Cytokines are a group of glycoproteins of 15–35 kDa molecular weights that play an important role in homeostasis particularly in immune-regulation. Studies in mice and humans suggest existence of T helper- 1(Th1) cells and Th2 cells based on cytokine secretion profile (Fiorentino et al., 1989). Th1 cells secret Interleukin-2 (IL-2), IL-12, Interferon-gamma (IFN-³) while Th2 cells secret IL-4, IL-6, IL-10 and TGF-b. Th1 cytokines favors cell mediate immune responses and pro-inflammatory responses, while Th2 cytokines favors humoral immune responses, allergies and asthma conditions. IL-10 promotes development of Th2 cytokine pattern by inhibiting IFN-³ production of T lymphocytes particularly through suppression of IL-12 synthesis thus acting as key regulator in skewing Th1 type of responses towards Th2 type (Fickenscher et al., 2002). IL-10 is mainly produced by monocytes and macrophages under various stimuli like endotoxin, TNF-α, catecholamines and cAMP elevating drugs (Platzer et al., 1995; Riese et al., 2000). While, other cells that secret IL-10 include subset of Th2 lymphocytes, B cells, mast cells, eosinophils and keratinocytes (Sabat et al., 2010). Antigen presenting cells and lymphocytes are main targets of IL-10. Studies in IL-10 knockout mice reveal lethal inflammation of intestine which can be stopped by application of IL-10 (Kuhn et al., 1993). Considering the importance of IL-10 in disease conditions and autoimmune disorders, a therapeutic potential has been explored. Neutralisation of IL-10 in over expressed conditions like systemic lupus erthymatous and lymphoma, application of IL-10 in inflammatory bowel disease and psoriasis have garnered considerable attention for therapeutic use of IL-10 in humans (Asadullah et al., 2003).

Although most of the work on IL-10 comes from human and mouse models much less work has been done in ruminant species. The gene encoding ovine IL-10 has been identified, cloned and sequenced (Martin et al., 1995; Dutia et al., 1994). It has been shown to contain 177 amino acid residues with 19 amino acid leader sequence. Mature peptide is of 18.36 kDa in molecular weight and consists of 158 amino acid residues. It is more than 77% similar to pig and human IL-10 and more than 68% similar to viral IL-10 homolog at amino acid level (Dutia et al., 1994). However, there are no similar reports on caprine IL-10 gene. Therefore present study is undertaken to identify IL-10 gene in goat and to elucidate sequence based information in relation to other species.

Interleukin-10 gene of Pashmina goat was amplified and cloned into TOPO-TA vector (Invitrogen) for sequencing. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood using density gradient (Histopaque 1.077) centrifugation. The isolated PBMCs were stimulated with 10µg/ml Escherichia coli Lipopolysaccharide (LPS) for 24hrs in 24 wells tissue culture plate. Total RNA was extracted using Trizol reagent (Sigma Aldrich) as per manufacturer's recommendations. The mRNA was transcribed into complementary DNA (cDNA) using oligo-dT primers and MMLV reverse transcriptase enzyme. Further, cDNA was subjected to PCR using cytokine specific primers designed using bovine IL-10 sequence. The 522 bp PCR product was gel extracted from 1% agarose using gel extraction kit (Promega) and cloned into TOPO-TA vector (Invitrogen). Recombinant plasmid was transformed into TOP-10 cells provided along with the vector as per manufacturer's recommendations. Plasmid was isolated and the insert was sequenced by di-deoxy chain termination method.- Nucleotide sequences of IL-10 from available species were retrieved from NCBI database for comparison with goat IL-10 sequence. Multiple sequence alignment and phylogenetic tree construction was performed using Megalign (DNAstar) software.

Nucleotide sequence of IL-10 was submitted to GenBank (Accession N0. DQ837159). The sequence was compared with the IL-10 sequences available in the NCBI database for other species. The percent similarity for both nucleotide and amino acid sequences were compared between goat IL-10 and other species (Table 1).

A high degree of homology between goat IL-10 sequence, and sequences of other species at nucleotide level (98.9%– 86.5%) and at amino acid level (98.1%–79.7%) was observed. Highest degree of similarity of goat IL-10 was seen with sheep IL-10 sequences. Ovine IL-10 gene has open reading frame of 531 bp and translatess to 177 amino acid residues (Martin et al., 1995). Nineteen amino acid residues towards N-terminal end form the leader sequence and is cleaved off by signal peptidase before yielding mature functional IL-10 protein of 158 amino acid residues. Human IL-10 is a non-glycosylated protein whereas mouse IL-10 is glycosylated. Glycosylation of IL-10 is not essential for functional activity of the molecule as recombinant Escherichia coli expressing mouse IL-10 is functionally active (Moore et al., 1993). Multiple sequence alignment was performed on amino acid sequences of various species using Clustal W of Megalign software (DNASTAR) and the results are shown in Figure 1.

From Figure 1 Sequence polymorphism of the gene across species is mostly confined to the first one third of sequence whereas the later two third sequences are relatively conserved. The conserved portion of sequence might imply common biological functions of the IL-10 across species. A total of three amino acid residues variations were seen between goat and sheep IL-10 sequences. These changes were at positions 25, 26 and 152. As depicted in Figure 1, caprine IL-10 has six alpha helices (A–F) that participate in receptor binding. One striking feature is that IL-10 has four cysteine residues at positions 12, 62, 108 and 114 that is conserved across the species. These cystiene residues take part in formation of disulphide bridges (Cys12-Cys108, Cys62-Cys114). These features are similar to those found in human IL-10. Each monomer of human IL-10 has two intra-chain disulphide bonds (Cys12-Cys108, Cys62-Cys114) and is composed of six helices named A,B,C,D,E and F (Zdanov et al.., 1995, 1996). Human IL-10 has been found to exist as homodimer in solutions and this structure is required for binding and activation of signal transduction through IL-10 receptor. The residues required for contact with the receptor lies in helix A, AB loop, helix F, N-terminal end and DE loop. Binding of IL-10 to IL-10 receptor induces activation of signal transduction possibly through JAK/STAT kinase leading to transcription of downstream genes (Josephson et al., 2001). Phylogenetic tree using Megalign (DNASTAR) was constructed from amino acid sequences of goat and other mammals obtained from the GenBank. The results are shown in Figure 2 which suggest that the goat IL-10 gene is a lot more related to small and large ruminants than that of monogastric animals.

In conclusion, the present work reports identification of IL-10 from goat (Capra hircus) by cloning and sequencing. Sequence of caprine IL-10 was remarkably similar to ovine IL-10 based on nucloetide/deduced amino acid sequence, signifying similar biological functionality for this cytokine.

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