Yuji Minai

Tumor Necrosis Factor-α (TNF-α) Regulates Shedding of TNF-α Receptor 1 by the Metalloprotease-Disintegrin ADAM8: Evidence for a Protease-Regulated Feedback Loop in Neuroprotection

Tumor necrosis factor α (TNF-α) is a potent cytokine in neurodegenerative disorders, but its precise role in particular brain disorders is ambiguous. In motor neuron (MN) disease of the mouse, exemplified by the model wobbler (WR), TNF-α causes upregulation of the metalloprotease-disintegrin ADAM8 (A8) in affected brain regions, spinal cord, and brainstem. The functional role of A8 during MN degeneration in the wobbler CNS was investigated by crossing WR with A8-deficient mice: a severely aggravated neuropathology was observed for A8-deficient WR compared with WR A8+/− mice, judged by drastically reduced survival [7 vs 81% survival at postnatal day 50 (P50)], accelerated force loss in the forelimbs, and terminal akinesis. In vitro protease assays using soluble A8 indicated specific cleavage of a TNF-α receptor 1 (p55 TNF-R1) but not a TNF-R2 peptide. Cleavage of TNF-R1 was confirmed in situ, because levels of soluble TNF-R1 were increased in spinal cords of standard WR compared with wild-type mice but not in A8-deficient WR mice. In isolated primary neurons and microglia, TNF-α-induced TNF-R1 shedding was dependent on the A8 gene dosage. Furthermore, exogenous TNF-α showed higher toxicity for cultured neurons from A8-deficient than for those from wild-type mice, demonstrating that TNF-R1 shedding by A8 is neuroprotective. Our results indicate an essential role for ADAM8 in modulating TNF-α signaling in CNS diseases: a feedback loop integrating TNF-α, ADAM8, and TNF-R1 shedding as a plausible mechanism for TNF-α mediated neuroprotection in situ and a rationale for therapeutic intervention.

NF-κB activation via MyD88-dependent Toll-like receptor signaling is inhibited by trichothecene mycotoxin deoxynivalenol

Macrophages induce the innate immunity by recognizing pathogens through Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Myeloid differentiation factor 88 (MyD88), which is an essential adaptor molecule for most TLRs, mediates the induction of inflammatory cytokines through nuclear factor κB (NF-κB). Trichothecene mycotoxin deoxynivalenol (DON) shows immunotoxic effects by interrupting inflammatory mediators produced by activated macrophages. The present study investigates the effect of DON on NF-κB in activated macrophages through MyD88-dependent pathways. DON inhibited NF-κB-dependent reporter activity induced by MyD88-dependent TLR agonists. In addition, lipopolysaccharide-induced phosphorylation of interleukin-1 receptor-associated kinase 1 and inhibitor κBα were attenuated by DON. Furthermore, DON downregulated the expression level of MyD88. These results suggest that DON inhibits NF-κB activation in macrophages stimulated with TLR ligands via MyD88-dependent TLR signals. Therefore exposure to DON may lead to the inhibition of MyD88-dependent pathway of TLR signaling.

Impaired Growth of Small Intestinal Epithelium by Adrenalectomy in Weaning Rats

Functional maturation of the small intestine occurs during the weaning period in rats. It is known that this development is facilitated by glucocorticoid. However, the effect of glucocorticoid on morphological development of small intestine has yet to be clarified. The present study evaluated the morphological development and cell proliferation of the small intestine in adrenalectomized (ADX) rat pups. To further understand the mechanism of glucocorticoid effects on intestinal development, we examined the localization of the glucocorticoid receptor in the small intestine. Microscopic analysis showed that growth of villi and crypts is age-dependent, and is significantly attenuated in ADX rats compared with sham-operated rats. BrdU-positive cells, i.e. proliferating cells, were primarily observed in crypt compartments and rapidly increased in number during the early weaning period. The increase in BrdU-positive cells could be attenuated by adrenalectomy. The morphological development of small intestine may be associated with increased proliferation of epithelial cells. On the other hand, glucocorticoid receptors were found in epithelial cells of the mid- and lower villi and not in crypts where BrdU-positive cells were localized. These results indicate that the growth of small intestine is attenuated by adrenalectomy, and that glucocorticoid indirectly acts on proliferation of epithelial cells during the weaning period.

Epigallocatechin gallate suppresses the cytotoxicity induced by trichothecene mycotoxins in mouse cultural macrophages.

Trichothecene mycotoxins are toxic secondary metabolites produced by a number of fungi including Fusarium species, which adversely affect lymphocytes. Deoxynivalenol (DON) and HT-2 toxin (HT-2) belong to the trichothecene group of mycotoxins and the occurrence of cereals and foodstuffs with these compounds are serious health problems. The aim of this study was to examine the effect of (−)-epigallocatechin gallate (EGCG), one of the main components in green tea catechins, on DON- or HT-2-induced cytotoxicity in mouse macrophages. EGCG had protective effects against the trichothecene-induced cytotoxicities of both mycotoxins. Additionally, EGCG suppressed the DON-induced activation of caspase-3/7, which is an indicator of apoptosis. These results indicate that EGCG might be useful in protection against DON- or HT-2-induced cell death, suggesting that EGCG could contribute to reducing the toxicities of trichothecenes.

Inhibitory effect of citrinin on lipopolisaccharide-induced nitric oxide production by mouse macrophage cells

The present study evaluated the immunotoxicity of citrinin (CIT), a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. Because nitric oxide (NO), a pro-inflammatory mediator, plays an important role in the protection from pathogens, we addressed the effect of CIT on NO production by a mouse macrophage-like cell line RAW264 activated with lipopolysaccharide (LPS). LPS-induced NO release from RAW264 cells was inhibited by CIT. Moreover, the transcription and expression of inducible NO synthase (iNOS) by LPS was suppressed by CIT. These results show that CIT suppressed the LPS-induced NO production and iNOS expression, which contribute to the host protection against invading pathogens. This suggests that CIT on LPS-induced NO release may exert adverse effects in macrophages, indicating immunotoxic effects of this toxin. .

Effect of monochromatic visible light on intracellular superoxide anion production and mitochondrial membrane potential of B16F1 and B16F10 murine melanoma cells

We have investigated the effect of visible light on animal cells using light‐emitting diodes to emit monochromatic visible light (red, yellow, green and blue light). To explore the relevant mechanism of apoptosis, we assessed the intracellular superoxide anion production and mitochondrial membrane potential (ΔΨm) of B16F1 and B16F10 murine melanoma cells after monochromatic light irradiation. Blue light caused ΔΨm depolarization subsequent to elevation of intracellular superoxide production. However, red and yellow light had no affect on both cell lines. Green light induced ΔΨm collapse only in B16F1 melanoma cells. ΔΨm is a key indicator of mitochondrial function, therefore its disruption causes mitochondria‐dependent apoptosis. Thus, blue light causes mitochondrial dysfunction and subsequent cell death.