REGULATORY T CELLS IN BOVINE PARATUBERCULOSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0220728
• Grant No.: 2010-65119-20409
• Proposal No.: 2009-01636
• Project Start Date: Dec 15, 2009
• Project End Date: Dec 14, 2012
• Grant Year: 2010
• Program Code: [92521]- Animal Health and Well-Being: Animal Health

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Animal Science

Non Technical Summary
Johnes disease represents one of the most economically significant diseases in the US dairy herd and has become a global problem. A report from the National Animal Health Monitoring Service estimated that in herds where 10 percent of culled cows showed clinical signs of Johnes disease, the average cost to producers was 227.00 US dollars per cow on the operation. Conservative estimates suggest that the U.S. dairy industry absorbs over 200 million dollars per year in losses due to Johne's disease (NAHMS, 1997), although some estimates place this figure closer to 1.5 billion dollars. Because many subclinical infections with MAP go undiagnosed for several years, the actual cost to producers, and National infection rates, may indeed be much higher than more conservative estimates suggest. A controversial link between MAP and exacerbation of some cases of human Crohns disease suggests that this pathogen may also be an important food safety concern. The burden of Johnes disease to the dairy industry was pointedly described when, in 2000, the industry asked congress to appropriate 1.3 billion dollars toward eradication of the disease. Testimony in the ensuing debate by dairy industry leaders clearly spelled out the negative economic impact of Johnes disease on the industry and highlighted the potential human health consequences of allowing Johnes disease positive cows to remain in the food chain. Despite the overwhelming importance of Johnes disease to the global dairy industry, it is clear that our knowledge of the bovine immune response to MAP (as well as other pathogens), although improving, is sparse. Much of the PDs recent work has been devoted to understanding how MAP interacts with the bovine immune system, both at a gross overall level in vivo, and in a mechanistic sense in cultured immune cells. Co-PD Davis has also been actively involved in these investigations. Completion of Objectives 1 and 2 outlined in this proposal will provide novel data that should shed considerable light on the role of regulatory T cells in MAP pathogenesis and host immune responses to MAP. Objective 3 will use this new information to discover when these cells arise during MAP infection and the potential effect of vaccination on their development. This information, combined with literature on other organisms (such as H. Pylori) will help guide MAP vaccine trials and may explain, finally, why the host immune response to MAP in unvaccinated cattle changes from an appropriate Th1-like activity to an ineffective Th2-like activity.

Animal Health Component

5%

Research Effort Categories

Basic

95%

Applied

5%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3499	1090	90%
311	3499	1100	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3499 - Dairy cattle, general/other;

Field Of Science
1090 - Immunology; 1100 - Bacteriology;

Keywords

johnes disease

paratuberculosis

mycobacteria

immunity

regulatory t cells

crohns disease

Goals / Objectives
Our overall hypothesis is that infection with MAP leads to development of regulatory T cells and that these cells limit peripheral and tissue-specific immune responses to MAP, allowing MAP to proliferate within intestinal tissues and develop to clinical disease. Based on a number of previous studies, we believe that as the number of MAP infected macrophages increases in intestinal tissues, T cells in infected tissues and local lymph nodes receive a constant low-level antigen stimulus, leading to development of regulatory T cells capable of dampening anti-MAP proinflammatory and cytotoxic immune activity. Another possibility is that intestinal macrophages selectively infected by MAP are deficient in co-stimulatory activity and thus T cell interactions with these cells leads to development of a regulatory phenotype instead of an effector proinflammatory phenotype. Another exciting possibility is accumulation of regulatory gamma-delta T cells at sites of MAP infection, which could turn naive T cells into regulatory T cells via expression of TGF-beta. Whatever the actual mechanism, it is clear that antigen reactive regulatory T cells are present in peripheral blood mononuclear cells (PBMCs) from MAP infected cows and that these cells severely limit proinflammatory responses to MAP antigens in vitro. Under this project our Specific Objectives are: 1) Examine MAP antigen specific regulatory T cells and measure their effects on antigen reactive effector cells and CD8+ cytotoxic cells. 2) Examine lesions and draining lymph nodes from MAP infected cattle for presence of regulatory T cells, including gamma-delta T cells. 3) Follow a cohort of calves naturally infected with MAP to determine the ontology of antigen-specific regulatory T cell development. Determine the effect of MAP vaccination on regulatory T cell development. Expected Outputs from the project are: 1) we anticipate that finding that there is a population of antigen-specific CD4+CD25high regulatory T cells in PBMCs from MAP infected cattle that quell proinflammatory activity of MAP reactive effector cells. 2)IOur studies will provide data on the percentage of T cells within lesions of MAP infected cattle that are of a regulatory phenotype, possibly providing linkage to previous experiments demonstrating a lackluster proinflammatory gene expression profile in MAP infected tissues. 3)We anticipate observing development of an initial proinflammatory response mediated by CD4+ effector cells (Th1 cells) during the first few months of MAP infection of calves. In accordance with our published model, we anticipate that regulatory T cell activity will expand and become detectable subsequent to the proinflammatory activity, perhaps coincident with early development of a Th2-like response. These outputs will enhance our basic understanding of the host response to M. paratuberculosis and help in the design of vaccines and vaccine trials.

