Sanguinarine modulates microglial function via PPARγ activation and protects against CNS demyelination.

Microglia aggregate in regions of active inflammation and demyelination in the CNS of multiple sclerosis (MS) patients and are considered pivotal in the disease process. Targeting microglia is a promising therapeutic approach for myelin repair. Previously, we identified two candidates for microglial modulation and remyelination using a Connectivity Map (CMAP)-based screening strategy. Interestingly, with results that overlapped, sanguinarine (SAN) emerged as a potential drug candidate to modulate microglial polarization and promote remyelination. In the current study, we demonstrate the efficacy of SAN in mitigating the MS-like experimental autoimmune encephalomyelitis (EAE) in a dose-dependent manner. Meanwhile, prophylactic administration of a medium dose (2.5 mg/kg) significantly reduces disease incidence and ameliorates clinical signs in EAE mice. At the cellular level, SAN reduces the accumulation of microglia in the spinal cord. Morphological analyses and immunophenotyping reveal a less activated state of microglia following SAN administration, supported by decreased inflammatory cytokine production in the spinal cord. Mechanistically, SAN skews primary microglia towards an immunoregulatory state and mitigates proinflammatory response through PPARγ activation. This creates a favorable milieu for the differentiation of oligodendrocyte progenitor cells (OPCs) when OPCs are incubated with conditioned medium from SAN-treated microglia. We further extend our investigation into the cuprizone-induced demyelinating model, confirming that SAN treatment upregulates oligodendrocyte lineage genes and increases myelin content, further suggesting its pro-myelination effect. In conclusion, our data propose SAN as a promising candidate adding to the preclinical therapeutic arsenal for regulating microglial function and promoting myelin repair in CNS demyelinating diseases such as MS.

Morphological and molecular alterations of reactive astrocytes without proliferation in cerebral cortex of an APP/PS1 transgenic mouse model and Alzheimer's patients.

Astrocytes are fundamental for maintaining brain homeostasis and are commonly involved in the progression of neurodegenerative diseases including Alzheimer's disease (AD). In response to injury or toxic material, astrocytes undergo activation that results in hypertrophy and process ramification. Although numerous studies have shown that reactive astrocytes are intimately related to the pathogenesis of AD, their characteristic features including morphological and molecular alterations that occur during different stages of AD progression remain to be elucidated. Here, we crossed astrocyte-specific reporter mice hGFAP-CreERT2;Rosa-tdTomato with APP/PS1 mice, and then used genetic tracing to characterize the morphological profiles and expression of molecular biomarkers associated with progressive ß-amyloid deposits in the cortical region of AD mice. Expression of glutamine synthetase (GS) was lower in cortical reactive astrocytes, in contrast to the higher expression of glial fibrillary acidic protein, of APP/PS1 mice and AD patients relative to that in cortical astrocytes of wild-type mice and age-matched controls, respectively. GS activity was also decreased obviously in the cortex of APP/PS1 mice at 6 and 12 months of age relative to that in the wild-type mice of the same ages. Furthermore, cortical reactive astrocytes in APP/PS1 mice and AD patients did not undergo proliferation. Finally, based on RNA-sequencing analysis, we identified differentially expressed transcripts of signal transduction molecules involved in early induction of reactive astrocytes in the cortex of APP/PS1 mice. These findings provide a morphological and molecular basis with which to understand the function and mechanism of reactive astrocytes in the progression of AD.

DNA Sensor IFI204 Contributes to Host Defense Against Staphylococcus aureus Infection in Mice.

Interferon-inducible protein (IFI204) (p204, the murine homolog of human IFI16) is known as a cytosolic DNA sensor to recognize DNA viruses and intracellular bacteria. However, little is known about its role during extracellular bacterial infection. Here we show that IFI204 is required for host defense against the infection of Staphylococcus aureus, an extracellular bacterial pathogen. IFI204 deficiency results in decreased survival, increased bacterial loads, severe organs damage, and decreased recruitment of neutrophils and macrophages. Production of several inflammatory cytokines/chemokines including IFN-ß and KC is markedly decreased, as well as the related STING-IRF3 and NF-κB pathways are impaired. However, exogenous administration of recombinant KC or IFN-ß is unable to rescue the susceptibility of IFI204-deficient mice, suggesting that other mechanisms rather than KC and IFN-ß account for IFI204-mediated host defense. IFI204 deficiency leads to a defect in extracellular bacterial killing in macrophages and neutrophils, although bacterial engulf, and intracellular killing activity are normal. Moreover, the defect of bactericidal activity is mediated by decreased extracellular trap formation in the absence of IFI204. Adoptively transferred WT bone marrow cells significantly protect WT and IFI204-deficient recipients against Staphylococcus infection compared with transferred IFI204-deficient bone marrow cells. Hence, this study suggests that IFI204 is essential for the host defense against Staphylococcus infection.

Liver X receptors agonists suppress NLRP3 inflammasome activation.

