Bilirubin induces microglial NLRP3 inflammasome activation in vitro and in vivo.

Despite current advancements in neonatal care, hyperbilirubinemia resulting in bilirubin-induced neurological dysfunction (BIND) continues to be one of the major reasons of mortality or lifelong disability. Although the exact mechanisms underlying brain injury upon bilirubin exposure remains unelucidated, inflammation is considered to be one of the major contributors to BIND. This study investigates the role of the NLRP3 inflammasome in bilirubin-induced injury using in vitro and in vivo models. We successfully demonstrated that the upregulation of NLRP3 expression is significantly associated with the release of active caspase-1 and IL-1ß in N9 microglial cells exposed to bilirubin. Functional in vitro experiments with NLRP3 siRNA confirms that bilirubin-induced inflammasome activation and cell death are mediated by the NLRP3 inflammasome. Following injection of bilirubin into the cisterna magna of a neonatal mouse, activation of the NLRP3 inflammasome and microglia were determined by double staining with Iba1-NLRP3 and Iba1-Caspase-1. Upon injection of bilirubin into the cisterna magna, neuronal loss was significantly higher in the wild-type mouse compared to Nlrp3-/- and Caspase-1-/- strains. Collectively, these data indicate that NLRP3 inflammasome has a crucial role in microglial activation and bilirubin-induced neuronal damage.

Genomewide Elucidation of Drug Resistance Mechanisms for Systemically Used Antifungal Drugs Amphotericin B, Caspofungin, and Voriconazole in the Budding Yeast.

There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.

In vitro effects of H2O2 on neural stem cell differentiation.

The development of the CNS is a complex and well-regulated process, where stem cells differentiate into committed cells depending on the stimuli from the microenvironment. Alterations of oxygen levels were stated to be significant in terms of brain development and neurogenesis during embryonic development, as well as the adult neurogenesis. As a product of oxygen processing, hydrogen peroxide (H2O2) has been established as a key regulator, acting as a secondary messenger, of signal transduction and cellular biological functions. H2O2 is involved in survival, proliferation, and differentiation of neural stem cells into committed cells of the CNS. Effects of different concentrations of exogenous H2O2 on neuronal differentiation and the molecular pathways involved are yet to be clearly understood. Here, we investigated the concentration-dependent effects of H2O2 on differentiation of neural stem cells using CGR8 embryonic mouse stem cell line. We have demonstrated that treated doses of H2O2 suppress neural differentiation; additionally, our study suggests that relatively high doses of exogenous H2O2 suppress the differentiation process of neural stem cells through AKT and p38 pathways.

Sulforaphane inhibits NLRP3 inflammasome activation in microglia through Nrf2-mediated miRNA alteration.

The NLRP3 inflammasome is a multiprotein complex that activates caspase-1 and triggers the release of the proinflammatory cytokines IL-1ß and IL-18 in response to diverse signals. Although inflammasome activation plays critical roles against various pathogens in host defense, overactivation of inflammasome contributes to the pathogenesis of inflammatory diseases, including acute CNS injuries and chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the current study, we demonstrated that Sulforaphane (SFN), a dietary natural product, inhibits NLRP3 inflammasome mediated IL-1ß and IL-18 secretion and pyroptosis in murine microglial cells. SFN decreased the secretion of IL-1ß and IL-18, and their mRNA levels in LPS primed microglia triggered by ATP. SFN suppressed the overexpression of cleaved caspase-1 and NLRP3 protein expressions as measured by caspase activity assay and western blot, respectively. SFN also prevented caspase-1 dependent pyroptotic cell death in microglia. Our data indicate that SFN suppresses NLRP3 inflammasome via the inhibition of NF-κB nuclear translocation and Nrf2 mediated miRNAs expression modulation in murine microglia.

Inhibitory effects of phytochemicals on NLRP3 inflammasome activation: A review.

BACKGROUND: The NLRP3 inflammasome formation and following cytokine secretion is a crucial step in innate immune responses. Internal and external factors may trigger inflammasome activation and result in inflammatory cytokine secretion. Inflammasome formation and activity play critical roles in several disease pathologies such as cardiovascular, metabolic, renal, digestive, and CNS diseases. Underlying pathways are not yet clear, but phytochemicals as alternative therapies have been extensively used for suppression of inflammatory responses. PURPOSE: In this review, we aimed to summarize in vivo and in vitro effects on NLRP3 inflammasome activation of selected phytochemicals. METHOD: Three phytochemicals; Sulforaphane, Curcumin, and Resveratrol were selected, and studies were reviewed to clarify their intracellular signaling mechanism in NLRP3 inflammasome activity. PubMed and Scopus databases are used for the search. For sulforaphane, 8 articles, for curcumin, 25 articles, and for resveratrol, 41 articles were included in the review. CONCLUSION: In vitro and in vivo studies pointed out that the selected phytochemicals have inhibitory properties on NLRP3 inflammasome activity. However, neither the mechanism is clear, nor the study designs and doses are standardized.

Oxygen exposure in early life activates NLRP3 inflammasome in mouse brain.

Despite widely known detrimental effects on the developing brain, supplemental oxygen is still irreplaceable in the management of newborn infants with respiratory distress. Identifying downstream mechanisms underlying oxygen toxicity is a key step for development of new neuroprotective strategies. Main purpose of this study is to investigate whether NLRP3 inflammasome activation has a role in the pathogenesis of hyperoxia-induced preterm brain injury. C57BL6 pups were randomly divided into either a hyperoxia group (exposed to 90 % oxygen from birth until postnatal day 7) or control group (maintained in room air; 21 % O2). At postnatal day 7, all animals were sacrificed. Immunohistochemical examination revealed that hyperoxic exposure for seven days resulted in a global increase in NLRP3 and IL-1ß immunopositive cells in neonatal mouse brain (p ≤ 0.001). There was a significant rise in Caspase-1 positive cell count in prefrontal and parietal area in the hyperoxia group when compared with controls (p ≤ 0.001). Western blot analysis of brain tissues showed elevated NLRP3, IL-1ß and Caspase-1 protein levels in the hyperoxia group when compared with controls (p ≤ 0.001). To the best of our knowledge, this is the first study that investigates an association between hyperoxia and establishment of NLRP3 inflammasome in preterm brain.

Peptide Derivatives of Erythropoietin in the Treatment of Neuroinflammation and Neurodegeneration.

During the past 35 years, recombinant DNA technology has allowed the production of a wide range of hematopoietic and neurotrophic growth factors including erythropoietin (EPO). These have emerged as promising protein drugs in various human diseases. Accumulated evidences have recently demonstrated the neuroprotective effect of EPO in preclinical models of acute and chronic degenerative disorders. Nevertheless, tissue protective effect of EPO could not be translated to the clinical trials because of common lethal thromboembolic events, erythropoiesis and hypertension. Although chemically modified nonerythropoietic analogs of EPO bypass these side effects, high expense, development of antidrug antibodies, and promotion of tumorigenicity are still concern especially in long-term use. As an alternative, nonerythropoietic EPO mimetic peptides can be used as candidate drugs with their high potency and selectivity, easy production, low cost, and immunogenicity properties. Recent experimental studies suggest that these peptides prevent ischemic brain injury and neuroinflammation. The results of clinical trial in patients with neuropathic pain are also promising. Herein, we summarize these studies and review advanced experimental and in silico methods in peptide drug discovery.