Regulation of immune responses by a tumor necrosis factor in pearl oysters: insights from PmTNF gene expression and function

Abstract

Tumor necrosis factor (TNF) is a multifunctional cytokine that regulates cellular processes such as inflammation, apoptosis, differentiation, and proliferation and activates various functions of the immune system. This article reports the discovery and characterization of a novel tumor necrosis factor gene in the pearl oyster Pinctada fucata martensii, which is named PmTNF. The deduced PmTNF protein sequence displays the typical structural characteristics of a TNF domain, and phylogenetic analysis of the sequences of PmTNF and its putative orthologs shows that they conform to the current taxonomy. Analysis of PmTNF mRNA expression via real-time PCR reveals its constitutive expression in all the examined tissues, with the highest expression in the gills. Furthermore, PmTNF expression in the gills varies upon exposure to pathogen-derived stimuli, with modest upregulation in response to lipopolysaccharides, but with significant downregulation in response to polyinosinic:polycytidylic acid. Nucleus insertion surgery induces an increase in PmTNF mRNA level in the gills at 12 h postoperation. Knocking down PmTNF through RNA interference significantly inhibits the expressions of immune-related genes in the NF-κB signaling pathway in the gills by 24 h (P < 0.05). The function of PmTNF is further characterized by studying the activity of an engineered recombinant PmTNF protein (rPmTNF) in vivo. Upon nuclear insertion, treatment with rPmTNF for 6 h upregulates several genes in the NF-κB pathway. Similarly, rPmTNF increases the activities of the antioxidant enzymes, including superoxide dismutase, glutathione and peroxidase, which reflect the total antioxidant capacity. Collectively, these results indicate that PmTNF participates in pearl oyster immunity by modulating the NF-κB pathway and activating the antioxidant defense system.

1. Introduction

TNF-α is a key member of the TNF ligand family that exists as membrane-bound and soluble cytokines, and it is released from the cell surface after proteolytic cleavage, mainly by metalloproteinases. Different metalloproteinases can be induced by various stimuli, and the resulting soluble TNF forms possibly play distinct physiological roles [1]. Members of the tumor necrosis factor (TNF) family and its closely related receptors constitute potent cytokine axes that are implicated in proinflammatory and immunological responses. They are involved in several crucial biological processes, such as cytotoxicity and antiviral activities [2].

Since TNF cDNA was first identified in humans in 1984 [3]. Nineteen TNF members have been characterized in mammals in recent decades [4] and are classified into two major types: TNF-α (also called cachectin) and TNF-β (also called lymphotoxin) [5]. The majority of TNF members are expressed by immune cells, such as monocytes, macrophages, T lymphocytes, and natural killer cells [6]. In fish, two types of TNF-α, termed shTNF-α1 and shTNF-α2, have been identified in snakehead (Channa argus), suggesting that they play important roles in bacterial infection and pathogen clearance [7]. Furthermore, a TNF gene (named RB-TNFN) in rock bream (Oplegnathus fasciatus) was cloned, and its functional expression was analyzed, revealing that RB-TNFN is highly conserved with TNF family signatures and mediates innate and adaptive immunity [8]. The TNF superfamily and its intricate regulatory processes have been studied extensively in vertebrates, and currently, an expanding array of TNF superfamily members has been discovered in various invertebrates. The initial discovery of an invertebrate TNF ligand (Eiger) isolated from Drosophila revealed that it plays a pivotal role in the modulation of extracellular pathogen growth, thereby contributing to the host immune defense mechanisms [9]. A TNF ligand (MjTNF), which contains a predicted transmembrane region and a TNF homology domain, has been characterized in the marine arthropod kuruma shrimp (Marsupenaeus japonicus). A high expression level of MjTNF was observed in vivo after stimulation with lipopolysaccharide [10]. A new member of the TNF superfamily (named EsTNFSF) was identified from the Chinese mitten crab (Eriocheir sinensis), which also contains a transmembrane region and a conserved extracellular C-terminal TNF domain [11]. Additionally, several TNF homologs have been identified and characterized in mollusks. The first molluscan TNF-α, AbTNF-α, was cloned from disk abalone (Haliotis discus discus) [12], and its mRNA expression in gill tissue was significantly induced by a mixture of pathogenic bacteria and lipopolysaccharide. The transcript level of ChTNF, a TNF homolog of oyster (Crassostrea hongkongensis), was significantly increased in blood cells following immune challenge [13]. Two novel TNF genes (CgTNF-1 and CgTNF-2) have been characterized in pacific oysters (Crassostrea gigas), both of which have been implicated in the immune response. CgTNF-1