Project Methods
We will use flow cytometric sorting to remove CD4+CD25high T cells from PBMCs of subclinical MAP infected cattle (n=4 to 6). Following removal, remaining PBMCs will be stimulated with live MAP antigens for 6 hours. Following stimulation, PBMCs will be harvested for RNA isolation and Q-RT-PCR. RNAs to be studied include IFN-gamma, IL-1alpha, IL-1beta, TNFalpha, IL-6 and IL-12p40. We will also measure IL-10 mRNA abundance. Controls will include: 1) intact PBMC populations from the same cows and 2) intact PBMCs from the same cows to which we have added the sorted CD4+CD25high cells, and 3) intact PBMCs and PBMCs with CD4+CD25high cells removed from healthy cattle (n=3). In addition, we will conduct stimulations with the mitogen ConA ato determine if, the putative regulatory cells are MAP antigen specific. To monitor outgrowth of effector cells, we will stimulate PBMCs from cattle naturally infected with MAP (n=4 to 6) with live MAP for up to 5 days in standard RPMI medium supplemented with 10% fetal bovine serum. Cells will be pre-loaded with CSFE, which remains in the cytoplasm. Each time the cells divide, CSFE becomes diluted by approximately one-half. Because CSFE fluoresces in the green, dilution can be detected by flow cytometry, which also allows us to target monitoring of specific cell types using our panel of cell surface primary antibodies, labeled with PE secondary antibodies. For effector cells, we will initially target the CD4+ cell population following stimulation For our cytotoxic T cell assays, we will utilize MAP infected monocyte-derived macrophages (MDMs) as target cells in combination with various populations of autologous regulatory and effector immune cells. We will prepare monocytes from 6 to 10 MAP infected cattle and allow these cells to differentiate into MDMs in culture for 5-7 days. MDMs will be infected with MAP at an MOI of 10:1 (MAP to cells). MAP infections will be allowed to proceed for 24 hours. During this time, autologous PBMCs will be prepared from the MAP infected cattle and stimulated ex vivo with live MAP. CD8+ T cells will be purified from the stimulated PBMC populations by magnetic cell sorting (MACS) or flow cytometry. Purified CD8+ T cells will be applied to the MAP infected MDMs and allowed to interact with these cells for various times including 6, 12 and 24 hours. Tissues from MAP infected cattle will also be examined directly by immunfluorescence. We will utilize confocal microscopy, offering the opportunity to multiply stain sections for expression of FoxP3, TGFbeta, IL-10 and various cell surface markers (i.e. CD3, CD4, CD25, and TCR1-N24 for gamma-delta T cells). A further advantage of fluorescence and confocal imaging is the ability to stain for MAP in conjunction with other antibodies, thus showing the spatial relationship between infected macrophages and potential regulatory T cells within lesions.

