Paul M. Coussens

Cytokine Gene Expression in Peripheral Blood Mononuclear Cells and Tissues of Cattle Infected with Mycobacterium avium subsp. paratuberculosis: Evidence for An Inherent Proinflammatory Gene Expression Pattern

ABSTRACT In cattle and other ruminants, infection with the intracellular pathogen Mycobacterium avium subsp. paratuberculosis results in a granulomatous enteritis (Johnes disease) that is often fatal. The key features of host immunity to M. avium subsp. paratuberculosis infection include an appropriate early proinflammatory and cytotoxic response (Th1-like) that eventually gives way to a predominant antibody-based response (Th2-like). Clinical disease symptoms often appear subsequent to waning of the Th1-like immune response. Understanding why this shift in the immune response occurs and the underlying molecular mechanisms involved is critical to future control measures and diagnosis. Previous studies have suggested that M. avium subsp. paratuberculosis may suppress gene expression in peripheral blood mononuclear cells (PBMCs) from infected cows, despite a continued inflammatory reaction at sites of infection. In the present study, we tested the hypothesis that exposure to M. avium subsp. paratuberculosis suppresses a proinflammatory gene expression pattern in PBMCs from infected cows. To do this, we examined expression of genes encoding interleukin-1α (IL-1α), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-16, and IL-18, as well as genes encoding gamma interferon (IFN-γ), transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α), in PBMCs, intestinal lesions, and mesenteric lymph nodes of cattle naturally infected with M. avium subsp. paratuberculosis. Cytokine gene expression in these cells and tissues was compared to expression in similar cells and tissues from control uninfected cattle. Our comprehensive results demonstrate that for most cytokine genes, including the genes encoding IFN-γ, TGF-β, TNF-α, IL-1α, IL-4, IL-6, IL-8, and IL-12p35, differential expression in PBMCs from infected and control cattle did not require stimulation with M. avium subsp. paratuberculosis. In fact, stimulation with M. avium subsp. paratuberculosis tended to reduce the differential expression observed in infected and uninfected cows for genes encoding IFN-γ, IL-1α, and IL-6. Only IL-10 gene expression was consistently enhanced by M. avium subsp. paratuberculosis stimulation of PBMCs from subclinically infected cattle. In ileal tissues from M. avium subsp. paratuberculosis-infected cattle, expression of the genes encoding IFN-γ, TGF-β, IL-5, and IL-8 was greater than the expression in comparable tissues from control uninfected cattle, while expression of the gene encoding IL-16 was lower in tissues from infected cattle than in control tissues. Mesenteric lymph nodes draining sites of M. avium subsp. paratuberculosis infection expressed higher levels of IL-1α, IL-8, IL-2, and IL-10 mRNA than similar tissues from control uninfected cattle expressed. In contrast, the genes encoding TGF-β and IL-16 were expressed at lower levels in lymph nodes from infected cattle than in tissues from uninfected cattle. Taken together, our results suggest that cells or other mechanisms capable of limiting proinflammatory responses to M. avium subsp. paratuberculosis develop in infected cattle and that a likely place for development and expansion of these cell populations is the mesenteric lymph nodes draining sites of infection.

Mycobacterium paratuberculosis and the bovine immune system.

Abstract Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a deadly intestinal ailment of ruminants. Johne’s disease is of tremendous economic importance to the worldwide dairy industry, causing major losses due to reduced production and early culling of animals. A highly controversial but developing link between exposure to M. paratuberculosis and human Crohn’s disease in some individuals has led to the suggestion that M. paratuberculosis is also a potential food safety concern. As with many other mycobacteria, M. paratuberculosis is exquisitely adapted to survival in the host, despite aggressive immune reactions to these organisms. One hallmark of mycobacteria, including M. paratuberculosis, is their propensity to infect macrophages. Inside the macrophage, M. paratuberculosisinterferes with the maturation of the phagosome by an unknown mechanism, thereby evading the host’s normal first line of defense against bacterial pathogens. The host immune system begins a series of attacks against M. paratuberculosis-infected macrophages, including the rapid deployment of activated γδ T cells, CD4+T cells and cytolytic CD8+ T cells. These cells interact with the persistently infected macrophage and with each other through a complex network of cytokines and receptors. Despite these aggressive efforts to clear the infection, M. paratuberculosis persists and the constant struggle of the immune system leads to pronounced damage to the intestinal epithelial cells. Enhancing our ability to control this important and tenacious pathogen will require a deeper understanding of how M. paratuberculosis interferes with macrophage action, the cell types involved in the immune response, the cytokines these cells use to communicate, and the host genetic factors that control the response to infection.

