An iodothyronine deiodinase from Chlamys farreri and its induced mRNA expression after LPS stimulation

Abstract

Iodothyronine deiodinase is responsible for the deiodination of thyroxine T4 to T3, and involved in the complex neuroendocrine-immune regulatory network to optimize the immune response in vertebrate. In this study, the full-length cDNA of an iodothyronine deiodinase (designated as CfDx) was cloned from scallop Chlamys farreri. The complete cDNA sequence of CfDx was of 1404 bp and contained an open reading frame of 900 bp encoding a polypeptide of 299 amino acids. The deduced amino acid sequence of CfDx contained an in-frame TGA stop codon probably encoding an essential selenocysteine (SeC), and there was a conserved region of about 15 amino acids surrounding the SeC residue. The CfDx mRNA transcripts were detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, gonad, gill and mantle, with the higher expression level in hepatopancreas and kidney. After LPS stimulation, the CfDx mRNA expression level in haemocytes increased significantly at 12 h (25.35-fold, P < 0.05) and 24 h (7.62-fold, P < 0.05), and the concentration of T3 in haemolymph increased significantly at 12 h (3.62 ng dL⁻¹, P < 0.05) even the concentration of T4 did not change significantly. After scallop received an injection of 50 μg CfDx dsRNA, the expression level of CfDx mRNA in haemocytes began to decrease significantly at 36 h and maintained the relative low level (about 0.3-fold of the PBS control group) from 36 to72 h, but the ratio of T4/T3 in haemolymph began to increase at 36 h (2.31-fold, P < 0.05) and kept increasing from 36 to 72 h comparing with that in the PBS control group. These results indicated that CfDx was a homologue of iodothyronine deiodinase in scallop C. farreri, and it might be involved in the immunomodulation via regulating the concentration of thyroid hormones T3 and T4 in the haemolymph of scallop.

Introduction

The neuroendocrine and immune systems constitute the neuroendocrine-immune regulatory network to maintain host homeostasis in vertebrates through neurotransmitter, hormone and cytokine [1]. In the network, the neuroendocrine system releases neurotransmitter and hormone to modulate the immune system, while the immune system influences the neuroendocrine system through cytokines [2]. In vertebrates, the central nervous system is able to modulate immune response to limit inflammation-induced damage [3]. To date, some analogous neuroendocrine-immune regulatory networks have been also reported in invertebrate [4], [5], [6], and the molecules and basic mechanism involved in the network are found to be well conserved and fundamentally similar throughout evolution.

In the neuroendocrine-immune regulatory network, thyroid hormones play important roles in regulating the immune response including inflammation, natural killer cell activity, antiviral action of IFN, and the proliferation of T and B lymphocytes [7], [8]. Thyroid hormones are released by the thyroid gland or immunocytes after immune stimulation to bind thyroid hormone receptors located in immunocytes to regulate the expression of the immune-related genes in vertebrates [9]. Thyroid hormones have also been detected in some invertebrates, such as echinoid and anthozoan [10]. There are abundant evidence to support that thyroid hormones could modulate metamorphose and development of invertebrates [10], [11]. However, the modulation of thyroid hormones to the immune response is far from well understood [12], [13].

The iodothyronine deiodinase is responsible for the deiodination of thyroid hormone T4 to T3, and plays central role for the regulation of thyroid hormones level. According to the biochemical characteristic of iodothyronine deiodinase, the iodothyronine deiodinase can be divided into three types (D1, D2, and D3). In all the reported three types, there is an in-frame UGA codon to encode a selenocysteine (SeC) codon in the catalytic site [14]. D1 and D2 produce receptor-active T3 in the outer ring of the T4 molecule, and inner ring deiodination is catalyzed by D3. D3 can also deiodinate T3 and thereby terminate thyroid hormone action [15]. Iodothyronine deiodinase has been considered to be involved in the immune responses of vertebrates [16]. For example, these iodothyronine deiodinases can be expressed in immunocytes, and their expression level is able to be modulated by a wide variety of cytokines and immune factors such as IL-1, IL-6 and NF-κB [17], [18]. Iodothyronine deiodinase genes have been identified in vertebrates such as fish, amphibians, and mammals [19], [20], whereas only a homologue of iodothyronine deiodinase (HrDx) has been found in invertebrate Halocynthia roretzi. The knowledge is quite meagre on the characteristics of iodothyronine deiodinase and its possible immunomodulation in invertebrates.

Zhikong scallop Chlamys farreri is one of the important cultured mollusc species in North China. Severe mortality of scallop has resulted from disease in recent years. The investigation of neuroendocrine and immune system could provide more efficient knowledge for the management of scallop health during stress or infection. The purposes of this study were to (1) clone iodothyronine deiodinase from C. farreri (designated as CfDx); (2) detect CfDx mRNA distribution in different tissues and its temporal expression in haemocytes as well as the change of T4, T3 concentration in haemolymph after LPS stimulation; (3) investigate the modulation of CfDx on T4 and T3, and to provide information about the function of CfDx in the neuroendocrine-immune regulatory network of scallops.