Progress 12/15/09 to 12/14/12

Outputs
OUTPUTS: In completion of this project, we have advanced techniques for identification, expansion, and characterization of regulatory T cells (Tregs) in cattle. These cells are a very small percentage of overall peripheral blood mononuclear cells (PBMCs) in most species and thus are difficult to study. Three markers and flow cytometry were used to isolate and quantify Tregs in both healthy cattle and cattle with Johnes disease. Tregs are CD4, CD25 and FoxP3 positive cells. One overall hypotheses for our project was that MAP reactive Tregs will develop over the long course of subclinical infection with mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johnes disease in ruminants. Our work has demonstrated that PBMCs from cattle infected with MAP contain significantly more Tregs than PBMCs from healthy cattle. These cells proliferate in response to MAP antigens in the presence of rapamycin, IL-2, and TGFβ. When Tregs are removed from PBMCs prior to MAP antigen stimulation, effector cell responses are significantly enhanced. Using high throughput cell sorting, we have developed techniques for separating Tregs from other cells to allow direct studies of Treg function and activity. CD25 is an activation marker for normal effector T cells, but is constitutively expressed on Tregs. CD25 upregulation on effector cells appears to be a simple, rapid, and cost effective method of diagnosing infection with MAP. Work on using CD25 in this manner continues in both MAP and bovine TB infected cattle, an unexpected offshoot of our Treg program. We have developed a Johnes disease lesion scoring system for cattle to aide our work in tissues from infected animals. Our work in tissues demonstrated that Tregs make up a significant proportion of T cells that are found in proximity to MAP-induced lesions. Of great interest was the finding that bovine leukemia virus (BLV) is a confounding factor in MAP infection. While normal healthy cattle have readily detectable Tregs in their mesenteric lymph nodes, BLV infection causes these cells to almost completely disappear. When MAP infection is overlaid on BLV infection, Tregs are again present in lymph nodes. It is not yet clear if these Tregs are MAP responsive. In addition, BLV infected cattle have significantly more IL-10 mRNA in their PBMCs than BLV negative cattle (200-400 fold more by qRT-PCR). IL-10 is an immune suppressive cytokine and would likely dampen any proinflammatory immune response, which is required for efficient control of MAP infection. Work in tissues and on BLV-MAP interactions continues under a USDA-NIFA Pre-doctoral Fellowship Grant awarded to Jon Roussey, a graduate student responsible for much of the work mentioned above. PARTICIPANTS: Undergraduate students who worked on this project included: Brooke Murphy, Merrick Coussens, Joseph Wallace, and Angela Crudington. Post-doctoral fellows who participated in this work included: Kelly Sporer, Patricia, deAlmedia, Patty Weber, and Monica Carvajal-Yepes. Technical support was provided by: Chris Colvin and Sue Sipkovsky. TARGET AUDIENCES: The primary target audiences for this project were other scientists interested in mycobacterial diseases of animals. Our work on links between MAP and BLV infections will be of general interest to practicing veterinarians and producers. The use of CD25 upregulation in response to MAP antigen stimulation should be of interest to those concerned with disease diagnosis in cattle. PROJECT MODIFICATIONS: None to report.

Impacts
Johnes disease represents one of the most economically significant diseases in the US dairy herd and has become a global problem. A report from the National Animal Health Monitoring Service estimated that in herds where 10 percent of culled cows showed clinical signs of Johnes disease, the average cost to producers was 227.00 US dollars per cow on the operation. Conservative estimates suggest that the U.S. dairy industry absorbs over 200 million dollars per year in losses due to Johne's disease (NAHMS, 1997), although some estimates place this figure closer to 1.5 billion dollars. A controversial, but developing link between MAP and exacerbation of some cases of human Crohns disease suggests that this pathogen may also be an important food safety concern. Despite the overwhelming importance of Johnes disease to the global dairy industry, it is clear that our knowledge of the bovine immune response to MAP (as well as other pathogens), although improving, is sparse. Regulatory T cell biology is a relatively new area, particularly important in long term chronic infections in mammalian species. As Johnes disease represents such an infection, we hypothesized that MAP reactive Tregs would likely develop in MAP infected cattle and could have a dramatic effect on development of clinical disease. Our program has helped to define bovine Tregs, demonstrated that MAP infected cattle have significantly more Tregs than healthy controls, that these cells respond to MAP antigens, and that Tregs are found in proximilty to MAP lesions in tissues. We uncovered a novel link between clinical Johnes disease and dual infection with bovine leukemia virus. Since BLV infection rates are high (>65% of US herds are positive) our work has the potential to change the way people think about these two infections.

