Browsing by Author "Sakai, Masahiro"

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    Pubmed
    Inflammatory immune response by lipopolysaccharide-responsive nucleotide binding oligomerization domain (NOD)-like receptors in the Japanese pufferfish (Takifugu rubripes).
    (2016-02-01T00:00:00Z) Biswas, Gouranga; Bilen, Soner; Kono, Tomoya; Sakai, Masahiro; Hikima, Jun-ichi
    Some of NOD-like receptors (NLRs), the cytosolic pattern recognition receptors form a multi-protein complex, inflammasome consisting of one or more NLRs, the adaptor protein ASC and inflammatory caspase to generate mature inflammatory cytokines, interleukin (IL)-1β and IL-18. However, inflammasome-mediated inflammatory cascade involving any NLR member is unknown in a lower vertebrate like fish. Also, inflammatory cytokine induction pathway in response to a specific ligand, namely bacterial lipopolysaccharide (LPS) has not yet been clarified. Therefore, 13 predicted NLR sequences of the Japanese pufferfish, Fugu (Takifugu rubripes) were retrieved in silico and categorized as NLR-C1∼13. Expression analysis of these genes in Fugu head kidney (HK) cells stimulated with a heat-killed Lactobacillus paracasei spp. paracasei (Lpp), LPS, nigericin and a combination of nigericin + LPS showed consistent up-regulations of NLR-C1, 5, 7, 10 and 12 genes in both Lpp and LPS stimulations and NLR-C9 gene in LPS stimulation only. However, nigericin and nigericin + LPS caused an increased expression of NLR-C10 and 12 in HK cells and leukocytes. Fugu treated with Lpp and LPS (in vivo), and infected with Vibrio harveyi had an elevated expression of NLR-C10 and 12. Increased transcription of caspase-1, ASC, IL-1β and IL-18 was recorded in nigericin-stimulated HK cells and leukocytes. Results suggested activation of probable inflammasome-mediated inflammatory cytokine response in Fugu. Moreover, LPS may be a key ligand that induces some of the Fugu NLR-Cs (NLR-C9, 10 and 12). Further characterization and functional analysis of Fugu NLR-C10 and 12 for ligand sensing, and processing of pro-inflammatory cytokine, IL-1β would elucidate the inflammasome evolution in fish.
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    Pubmed
    Inflammatory responses in the Japanese pufferfish (Takifugu rubripes) head kidney cells stimulated with an inflammasome-inducing agent, nigericin.
    (2014-10-01T00:00:00Z) Bilen, Soner; Biswas, Gouranga; Otsuyama, Shohei; Kono, Tomoya; Sakai, Masahiro; Hikima, Jun-ichi
    A cytosolic receptor complex called inflammasome is responsible for mounting inflammatory response by releasing pro-inflammatory cytokines, interleukin (IL)-1β and IL-18. However, inflammatory cascades mediated by the inflammasome are unknown in a lower vertebrate like fish. Therefore, in an in vitro experiment, in order to obtain a preliminary information, we conducted transcriptomic analysis of 18 cytokines including pro-inflammatory cytokines in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells stimulated with an inflammasome-inducing agent, nigericin, and a combination of nigericin and LPS by a multiplex RT-PCR assay (GenomeLab Genetic Analysis System, GeXPS; Beckman Coulter Inc.). Furthermore, expression of IL-1β, IL-6, IL-18, nuclear factor (NF)-κB, nucleotide-binding oligomerization domain 2 (NOD2) and NOD-like receptor X1 (NLRX1) genes was examined in HK cells by a quantitative real-time PCR. Additionally, to confirm functionality of activated inflammatory immunity, we also assessed phagocytic activity, superoxide anion production (NBT assay) and lysozyme activity in the nigericin-stimulated HK cells. An increased gene expression of pro-inflammatory cytokines (IL-1β and IL-18), NF-κB and NOD2 was recorded in nigericin and combined nigericin+LPS- stimulated HK cells. Enhanced cellular (phagocytic activity and NBT assay) and humoral (lysozyme activity) immune parameters in the stimulated cells confirmed induction of inflammatory response. Results suggested probable activation of inflammasome components for processing of the inflammatory cytokines in the Japanese pufferfish.