Kudoa septempunctatawas recognised by Toll-like receptor 2 produced by a RAW 264 macrophage-like cell line

Abstract

Kudoa septempunctata is a myxosporean parasite that infects Paralichthys olivaceus (olive flounder). Previously, we reported that the consumption of raw P. olivaceus meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhoea and emesis. In this study, we investigated the cytokine production of mouse macrophage-like RAW 264 cells stimulated with K. septempunctata. When the RAW 264 cells were incubated with the spores of K. septempunctata for 24 h, they secreted tumour necrosis factor α (TNF-α) and several chemokines, such as IP-10, MIP-1β, and MIP-2. The secretion of TNF-α was induced in a dose-dependent manner in a bioassay using L929 cells and mouse TNF-α-specific enzyme-linked immunosorbent assay (ELISA). To identify the macrophage receptor of K. septempunctata, activation of HEK 293 cells expressing one of the Toll-like receptors (TLR) was measured using an NF-κB-dependent reporter assay. TLR2-expressing HEK 293 cells were strongly activated following stimulation with the spores. These results suggested that K. septempunctata was recognised by TLR2 on the macrophages, which were then activated and produced TNF-α.

Immune-triggering effect of the foodborne parasite Kudoa septempunctata through the C-type lectin Mincle in HT29 cells

“…Moreover, our findings raise the possibility of an interaction between Mincle and TLR2 signalling in the production of inflammatory cytokines based on a previous in vitro study of Kudoa ( 9 ). Although only limited early stage studies have been conducted, Kudoa appears to stimulate macrophages through TLR2 signalling, and TLR2 and Mincle can cooperatively sense pathogens through the cooperative action of both receptors ( 9 , 18 ). Most importantly, Mincle-mediated innate immunity can induce sustained inflammatory responses, especially those involving IL-1 and TNF-α, and it can promote allergic responses ( 12 , 19 ).…”

“…The inflammatory responses observed in this study firmly indicate the possibility that Kudoa infection or KsAg treatment can induce inflammatory reactions in HT29 cells. In contrast, previous studies using other cell lines such as Caco-2 human intestinal and RAW264.7 macrophage-like cells did not clearly indicate the cytotoxicity of Kudoa (8,9). Meanwhile, other effort to determine the pathogenicity of K. septempunctata infection focused on the loss of cell monolayer integrity in intestinal cells after infection (7).…”

“…Several studies have debated the clinical significance of K. septempunctata and pathogenicity in in vivo test using mouse model (2)(3)(4)(5)(6). The pathogenicity of K. septempunctata was also controversial in in vitro assays using Kudoa spores (7)(8)(9). Ohnishi et al insisted that the invasion of Caco-2 human intestinal cells by Kudoa sporoplasm induced damage through the loss of cell monolayer integrity via the elimination of transepithelial electrical resistance, suggesting their role as the causative agent of food poisoning in humans (7).…”

“…Several studies have debated the clinical significance of K. septempunctata and pathogenicity in in vivo test using mouse model ( 2 - 6 ). The pathogenicity of K. septempunctata was also controversial in in vitro assays using Kudoa spores ( 7 - 9 ). Ohnishi et al .…”

“…insisted that the invasion of Caco-2 human intestinal cells by Kudoa sporoplasm induced damage through the loss of cell monolayer integrity via the elimination of transepithelial electrical resistance, suggesting their role as the causative agent of food poisoning in humans ( 7 ). Conversely, another study reported that infection of Caco-2 cells by K. septempunctata spores did not result in inflammation ( 8 ), and another report found that K. septempunctata activated TLR-2-expressing HEK293 cells and induced TNF-α production ( 9 ). As mentioned previously, the no definite conclusion can be drawn regarding the pathogenicity of K. septempunctata or the mechanism of food poisoning.…”