Publications

• Coussens, PM., S. Sipkovsky, B. Murphy, J. Roussey, and C. Colvin. 2012. Regulatory T cells in Cattle and their potential role in bovine paratuberculosis. Comparative Immunology, Microbiology and Infectious Diseases. 35: 233-9.
• Steibel, J.P., R. Poletto, P.M. Coussens, and G.J.M. Rosa. 2009. A powerful and flexible linear mixed model framework for the analysis of relative quantification RT-PCR data. Genomics, 94:146-52.
• F.B. Rosa, J. Roussey, P. M. Coussens, I. M. Langohr. 2011. Pathology in Practice: Cardiac and Aortic Mineralization in a Holstein Cow with Granulomatous Ileitis. JAVMA, In Press.
• Kabara, E., Kloss, C.C., Wilson, M.R., Sreevatsen, S., Janagama, H., and P.M. Coussens. 2010. A Large-Scale Study of Differential Gene Expression in Macrophages Infected with several strains of Mycobacterium avium subspecies paratuberculosis. Briefings in Functional Genomics and Proteomics. 9(3):220-37.
• Kabara, E.A. and P.M. Coussens. 2012. Infection of primary bovine macrophages with Mycobacterium avium subspecies paratuberculosis suppresses host cell apoptosis. Frontiers in Microbiology. 3:215.
• Lim, A., J.P. Steibel, P.M. Coussens, D.L. Grooms, and S.R. Bolin. 2012. Differential gene expression segregates cattle confirmed positive for bovine tuberculosis from antemortem tuberculosis test-false positive cattle originating from herds free of bovine tuberculosis. Vet. Med. Int. 192926 Epub

Progress 12/15/10 to 12/14/11

Outputs
OUTPUTS: We have developed techniques for identification and characterization of regulatory T cells (Tregs) in cattle. These cells are a very small percentage of overall peripheral blood mononuclear cells (PBMCs) in most species and thus are difficult to study. We have used three markers and flow cytometry to isolate and quantify Tregs in both healthy cattle and cattle with Johnes disease. The three markers are CD4, CD25 and a transcription factor relatively specific for Tregs, FoxP3. One of the overall hypotheses for our project was that MAP reactive Tregs will develop over the long course of subclinical infection with mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johnes disease in ruminants. Indeed, our work has demonstrated that PBMCs from cattle infected with MAP contain significantly more Tregs than PBMCs from healthy cattle. We are now working to develop assays to examine the functional characteristics of these cells, including dampening of Th1-like and cytotoxic immune responses. Our work in tissues of MAP infected and healthy cattle has demonstrated that Tregs make up a significant proportion of the T cells that are found in proximity to MAP-induced lesions. Of great interest was the finding that bovine leukemia virus (BLV) is a confounding factor in MAP infection. While normal healthy cattle have readily detectable Tregs in their mesenteric lymph nodes, BLV infection causes these cells to almost completely disappear. When MAP infection is overlaid on BLV infection, Tregs are again present in lymph nodes. In addition, BLV infected cattle have significantly more IL-10 mRNA in their PBMCs than BLV negative cattle (200-400 fold more by qRT-PCR). IL-10 is an immune suppressive cytokine and would likely dampen any proinflammatory immune response, which is required for efficient control of MAP infection. PARTICIPANTS: Paul M. Coussens, PhD. Overall project director and PD. Sue Sipkovsky, B.S., Laboratory technician flow cytometry and cell preparations. Christopher Colvin, B.S., Laboratory manager, cell preparation, qRT-PCR. Jonathan Roussey, B.S., Graduate student. Confocal imaging, immunohistochemistry, flow cytometry Brooke Murphy, Undergraduate research assistant. Flow cytometry, cell preparations, tissue culture. TARGET AUDIENCES: Other scientists in immunology and infectious disease research. Veterinarians who work with cattle. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Johnes disease represents one of the most economically significant diseases in the US dairy herd and has become a global problem. A report from the National Animal Health Monitoring Service estimated that in herds where 10 percent of culled cows showed clinical signs of Johnes disease, the average cost to producers was 227.00 US dollars per cow on the operation. Conservative estimates suggest that the U.S. dairy industry absorbs over 200 million dollars per year in losses due to Johne's disease (NAHMS, 1997), although some estimates place this figure closer to 1.5 billion dollars. Because many subclinical infections with MAP go undiagnosed for several years, the actual cost to producers, and National infection rates, may indeed be much higher than more conservative estimates suggest. A controversial link between MAP and exacerbation of some cases of human Crohns disease suggests that this pathogen may also be an important food safety concern. Despite the overwhelming importance of Johnes disease to the global dairy industry, it is clear that our knowledge of the bovine immune response to MAP (as well as other pathogens), although improving, is sparse. Regulatory T cell biology is a relatively new area, particularly important in long term chronic infections in mammalian species. As Johnes disease represents such an infection, we hypothesized that MAP reactive Tregs would likely develop in MAP infected cattle and could have a dramatic effect on development of clinical disease. In uncovering a novel link between clinical Johnes disease and dual infection with bovine leukemia virus, our work has the potential to change the way people think about these infections. Since very few cattle with BLV develop tumors associated with this infection, control measures are not typically practiced. Our work suggests that BLV infection may have a dramatic impact on the outcome of MAP infections and, perhaps, other viral or intracellular bacterial infections.

