Cytokine Gene Expression in Response to SnSAG1 in Horses with Equine Protozoal Myeloencephalitis

Equine protozoal myeloencephalitis (EPM) is a neurologic syndromeseen in horses from the Americas and is mainly caused by Sarcocystisneurona. Recently, a 29-kDa surface antigen from S. neuronamerozoites was identified as being highly immunodominant ona Western blot. This antigen has been sequenced and cloned,and the expressed protein has been named SnSAG1. In a previousstudy, cell-mediated immune responses to SnSAG1 were shown tobe statistically significantly reduced in horses with EPM incomparison to EPM-negative control horses. It therefore appearsas though the parasite is able to induce immunosuppression towardsparasite-derived antigens as parasite-specific responses aredecreased. Isolated peripheral blood lymphocytes from 21 EPM(cerebrospinal fluid [CSF] Western blot)-negative horses withno clinical signs and 21 horses with clinical signs of EPM (CSFWestern blot positive) were cocultured with SnSAG1 for 48 and72 h, and the effect on cytokine production was investigatedby means of reverse transcriptase PCR. Cytokines assayed includegamma interferon (IFN- ${gamma}$ ), tumor necrosis factor alpha, interleukin(IL)-2, IL-4, and IL-6. ß-Actin was used as the housekeepinggene. A Wilcoxon signed-rank test of the findings indicatedthat there was a statistically significant decrease in IFN- ${gamma}$ production after 48 h in culture for samples from horses withclinical disease. There was also a statistically significantincrease in IL-4 production after 72 h in culture for samplesfrom horses with EPM. These results further support the notionthat this parasite is able to subvert the immune system in horseswith clinical disease.

INTRODUCTION

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Introduction

Equine protozoal myeloencephalitis (EPM) was first recognizedin the early 1970s (2, 6, 8) and has subsequently grown in importanceas our knowledge of this disease has increased. It is now consideredthe most important protozoal disease of horses in the Americas(16) and is caused mainly by the apicomplexan parasite Sarcocystisneurona (9).

Sporocysts excreted by opossums (Didelphis virginiana) are thesource of S. neurona infection (10), which explains why thisdisease is observed only in the Americas in the geographic rangeof the opossum. The natural route of infection for S. neuronais via the gastrointestinal tract, as infection occurs throughoral ingestion of sporocysts on contaminated pastures, feed,or drinking water.

Serological surveys of horses have shown that up to 53% haveantibodies to S. neurona (3, 4, 18), and up to 38% of healthyhorses presenting at Auburn University's College of VeterinaryMedicine (donations to the teaching program) have S. neuronaantibodies in the cerebrospinal fluid (unpublished data). Thesestudies indicate that exposure to this parasite is widespread.However, only a small percentage of infected horses developovert clinical disease, and this is thought to be related tofactors such as the strain of parasite, the infective dose,concurrent infections, and maybe most importantly the immunestatus of the horse. Recently it was shown that cell-mediatedimmune responses to a highly immunodominant 29-kDa surface antigenfrom S. neurona merozoites (SnSAG1) (12, 13) are statisticallysignificantly reduced in horses with EPM (19), and it appearsas though the parasite exacerbates this immunosuppression.

Knowledge of the full immune response to infectious agents inequids has been severely hampered by the lack of available reagents.As there is a correlation between gene expression and proteinproduction (5), it can be assumed that detection of gene expressionrelates to cytokine protein production (14). This paper describesin vitro cytokine induction by the immunodominant protein SnSAG1as measured by reverse transcriptase PCR (RT-PCR).

MATERIALS AND METHODS

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Materials and Methods

Horses. The present study included 21 EPM-negative horses (with Westernblot-negative cerebrospinal fluid [CSF] samples; EBI, Lexington,KY) and 21 horses with clinical signs of EPM and Western blot-positiveCSF samples from Alabama and Florida (19).

Lymphoblastogenesis. Peripheral blood samples were collected by jugular venipunctureinto sterile tubes containing EDTA, and lymphocytes were isolatedby differential centrifugation on Histopaque-1083 (Sigma, St.Louis, Missouri). All in vitro procedures were carried out underaseptic conditions. Lymphocyte numbers were assessed in Turk'swhite blood cell counting fluid (19), and the concentrationwas adjusted to 2 x 10⁶ lymphocytes/ml in RPMI 1640 medium (containing10% heat-inactivated fetal calf serum, antibiotics, and L-glutamine)(Atlanta Biologicals, Norcross, Georgia). Lymphocytes (containingless than 10% contaminating monocytes or neutrophils) were placedin the wells of 96-well (100 µl/well) round-bottom platescoated with 10 µg/ml of SnSAG1 and incubated at 37°Cin a humidified atmosphere containing 5% CO₂ and 95% air for48 h and 72 h (17). The cells were then harvested and pelletedin a microcentrifuge, resuspended in RNAlater (QIAGEN, Valencia,California), and stored frozen until assayed by RT-PCR.

RT-PCR. mRNA was isolated by using RNA STAT-60 total RNA/mRNA isolationreagent (Tel-Test, Friendswood, Texas) according to the manufacturer'sinstructions. The RT-PCR was performed using an Access RT-PCRsystem kit (Promega, Madison, Wisconsin), also in accordancewith the manufacturer's instructions. Approximately 500 ng ofcDNA was used per PCR. The following cytokines were assayedunder conditions previously described: gamma interferon (IFN- ${gamma}$ ),tumor necrosis factor alpha (TNF- ${alpha}$ ), interleukin (IL)-2, IL-6with ß-actin as the housekeeping gene (14), and IL-4(7). These cytokines were chosen as they are representativeof T helper 1 (Th1) or T helper 2 (Th2) immune responses. Thelevel of gene expression was not quantified, and the resultswere expressed as the presence or the absence of gene transcriptsas visualized on a 1.5% agarose gel (Fig. 1).

