the two groups. The results showed that two of the seven genes examined (TLR2 and IL10) were differentially expressed in both the stimulated tuberculosis subjects and the stimulated healthy control subjects (P≤0.05 by t-test). Although TLR2 showed increased expression in both stimulated groups, it had a greater fold increase in the stimulated control group (6.54-fold) over the stimulated tuberculosis group (2.64-fold). This may indicate that TLR2 plays a larger role in regulation in healthy animals. In contrast, IL10 expression in stimulated tuberculosis animals (8.74-fold) was greater than that seen in the stimulated control group (2.90-fold). Thus, TLR2 may play a key role in the response of MDMs from healthy cattle to M. bovis stimulation, while IL10 may play a similar key role during M. bovis stimulation of MDMs from tuberculosis cattle.
CPE of M. bovis
The CPE and the relationship between M. bovis and MDMs cells were observed directly by microscopy (Fig. 2) and Ziehl–Neelsen stain (Fig. 3). The present findings demonstrate that the CPE could be seen under microscopy after 3 h of stimulation, and it became more severe over time. Necrosis and detachment of cells were caused by the intrusion and adherence of bacteria. At 3 h, the medium incubated with cells was almost clear and intracellular fast-acid bacteria were seldom seen. However, by 10 h, the medium became unclear due to cellular debris and dead cell granules and bacteria could be observed inside the cytoplasm and the nucleus of some cells. Twenty-four hours after stimulation, the massive cellular death made microscopy images obscure and only a small number of cells survived. The M. bovis used in this study was a virulent strain, triggering a strong interaction and quickly leading to massive cellular death. Based on the observations made by microscopy and fast-acid stain, there are no obvious differences in CPE of M. bovis between MDMs from tuberculosis and healthy control cattle.
CFU of intracellular M. bovis
The growth and survival status of intracellular M. bovis were assessed by bacterial CFU in the MDMs from tuberculosis and healthy control cattle (Fig. 4). Our data indicate that at 3 h, the CFU of intracellular survival of M. bovis is very low and difficult to measure in several subjects (less than three bacterial clones on a 7H10 agar plate). This result is consistent with the previous observation, by microscopy and fast-acid stain, that M. bovis grows poorly in cells after 3 h of infection. This study also shows that 10 h after stimulation, CFUs of M. bovis significantly increased compared with that seen at 3 h in MDMs from both tuberculosis and control cattle. However, intracellular M. bovis CFU decreases drastically after 24 h, which could be attributed to the massive cellular death observed. The CFU assessment shows no significant difference in the intracellular bacterial load of M. bovis between MDMs from tuberculosis and healthy control cattle.
BTB is a chronic infectious disease caused by the pathogen M. bovis and continues to pose a threat to livestock worldwide. Mycobacterium bovis is the causative agent of most cases of tuberculosis in cattle and M. bovis Beijing strains cause a substantial proportion of tuberculosis cases worldwide (Chen et al., 2009; Kremer et al., 2009). Understanding the specific immune response to BTB will aid in developing improved control and diagnostic strategies. Studies on tuberculosis in humans indicate that innate immunity, TLR signaling and the Th1/Th2 bias of the immune response are essential for host defense against tuberculosis (Doherty & Arditi, 2004; Winek et al., 2009; Ahmad, 2011). However, these specific cell signal pathways and immune responses are poorly defined in cattle.
Meade et al. (2006) examined the gene expression profiles of PBMCs from BTB-infected and healthy cattle and demonstrated the differential expression of innate immunity-related genes. In this study, gene expressions of MDMs cells from tuberculosis and healthy groups stimulated with M. bovis were detected. Seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in immune responses were examined. In MDMs, the expression of the seven examined genes was increased in both stimulated tuberculosis and stimulated healthy cattle. The expression of the proinflammatory cytokine TNF-α, IL1β and its receptor IL1R1 markedly increased, indicating that these genes may play a key role in the early interaction of host cells and M. bovis. The expression of these three genes, although elevated in response to M. bovis stimulation, showed no significant difference between the two groups. This finding may indicate that the macrophages from tuberculosis cattle have a capability similar to healthy cattle in generating proinflammatory cytokine (IL1β and TNF-α) during early immune response to M. bovis stimulation. In agreement, it is frequently reported that the tuberculosis infection could induce a burst of inflammatory cytokines IL1β and TNF-α in the infected location (Arcila et al., 2007; Qiu et al., 2008; Winek et al., 2009).
Two Toll-like receptor genes (TLR2 and TLR4) were examined. The two genes have been studied widely, because they are very important in innate immunity and TLR signaling aids the activation of antigen-specific T cells (Cooper, 2009). Previous studies demonstrated that M. tuberculosis products can be recognized by TLR2 or TLR4 (Aliprantis et al., 1999; Underhill et al., 1999; Abel et al., 2002). The Th1/Th2 bias of the immune response can be mediated by TLRs, and it contributes to the outcome of BTB infection in cattle (Jones et al., 2001; Werling & Jungi, 2003; Doherty & Arditi, 2004). In our study, the expressions of TLR2 and TLR4 were increased in both tuberculosis and healthy control groups 3-h poststimulation; however, only TLR2 expression showed a significant difference (P≤0.05) between the two groups. The increase in the expression of the TLR2 gene is more significant in healthy cattle (6.54-fold) response to stimulation than the tuberculosis group (2.64-fold). This demonstrates that the TLR2 signal pathway may play a larger role in healthy control cattle than the tuberculosis group, possibly resulting in a more efficient proinflammatory gene activation.
An important anti-inflammatory cytokine, IL10, was also examined in our study. IL10 downregulates the Th1 type immune response and upregulates the Th2 type in pathogen–host interaction (Jacobs et al., 2000). It has been shown in previous studies, in both humans and mice, that increased expression of IL10 is associated with the decreased ability of macrophages to restrict the growth of intracellular Mycobacterium (Jamil et al., 2007; Bilenki et al., 2010). Our results show that IL10 expression is more increased in tuberculosis cattle (8.74-fold) than the healthy group (2.90-fold) relative to 3-h poststimulation compared with nonstimulated cells. The differential regulation of IL10 in tuberculosis cattle may reflect vulnerability in the defense of macrophages against M. bovis.
The development trend of gene expressions in this study is consistent with that seen by Meade and colleagues, while the different levels of gene expression seen could be attributed to cell populations (PBMCs vs. MDMs), stimulator (bovine tuberculin vs. M. bovis) or comparison of different clinical phenotypes [active BTB infection vs. Latent TB (LTB)]. Our study provides evidence of differences in gene expression between tuberculosis and healthy cattle, which confirms that the innate immune response, TLRs signal pathway and Th1/Th2 bias are important in BTB infection. The current techniques cannot predict the risk of an individual LTB animal developing into the active disease, and genes implicated in susceptibility and resistance of tuberculosis in cattle cannot point to clear solutions. Building on the differences in gene expression regulation demonstrated in this study, it may provide insights into the diagnosis and treatment of tuberculosis cattle and lead to diagnostics that may characterize the immune response prognostic information in BTB infection.
This work was supported by the key project of Ministry of Agriculture, China (Project no. 2009ZX08009-183B), the Beijing Science Foundation of China (Project no. 6101002) and the Natural Science Foundation of China (Project no. 30972164).