Publications

• Coussens, PM., S. Sipkovsky, B. Murphy, J. Roussey, and C. Colvin. 2011. Regulatory T cells in bovine paratuberculosis. Comparative Immunology, Microbiology and Infectious Diseases. In Press

Progress 12/15/09 to 12/14/10

Outputs
OUTPUTS: This project focuses on determining the role regulatory T cells (Tregs) play in progression of Johne's disease in cattle. Tregs have not been well characterized in cattle and, even in the best of circumstances, comprise an very small proportion of total lymphocytes. We first wished to determine an optimal time of MAP stimulation. PBMCs were cultured for 0, 24, 48, 72, or 96 hours in RPMI 1640 media with and without MAP. After stimulation cells were collected and labeled with fluorescent antibodies for anti-CD3+, Annexin V and 7- amino-actinomycin (7-AAD) to gauge cell death. Cells incubated for more than 72 hrs in the presence of MAP were highly apoptotic. In all subsequent studies, PBMCs were incubated for 72 hours before being collected and washed and stained for flow cytometry. MAP stimulation enhanced relative percent CD25high cells in lymphocytes from MAP-infected cattle. All FoxP3+ cells are also CD25+ and >80% of FoxP3+ cells are in the CD25high cell gate. Between 30% and 80% of CD25high cells are FoxP3+, higher in MAP-infected cattle. MAP stimulation enhanced CD25 expression, relative to Nil stimulation, in lymphocytes from MAP-infected cows. The CD25low population consistently followed this general scheme. The CD25high cell population was more variable. We thus investigated CD25high expression following MAP stimulation of a large number of test positive cows (n=11). MAP stimulation caused a significant increase in relative number of CD25high cells relative to Nil stimulation (p < 0.01). It will be of interest to determine if these results correlate with changes in FoxP3 expression. Peripheral regulatory T cells generally produce IL-10 to dampen inflammatory immune responses. Expression of this cytokine in PBMCs from our original two MAP infected cows mirrored the CD25high profiles following MAP stimulation. Our data suggest that MAP stimulation causes lymphocytes from uninfected cows to become CD25high, but FoxP3 negative. In MAP-infected cows, MAP stimulation causes cells to become CD25high/FoxP3+. One possible explanation for our results is that MAP, like many other mycobacteria, contains potent antigens that cause an increase in CD25 (IL-2 receptor) expression on T cells from both Johnes test negative and Johnes test positive cows. Johnes test negative cows contain FoxP3+ regulatory cells, but these are not responsive to MAP and do not proliferate. In contrast, FoxP3+ T cells from Johnes test positive cows ARE reactive to MAP antigens and do proliferate in response to MAP antigen stimulation. While it is also possible that MAP stimulation causes apoptosis of FoxP3+ cells in CD3+ T cells from test negative cows, but NOT in CD3+ T cells from Johnes test positive cows, we believe this is unlikely and not supported by our data. PARTICIPANTS: Paul M. Coussens, Professor, Michigan State University Ms. Sue Sipkovsky, Research Assistant I, Michigan State University Ms. Brooke Murphy, Undergraduate research Assist., Michigan State University Mr. Jon Roussey, Graduate student, Michigan State University Dr William Davis, Professor, Washington State University TARGET AUDIENCES: The main target audience for our work is other scientists working on M. paratuberculosis immunopathogenesis. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
To date our work on this project has shown that: MAP induces significant apoptosis in CD3 positive and CD3 negative cells by 72 hours post-treatment. Most FoxP3 positive cells are also CD3/CD25high/CD45RO positive. MAP stimulation significantly increases relative percent of CD3 positive cells that are CD25high, but there is considerable animal variation. Expression of IL-10 mRNA may mirror CD25high expression.

Publications

• No publications reported this period