A powerful and flexible linear mixed model framework for the analysis of relative quantification RT-PCR data

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently viewed as the most precise technique to quantify levels of messenger RNA. Relative quantification compares the expression of a target gene under two or more experimental conditions normalized to the measured expression of a control gene. The statistical methods and software currently available for the analysis of relative quantification of RT-PCR data lack the flexibility and statistical properties to produce valid inferences in a wide range of experimental situations. In this paper we present a novel method for the analysis of relative quantification of qRT-PCR data, which consists of the analysis of cycles to threshold values (C(T)) for a target and a control gene using a general linear mixed model methodology. Our method allows testing of a broader class of hypotheses than traditional analyses such as the classical comparative C(T). Moreover, a simulation study using plasmode datasets indicated that the estimated fold-change in pairwise comparisons was the same using either linear mixed models or a comparative C(T) method, but the linear mixed model approach was more powerful. In summary, the method presented in this paper is more accurate, powerful and flexible than the traditional methods for analysis of qRT-PCR data. This new method is especially useful for studies involving multiple experimental factors and complex designs.

Model for Immune Responses to Mycobacterium avium Subspecies paratuberculosis in Cattle

Infection of ruminant species with the facultative intracellular bacterium Mycobacterium avium subsp. paratuberculosis causes a fatal granulomatous enteritis known as Johnes disease or paratuberculosis. In cattle, Johnes disease is responsible for significant losses to the U.S. dairy industry (an

Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle

Abstract We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 ± 1.0 versus 7.6 ± 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 ±3 8.5 versus 24.1 ± 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor β, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for β glycan, cyclo-oxygenase-1, tumor necrosis factor α, caspase-activated DNase, and DRAK-2, and in theca cells for β glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.

Molecular Evidence That Growth of Dominant Follicles Involves a Reduction in Follicle-Stimulating Hormone Dependence and an Increase in Luteinizing Hormone Dependence in Cattle

Abstract The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.

Gene Expression Profiling of Peripheral Blood Mononuclear Cells from Cattle Infected with Mycobacterium paratuberculosis

ABSTRACT A bovine-specific cDNA microarray system containing 721 unique leukocyte expressed sequence tags (ESTs) and amplicons representing known genes was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from clinical and subclinical Johnes disease-positive Holstein cows (n = 2 per group). Stimulation of PBMCs from clinically infected cows with Mycobacterium paratuberculosis tended to decrease expression of 83 genes (fold change, >1.5). Of these 83 genes, 16 displayed significant down regulation across both clinical cows (P < 0.1), including genes encoding microspherule protein 1, fibroblast growth factor, and the Lyn B protein kinase. Only eight genes from PBMCs of clinically infected cows exhibited a modest up regulation following stimulation with M. paratuberculosis, including those encoding bovine CD40L, gamma interferon, interleukin-10 (IL-10), and tissue inhibitor of matrix metalloproteinases (TIMP) 4. In contrast, stimulation of PBMCs from subclinically infected cows with M. paratuberculosis tended to up regulate expression of 71 genes representing 68 unique transcripts. Of these, 11 genes showed significant up regulation (fold change, >1.5; P < 0.1) across both animals, including those encoding bovine CD40L, several matrix metalloproteinases, and SPARC (secreted protein, acidic and rich in cystine). Repression of gene expression was also observed in PBMCs from the subclinical cows, with 16 genes being significantly down regulated (fold change, >1.5; P < 0.1) across both animals, including those encoding the bovine orthologs of cytochrome oxidase subunit III, IL-1 receptor type I, and fibrinogen-like 2 protein. Only one clone, representing an unknown bovine EST, was similarly down regulated in PBMCs from both the clinical and subclinical cows. Thus, the most prominent change induced by exposure of PBMCs from clinical cows to M. paratuberculosis in vitro tended to be repression of gene expression, while changes in similarly treated PBMCs from subclinical cows was balanced between gene activation and repression. Comparison of gene expression profiles between PBMCs from clinical and uninfected (control) cows stimulated with the general mitogen concanavalin A were highly similar (overall r = 0.84), suggesting that M. paratuberculosis-induced gene repression in clinically infected cow PBMCs was not due to a general failure of the immune response in these animals.

