Human Interleukin 23 Receptor Induces Cell Apoptosis in Mammalian Cells by Intrinsic Mitochondrial Pathway Associated with the Down-Regulation of RAS/Mitogen-Activated Protein Kinase and Signal Transducers and Activators of Transcription factor 3 Signaling Pathways

The composition of IL-23R complex is similar to that of the IL-12 receptor (IL-12R) complex with a shared IL-12R-β1 chain. The IL-12R-β1 heterodimerizes with IL-23R and IL-12R-β2 to form IL-23R and IL-12R complexes, respectively. The IL-12R-β2 has been shown to function as a tumor suppressor gene and apoptotic inducer. However, whether IL-23R also functions in cell apoptosis is currently unknown. In this study, we demonstrate for the first time that overexpression of IL-23R markedly induces cell apoptosis in both 293ET and HeLa cells. The activations of caspase 3 and caspase 9 are induced by IL-23R. Mechanistic study reveals that IL-23R markedly inhibits RAS/MAPK and STAT3 but not STAT1 and PI-3K/Akt signaling pathways in both 293ET and HeLa cells. Overexpression of IL-23R significantly up-regulates IL-12Rβ1 expression but not IL-23α and IL-12β expressions in both cell lines. Therefore, our data strongly indicates that IL-23R is able to induce cell apoptosis by activating the intrinsic mitochondrial pathways associated with the inhibition in RAS/MAPK and STAT3 activations in mammalian cells.     

Takinib Inhibits Inflammation in Human Rheumatoid Arthritis Synovial Fibroblasts by Targeting the Janus Kinase-Signal Transducer and Activator of Transcription 3 (JAK/STAT3) Pathway

TGF β-activated kinase 1 (TAK1) is an important participant in inflammatory pathogenesis for diseases such as rheumatoid arthritis (RA) and gouty arthritis. The central position it occupies between the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways makes it an attractive therapeutic target. As this field has developed in recent years, several novel inhibitors have been presented as having specific activity that reduces the TAK1 function either covalently as in the case of 5Z-7-oxozeanol (5Z7O) or reversibly (NG-25). However, the mechanism through which takinib elicits its anti-inflammatory activity remains elusive. While this inhibitor shows great promise, a thorough analysis of its inhibitor function and its potential off-target effects is necessary before addressing its clinical potential or its use in inflammatory conditions. An analysis through Western blot showed an unexpected increase in IL-1β-induced TAK1 phosphorylation—a prerequisite for and indicator of its functional potential—by takinib while simultaneously demonstrating the inhibition of the JAK/STAT pathway in human rheumatoid arthritis synovial fibroblasts (RASFs) in vitro. In THP-1 monocyte-derived macrophages, takinib again led to the lipopolysaccharide-induced phosphorylation of TAK1 without a marked inhibition of the TAK1 downstream effectors, namely, of c-Jun N-terminal kinase (JNK), phospho-c-Jun, NF-κB phospho-p65 or phospho-IκBα. Taken together, these findings indicate that takinib inhibits inflammation in these cells by targeting multiple signaling pathways, most notably the JAK/STAT pathway in human RASFs.     

Long,Noncoding RNA SRA Induces Apoptosis of β-Cells by Promoting the IRAK1/LDHA/Lactate Pathway

Long non-coding RNA steroid receptor RNA activators (LncRNA SRAs) are implicated in the β-cell destruction of Type 1 diabetes mellitus (T1D), but functional association remains poorly understood. Here, we aimed to verify the role of LncRNA SRA regulation in β-cells. LncRNA SRAs were highly expressed in plasma samples and peripheral blood mononuclear cells (PBMCs) from T1D patients. LncRNA SRA was strongly upregulated by high-glucose treatment. LncRNA SRA acts as a microRNA (miR)-146b sponge through direct sequence–structure interactions. Silencing of lncRNA SRA increased the functional genes of Tregs, resulting in metabolic reprogramming, such as decreased lactate levels, repressed lactate dehydrogenase A (LDHA)/phosphorylated LDHA (pLDHA at Tyr10) expression, decreased reactive oxygen species (ROS) production, increased ATP production, and finally, decreased β-cell apoptosis in vitro. There was a positive association between lactate level and hemoglobin A1c (HbA1c) level in the plasma from patients with T1D. Recombinant human interleukin (IL)-2 treatment repressed lncRNA SRA expression and activity in β-cells. Higher levels of lncRNA-SRA/lactate in the plasma are associated with poor regulation in T1D patients. LncRNA SRA contributed to T1D pathogenesis through the inhibition of miR-146b in β-cells, with activating signaling transduction of interleukin-1 receptor-associated kinase 1 (IRAK1)/LDHA/pLDHA. Taken together, LncRNA SRA plays a critical role in the function of β-cells.     

