Characterisation of macrophage migration inhibitory factor from Eimeria species infectious to chickens

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Abstract

Macrophage migration inhibitory factor (MIF) was the first cytokine to be identified almost 40 years ago. Homologues of MIF have been isolated recently from invertebrates, making it an interesting molecule from an evolutionary as well as functional perspective. The present study represents the first report of MIF homologues in apicomplexan parasites, belonging to the genus Eimeria. A single full-length clone was isolated from Eimeria acervulina that shared between 35 and 38% amino acid identity with MIFs of vertebrates. A MIF cDNA from Eimeria tenella shared 64% amino acid identity with E. acervulina MIF. The mRNA expression was highest in merozoites, whereas developing oocysts and sporozoites expressed low to undetectable levels. Protein expression patterns were nearly identical to that observed by reverse transcriptase polymerase chain reaction (RT-PCR), suggesting strong developmental regulation. Immunofluorescence staining and co-localisation studies of E. acervulina merozoites indicated that MIF is distributed throughout the cytosol, and appears to be concentrated in the apical end of the parasite. The presence of MIF was detected in excretory/secretory (ES) products collected from E. acervulina merozoites, and isoelectric focusing indicated that three MIF isoforms are present in this stage. Phylogenetic analysis revealed that apicomplexan MIF sequences form a sister relationship to MIF-like molecules from Arabidopsis thaliana.

Introduction

Macrophage migration inhibitory factor (MIF) was one of the first cytokines described. It was identified as a soluble protein secreted from sensitised peritoneal lymphocytes, which inhibited the migration of macrophages [1]. MIF is essential in adaptive immune responses as well as innate immunity. Mammalian MIF is an immunoregulatory molecule that affects macrophage function [2], [3] resulting in inflammatory responses, inducible by pro-inflammatory molecules such as bacterial lipopolysaccharide (LPS) [4]. Because of its role in inflammation and subsequent conditions such as sepsis, shock and arthritis, the possibility of MIF as a therapeutic target is being explored [5]. The effect of MIF on adaptive immune responses is clear, since MIF antagonism suppresses T-cell activation [6] as well as delayed type hypersensitivity (DTH) responses [7]. Since its discovery, many additional functions have been ascribed to this molecule that distinguish it from other cytokines. MIF has been found to be a regulator of endocrine function [2], [8], and its expression has been observed in many different cell types, including brain [9] and the β islet cells of the pancreas [10]. One of MIF's unusual features is the possession of an enzymatic thiol-protein oxidoreductase activity [11], and a tautomerase/isomerase activity [12]. The secretion of MIF also appears to be regulated by an unconventional leaderless pathway [4], [13] and no MIF surface receptor has yet been found.

In recent years, MIF orthologues have been isolated from non-vertebrate organisms, such as nematodes, Caenorhabditis elegans [14], Brugia spp. [15], [16], Trichinella spp., Trichuris spp. [17], [18], Wuchereria bancrofti and Onchocerca volvulus [15]. In these organisms MIF appears to have tautomerase activity as well as the ability to chemotactically induce macrophage migration [16], [17], [18]. It is unknown why a pro-inflammatory cytokine is actively expressed by parasitic nematodes, since this would not seem to be advantageous to parasites that induce a counter-inflammatory phenotype in the host [19], [20]. However, there is some evidence that high MIF expression can produce anti-inflammatory effects [21], [22], hypothetically resulting in MIF-mediated inflammatory suppression and subsequent immune evasion. In the free-living nematode C. elegans, MIF appears to be involved in homeostatic mechanisms during times of adverse conditions and stress [14]. Thus, MIF likely has multiple functions in invertebrates as well as vertebrates. An evolutionary analysis of MIF indicated that sequences from nematodes form a sister relationship to vertebrate MIFs or a sister relationship to mammalian d-dopachrome tautomerase (DDT) (an enzyme which is considered to be part of the MIF family based on sequence identity and conservation of enzymatic activity), suggesting that MIF and DDT diverged from a single ancestral gene prior to the separation of nematodes from other metazoans [23].

The present research describes the isolation and characterisation of MIF from single celled protozoans belonging to phylum Apicomplexa. Members of this phylum are all parasitic protozoa that are important in human as well as veterinary health. This study was carried out in coccidia that infect chickens, belonging to the genus Eimeria. These parasites are endemic and cause losses of over US$ 800 million to the poultry industry, in the US alone [24]. Characterising MIF in these protozoa allows us to begin the investigation of the biological and immunomodulatory roles that these parasites exert on the host, as well as permitting a more complete analysis of MIF evolution.

Section snippets

Animals, parasites and experimental infections

Fertilised eggs of TK chickens were obtained from Hyline International Production Center (Dallas Center, IA, USA) and hatched at Animal Parasitic Diseases Laboratorie's Facilities, Agricultural Research Service, US Department of Agriculture (Beltsville, MD, USA). Chickens were kept in brooders until 3 weeks of age, at which time they were transferred to wire colony cages in separate housing for parasite infection. Chickens were inoculated per os with 2 × 106 sporulated oocysts of Eimeria

Characterisation of Eimeria MIF gene

Random screening of an E. acervulina merozoite cDNA library identified a single clone (L24) that shared high sequence similarity (E value of 2e−12) with MIF described from Petromyzon marinus (sea lamprey) [23]. Clone L24 contained an entire open reading frame (ORF) consisting of 348 nts as well as 89 bp of 5′-untranslated region (UTR) and 224 bp of a 3′-UTR. By comparing the E. acervulina MIF sequence against the E. tenella genome using BLASTN and BLASTX algorithms (//www.sanger.ac.uk/cgi-bin/blast/submitblast/e_tenella/omni

Discussion

Presence of MIF in organisms that do not possess adaptive or combinatorial immune responses raises many interesting questions regarding the function and evolution of this molecule. In the present study MIF homologues were described for the first time from single-celled parasitic protozoa belonging to phylum Apicomplexa. The full-length cDNA clone was initially discovered during a random screening of ESTs from an E. acervulina cDNA library. Through subsequent use of bioinformatics MIF sequences

Acknowledgements

The authors wish to thank Jenifer Herrmann, Ruth Barfield and Gary Wilkins for expert technical assistance, Matt Yamage for construction of the E. acervulina cDNA library, and Dr. James Trout for critical review of the manuscript. We are also indebted to Dr. Martin Shirley for granting permission to use the E. tenella genome sequence available at the Sanger Institute website. This work was supported by USDA/ARS CRIS project number 1265-31320-070-00D.

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    Note: Nucleotide sequences reported in this paper have been submitted to GenBank™, EMBL and DDBJ Databases with the accession number(s) DQ323515 and DQ323516.

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