Inflammasomes are multiprotein complexes that control the production of IL-1ß and IL-18. NLRP3 inflammasome, the most characterized inflammasome, plays prominent roles in defense against infection, however aberrant activation is deleterious and leads to diseases. Therefore, its tight control offers therapeutic promise. Liver X receptors (LXRs) have significant anti-inflammatory properties. Whether LXRs regulate inflammasome remains unresolved. We thus tested the hypothesis that LXR's anti-inflammatory properties may result from its ability to suppress inflammasome activation. In this study, LXRs agonists inhibited the induction of IL-1ß production, caspase-1 cleavage and ASC oligomerization by NLRP3 inflammasome. The agonists also inhibited inflammasome-associated mtROS production. Importantly, the agonists inhibited the priming of inflammasome activation. In vivo data also showed that LXRs agonist prevented NLRP3-dependent peritonitis. In conclusion, LXRs agonists are identified to potently suppress NLRP3 inflammasome and the regulation of LXRs signaling is a potential therapeutic for inflammasome-driven diseases.

SAMD9L inactivation promotes cell proliferation via facilitating G1-S transition in hepatitis B virus-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant cancer with poor prognosis, and driver genes harboring genetic lesions and/or expression dysregulation contribute to hepatocarcinogenesis. Sterile Alpha Motif Domain-containing 9-like (SAMD9L) was a novel identified mutated gene in our previous study on exome sequencing of hepatitis B virus (HBV)-associated HCC, but its expression and role in HCC remain unknown. Here, we demonstrated that SAMD9L was frequently inactivated by somatic mutations, and that its expression was deregulated in HCC patients with hepatitis B virus (HBV) infection. SAMD9L knockdown significantly promoted cell proliferation, colony formation of SK-hep-1, QGY-7701, BEL-7721 and MHCC-97H HCC cells. Furthermore, SK-hep-1 and MHCC-97H cells with stable SAMD9L knockdown exhibited enhanced tumorigenicity in athymic mice. Interestingly, SAMD9L silence facilitated G1-S transition of cell cycle progression and led to the elevated activity of Wnt/ß-catenin pathway. Collectively, these findings highlight a novel tumor-suppressive role of SAMD9L inactivation by somatic mutation and decreased expression in human HBV-related HCC.

RNA interference against cancer/testis genes identifies dual specificity phosphatase 21 as a potential therapeutic target in human hepatocellular carcinoma.

UNLABELLED: Cancer/testis (CT) antigens have been considered therapeutic targets for treating cancers. However, a central question is whether their expression contributes to tumorigenesis or if they are functionally irrelevant by-products derived from the process of cellular transformation. In any case, these CT antigens are essential for cancer cell survival and may serve as potential therapeutic targets. Recently, the cell-based RNA interference (RNAi) screen has proven to be a powerful approach for identifying potential therapeutic targets. In this study we sought to identify new CT antigens as potential therapeutic targets for human hepatocellular carcinoma (HCC), and 179 potential CT genes on the X chromosome were screened through a bioinformatics analysis of gene expression profiles. Then an RNAi screen against these potential CT genes identified nine that were required for sustaining the survival of Focus and PLC/PRF/5 cells. Among the nine genes, the physiologically testis-restricted dual specificity phosphatase 21 (DUSP21) encoding a dual specificity phosphatase was up-regulated in 39 (33%) of 118 human HCC specimens. Ectopic DUSP21 had no obvious impact on proliferation and colony formation in HCC cells. However, DUSP21 silencing significantly suppressed cell proliferation, colony formation, and in vivo tumorigenicity in HCC cells. The administration of adenovirus-mediated RNAi and an atelocollagen/siRNA mixture against endogenous DUSP21 significantly suppressed xenograft HCC tumors in mice. Further investigations showed that DUSP21 knockdown led to arrest of the cell cycle in G1 phase, cell senescence, and expression changes of some factors with functions in the cell cycle and/or senescence. Furthermore, the antiproliferative role of DUSP21 knockdown is through activation of p38 mitogen-activated protein kinase in HCC. CONCLUSION: DUSP21 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment.

Exome sequencing of hepatitis B virus-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and shows a propensity to metastasize and infiltrate adjacent and more distant tissues. HCC is associated with multiple risk factors, including hepatitis B virus (HBV) infection, which is especially prevalent in China. Here, we used exome sequencing to identify somatic mutations in ten HBV-positive individuals with HCC with portal vein tumor thromboses (PVTTs), intrahepatic metastases. Both C:G>A:T and T:A>A:T transversions were frequently found among the 331 non-silent mutations. Notably, ARID1A, which encodes a component of the SWI/SNF chromatin remodeling complex, was mutated in 14 of 110 (13%) HBV-associated HCC specimens. We used RNA interference to assess the roles of 91 of the confirmed mutated genes in cellular survival. The results suggest that seven of these genes, including VCAM1 and CDK14, may confer growth and infiltration capacity to HCC cells. This study provides a view of the landscape of somatic mutations that may be implicated in advanced HCC.

Optimal minimax designs over a prespecified interval in a heteroscedastic polynomial model.

Minimax optimal designs can be useful for estimating response surface but they are notoriously difficult to study analytically. We provide formulae for three types of minimax optimal designs over a user-specified region. We focus on polynomial models with various types of heteroscedastic errors but the design strategy is applicable to other types of linear models and optimality criteria. Relationships among the three types of minimax optimal designs are discussed.