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FIG. 1. Example of RT-PCR amplicons separated in a 1% agarose gel and stained with ethidium bromide. PCR products were produced by lymphocytes in response to SnSAG1 incubation. Lane 1, 1-kb molecular weight marker; lane 2, RT-PCR positive control; lane 3, ß-actin amplicon; lane 4, IFN- ${gamma}$ amplicon; lane 5, TNF- ${alpha}$ amplicon; lane 6, IL-2 amplicon; lane 7, IL-4 amplicon; lane 8, IL-6 amplicon.

Statistics. A Wilcoxon signed-rank test was performed to evaluate any statisticallysignificant increased or decreased differences between the cytokinetranscripts expressed by EPM-negative and EPM-positive horsesin response to SnSAG1 incubation after 48 h or 72 h.

RESULTS

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Results

The results of this study indicate a trend towards an inductionof proinflammatory cytokines in response to Sarcocystis neuronainfection. A statistically significant difference was reportedfor IFN- ${gamma}$ for both the 48-h (P = 0.005) and 72-h (P = 0.046)incubation periods, with a decrease in the expression of thiscytokine gene in horses with clinical EPM compared with theexpression of this gene in EPM-negative horses at 48 h. Therewas significantly less IFN- ${gamma}$ expression after 48 h of incubationthan after 72 h of incubation, indicating a significant lagin expression of this very important cytokine by horses infectedwith S. neurona. In EPM-negative control horses, there was noevidence of IFN- ${gamma}$ expression after 72 h (Table 1), indicatingthat there is an early induction of this cytokine in responseto this parasite (P > 0.005) in normal horses.

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TABLE 1. Presence of gene transcripts in response to coculture with SnSAG1 for 48 or 72 h as visualized on a 1.5% agarose gel

A statistically significant increase in IL-4 gene expressionwas also observed after 72 h (P = 0.102) of incubation in horsesthat were positive for EPM (Table 1). There was no statisticallysignificant difference in the amount of IL-4 gene transcriptionafter 48 or 72 h of incubation in the presence of SnSAG1 inEPM-negative horses.

There were no statistically significant differences in geneexpression of any of the other cytokines regardless of the horses'EPM status, although certain trends appeared to be developing.It would appear as though TNF- ${alpha}$ , IL-2, and IL-6 expression levelsdecrease with time in EPM-negative horses but increase withtime in EPM-positive horses.

DISCUSSION

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Discussion

S. neurona is able to induce immunosuppression towards parasite-derivedantigens as parasite-specific blastogenic responses are decreased(19). The results of this study indicate that this immunosuppressionis due, in part, to a trend towards Th1 cytokine gene suppressionand Th2 cytokine gene induction. This supports other studiesin which it has been shown that levels of cytokines that areimportant in stimulating cell-mediated responses to protozoanantigens such as Toxoplasma gondii and Leishmania spp. are decreased(17, 20). It is possible that local immunosuppression is beinginduced by the presence of S. neurona merozoites in horses withclinical EPM, and the extent of this suppression could be relatedto host genetic factors. This possibility could explain whynot all infected horses develop clinical disease.

In the current study, IFN- ${gamma}$ expression was shown to be suppressedin horses with clinical signs of EPM, further supporting thesuggestion that S. neurona is able to depress expression ofthis very important cytokine. From previous studies (19), ithas been shown that lymphocyte blastogenesis still occurs after72 h in response to SnSAG1, thus eliminating the possibilitythat this protein induces cell death. Infection of immunocompetentmice with S. neurona merozoites does not result in parasitemiaor any signs of disease. However, IFN- ${gamma}$ knockout mice succumbto disease, and parasites can be recovered from severe combinedimmunodeficient Arabian foals, further indicating the importanceof this cytokine in disease prevention (11, 15).

IFN- ${gamma}$ is critical in the development of a Th1 response and isproduced mainly by T cells and natural killer cells. It hasa wide range of biological functions, including antiviral functions,influencing cell-mediated cytotoxicity, priming macrophages,and augmenting production of IL-12, a cytokine which has a pivotalrole in upregulation of various immune responses (1).

Interleukin-4 is produced primarily by Th2 cells and also hasmany biological effects, such as inducing B lymphocytes to undergoimmunoglobulin isotype switching to produce immunoglobulin E,suppressing production of TNF- ${alpha}$ , and inhibiting macrophage synthesisof IL-12 (1). The fact that S. neurona is able to upregulateIL-4 gene expression suggests another pathway by which thisparasite may be subverting the immune response, resulting inclinical disease.

The observed trend of TNF- ${alpha}$ , IL-2, and IL-6 expression decreasingwith time in EPM-negative horses, while increasing with timein EPM-positive horses, could be because these cytokines areproduced by macrophages, and the increased production couldbe due to an anamnestic type of response in EPM-positive horsesalready exposed to this parasite. Further study examining differentincubation times may shed some light in this area.

In conclusion, there are many ways in which protozoan parasitesevade host immune responses, and these include changes in cytokinepatterns as seen in the present study (20). One can assume thatthere are many more underlying factors involved, such as geneticsof the host, inoculum dose, concurrent infections, and strainof the parasite, as not all infected horses develop disease.Whatever predisposes horses towards disease development maybe further exacerbated by the presence of the parasite. Furtherwork in this field is ongoing.

ACKNOWLEDGMENTS

This study was supported in part by an innovation grant fromFort Dodge Animal Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849. Phone: (334) 844-2701. Fax: (334) 844-2652. E-mail: spencja{at}vetmed.auburn.edu.

REFERENCES

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