NBM-BMX,an HDAC8 Inhibitor,Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the β-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.     

Analysis of PI3K Pathway Associated Molecules Reveals Dysregulated Innate and Adaptive Functions of B Cells in Early Diffuse Cutaneous Systemic Sclerosis

B cell activation is an early event in the development of systemic sclerosis (SSc). The classical activation of B cells downstream of the B-cell receptor (BCR) involves the phosphatidylinositol-3 kinase (PI3K) pathway that integrates the effects of multiple co-stimulatory receptors. Our analysis of PI3K pathway associated molecules in peripheral blood B cells of early diffuse cutaneous SSc (dcSSc) patients showed altered mRNA expression of Toll-like receptor (TLR) homolog CD180, TLR4, complement component 3, IL-4 receptor and secreted phosphoprotein 1 (SPP1). Parallel to this, we found elevated basal SPP1 secretion in dcSSc B cells, but, with BCR + IL-4 receptor co-stimulation, we could not induce further secretion. CD180 stimulation alone resulted in NF-κB activation in more B cells than CD180 + BCR co-stimulation both in dcSSc and healthy control (HC), but the co-engagement increased the phosphorylation of NF-κB only in dcSSc B cells. Additionally, in contrast with HC B cells, the lower basal production of IL-10 by dcSSc B cells could not be elevated with CD180 stimulation. Furthermore, activation via CD180 increased the percentage of CD86+ switched memory (CD27+IgD−) B cells in dcSSc compared to HC. Our results suggest that alternative B cell activation and CD180 dysfunction cause imbalance of regulatory mechanisms in dcSSc B cells.     

Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8⁺ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8⁺ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.     

Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC₅₀ values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC₅₀ values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca²⁺ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.     

IL-33 Enhances IFNγ and TNFα Production by Human MAIT Cells: A New Pro-Th1 Effect of IL-33

Mucosal-associated invariant T (MAIT) cells represent a distinct T cell population restricted by the MHC-class-I-related molecule, MR1, which recognizes microbial-derived vitamin B2 (riboflavin) metabolites. Their abundance in humans, together with their ability to promptly produce distinct cytokines including interferon γ (IFNγ) and tumor necrosis factor α (TNFα), are consistent with regulatory functions in innate as well as adaptive immunity. Here, we tested whether the alarmin interleukin 33 (IL-33), which is secreted following inflammation or cell damage, could activate human MAIT cells. We found that MAIT cells stimulated with IL-33 produced high levels of IFNγ, TNFα and Granzyme B (GrzB). The action of IL-33 required IL-12 but was independent of T cell receptor (TCR) cross-linking. MAIT cells expressed the IL-33 receptor ST2 (suppression of tumorigenicity 2) and upregulated Tbet (T-box expressed in T cells) in response to IL-12 or IL-33. Electronically sorted MAIT cells also upregulated the expression of CCL3 (Chemokine C-C motif ligand 3), CD40L (CD40 Ligand), CSF-1 (Colony Stimulating Factor 1), LTA (Lymphotoxin-alpha) and IL-2RA (IL-2 receptor alpha chain) mRNAs in response to IL-33 plus IL-12. In conclusion, IL-33 combined with IL-12 can directly target MAIT cells to induce their activation and cytokine production. This novel mechanism of IL-33 activation provides insight into the mode of action by which human MAIT cells can promote inflammatory responses in a TCR-independent manner.