Tuberculosis immunity: Opportunities from studies with cattle

Mycobacterium tuberculosis and M. bovis share >99% genetic identity and induce similar host responses and disease profiles upon infection. There is a rich history of codiscovery in the development of control measures applicable to both human and bovine tuberculosis (TB) including skin-testing procedures, M. bovis BCG vaccination, and interferon-γ release assays. The calf TB infection model offers several opportunities to further our understanding of TB immunopathogenesis. Recent observations include correlation of central memory immune responses with TB vaccine efficacy, association of SIRPα + cells in ESAT-6:CFP10-elicited multinucleate giant cell formation, early γδ T cell responses to TB, antimycobacterial activity of memory CD4+ T cells via granulysin production, association of specific antibody with antigen burden, and suppression of innate immune gene expression in infected animals. Partnerships teaming researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in man and animals.

Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

ABSTRACT A bovine-specific cDNA microarray system was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from control uninfected (n = 4) and Johnes disease-positive (n = 6) Holstein cows. Microarray experiments were designed so that for each animal, a direct comparison was made between PBMCs stimulated in vitro with Mycobacterium avium subsp. paratuberculosis and PBMCs stimulated with phosphate-buffered saline (nil-stimulated PBMCs). As expected, M. avium subsp. paratuberculosis stimulation of infected cow PBMCs enhanced expression of gamma interferon transcripts. In addition, expression of 15 other genes was significantly affected (>1.25-fold change; P < 0.05) by in vitro stimulation with M. avium subsp. paratuberculosis. Similar treatment of control cow PBMCs with M. avium subsp. paratuberculosis resulted in significant changes in expression of 13 genes, only 2 of which were also affected in PBMCs from the infected cow PBMCs. To compare gene expression patterns in the two cow infection groups (infected cows and uninfected cows), a mixed-model analysis was performed with the microarray data. This analysis indicated that there were major differences in the gene expression patterns between cells isolated from the two groups of cows, regardless of in vitro stimulation. A total of 86 genes were significantly differentially expressed (P < 0.01) in M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows compared to expression in similarly treated PBMCs from control cows. Surprisingly, a larger number of genes (110 genes) were also found to be significantly differentially expressed (P < 0.01) in nil-stimulated cells from the two infection groups. The expression patterns of selected genes were substantiated by quantitative real-time reverse transcriptase PCR. Flow cytometric analysis indicated that there were no gross differences in the relative populations of major immune cell types in PBMCs from infected and control cows. Thus, data presented in this report indicate that the gene expression program of PBMCs from M. avium subsp. paratuberculosis-infected cows is inherently different from that of cells from control uninfected cows.

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

BackgroundMycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johnes disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.ResultsIntracellular survival studies showed that a bovine MAP isolate (B1018) and a human MAP isolate (Hu6) persisted in relatively higher numbers when compared with a sheep MAP isolate (S7565) at 24-hr, 48-hr and 96-hr post infection (PI). MDMs stimulated with B1018 up-regulated IL-10 at the transcript level and down-regulated TNFα at the protein and transcript levels compared with stimulations by the S7565 and Hu6. MDMs infected with Hu6 showed a down regulatory pattern of IL-10 and TNFα compared to stimulations by S7565. Cells stimulated with B1018 and Hu6 had low levels of matrix metalloprotease-3 (MMP3) and high levels of tissue inhibitor of metalloprotease-1 (TIMP1) at 96-hr PI relative to MDMs stimulated by S7565.ConclusionTaken together, results suggest that the bovine (B1018) and the human (Hu6) MAP isolates lead to anti-inflammatory and anti-invasive pathways in the macrophage environment whereas the sheep (S7565) MAP isolate induces a pro-inflammatory pathway. Thus the infecting strain genotype may play a role in polarizing the host immune responses and dictate the clinicopathological outcomes in this economically important disease.