Yi Lin Cheng

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.

Anesthetic Propofol Reduces Endotoxic Inflammation by Inhibiting Reactive Oxygen Species-regulated Akt/IKKβ/NF-κB Signaling

Background Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages. Methodology/Principal Findings Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages. Conclusions/Significance These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

IFN‐γ synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase‐3‐inhibited IL‐10

Interferon‐γ (IFN‐γ) plays a crucial role in innate immunity and inflammation. It causes the synergistic effect on endotoxin lipopolysaccharide (LPS)‐stimulated inducible nitric oxide synthase (iNOS)/NO biosynthesis; however, the mechanism remains unclear. In the present study, we investigated the effects of glycogen synthase kinase‐3 (GSK‐3)‐mediated inhibition of anti‐inflammatory interleukin‐10 (IL‐10). We found, in LPS‐stimulated macrophages, that IFN‐γ increased iNOS expression and NO production in a time‐dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor‐α and regulated on activation, normal T expressed and secreted, and the downregulation of IL‐10. RT‐PCR further showed changes in the IL‐10 mRNA level as well. Treating cells with recombinant IL‐10 showed a decrease in IFN‐γ/LPS‐induced iNOS/NO biosynthesis, whereas anti‐IL‐10 neutralizing antibodies enhanced this effect, suggesting that IL‐10 acts in an anti‐inflammatory role. GSK‐3‐inhibitor treatment blocked IFN‐γ/LPS‐induced iNOS/NO biosynthesis but upregulated IL‐10 production. Inhibiting GSK‐3 using short‐interference RNA showed similar results. Additionally, treating cells with anti‐IL‐10 neutralizing antibodies blocked these effects. We further showed that inhibiting GSK‐3 increased phosphorylation of transcription factor cyclic AMP response element binding protein. Inhibiting protein tyrosine kinase Pyk2, an upstream regulator of GSK‐3β, caused inhibition on IFN‐γ/LPS‐induced GSK‐3β phosphorylation at tyrosine 216 and iNOS/NO biosynthesis. Taken together, these findings reveal the involvement of GSK‐3‐inhibited IL‐10 on the induction of iNOS/NO biosynthesis by IFN‐γ synergized with LPS. J. Cell. Biochem. 105: 746–755, 2008.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis

Inactivation of glycogen synthase kinase (GSK)‐3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK‐3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr‐Abl tyrosine kinase. In Bcr‐Abl signaling, the role of GSK‐3 is not well defined. Here, we report that enforced expression of constitutively active GSK‐3 reduced proliferation and increased Bcr‐Abl inhibition‐induced apoptosis by nearly 1‐fold. Bcr‐Abl inhibition activated GSK‐3 and GSK‐3‐dependent apoptosis. Inactivation of GSK‐3 by Bcr‐Abl activity is, therefore, confirmed. To reactivate GSK‐3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor‐suppressor sphingo‐lipid and a potent GSK‐3 activator. We found that either PDMP or silence of GCS increased Bcr‐Abl inhibition‐induced GSK‐3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug‐resistant CML T315I mutant to Bcr‐Abl inhibitor GNF‐2‐, imatinib‐, or nilotinib‐induced apoptosis by >5‐fold. Combining PDMP and GNF‐2 eliminated transplanted‐CML‐T315I‐mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF‐2‐induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr‐Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK‐3‐mediated apoptosis. This study suggests a feasible novel anti‐CML strategy by accumulating endogenous ceramide to reactivate GSK‐3 and abrogate drug resistance.—Huang, W.‐C., Tsai, C.‐C., Chen, C.‐L., Chen, T.‐Y., Chen, Y.‐P., Lin, Y.‐S., Lu, P.‐J., Lin, C.‐M., Wang, S.‐W., Tsao, C.‐W., Wang, C.‐Y., Cheng, Y.‐L., Hsieh, C.‐Y., Tseng, P.‐C., Lin, C.‐F. Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr‐Abl inhibitor and cooperatively induces glycogen synthase kinase‐3‐regulated apoptosis. FASEB J. 25, 3661–3673 (2011). www.fasebj.org

Staphylococcus aureus induces microglial inflammation via a glycogen synthase kinase 3β-regulated pathway

ABSTRACT A proinflammatory role for glycogen synthase kinase 3β (GSK-3β) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-α largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3β, and inhibiting GSK-3β reduced TNF-α production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-κB was essential for heat-inactivated S. aureus-induced TNF-α and NO, inhibiting GSK-3β blocked heat-inactivated S. aureus-induced NF-κB p65 nuclear translocation. Additionally, inhibiting GSK-3β enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-α production). Neutralization of IL-10 reduced TNF-α downregulation caused by GSK-3β inhibition. These results suggest that GSK-3β regulates heat-inactivated S. aureus-induced TNF-α and NO production in microglia mainly by activating NF-κB and probably by inhibiting IL-10.

Increased galectin-3 facilitates leukemia cell survival from apoptotic stimuli

Galectin-3 is regulated for cancer cell survival and apoptosis depending upon the cell type and stimulus. We investigated a glycogen synthase kinase (GSK)-3β/galectin-3-regulated mechanism used by leukemia cells to escape from apoptotic stimuli. Galectin-3 expression was time- and transcription-dependently deregulated in K562 chronic myeloid leukemia cells stimulated for apoptosis by cisplatin (a platinum-based chemotherapy drug), sphingolipid ceramide analog C(2)-ceramide, and LY294002 (a phosphatidylinositol 3-kinase inhibitor). Notably, galectin-3 was upregulated in survival cells. Forced galectin-3 expression caused resistance to apoptosis, whereas knockdown galectin-3 expression increased susceptibility to apoptosis. Sub-cellular distribution of inducible galectin-3 was mitochondria-specific. Apoptotic stimuli decreased pro-survival Bcl-2 family protein expression (especially Mcl-1), whereas galectin-3 overexpression reversed but it was enhanced by a galectin-3 expression knockdown. Under apoptotic stimulation, GSK-3β was activated after Akt was inactivated and GSK-3β was inhibited-either pharmacologically or using short hairpin RNA to abolish galectin-3, increase apoptosis, and inhibit colony formation-which suggests a pro-survival role for GSK-3β. We found that GSK-3β upregulated galectin-3 and stabilized anti-apoptotic Bcl-2 family proteins, which is important for the escape of leukemia cells from apoptotic stimuli.

Microglia retard dengue virus-induced acute viral encephalitis

Patients with dengue virus (DENV) infection may also present acute viral encephalitis through an unknown mechanism. Here, we report that encephalitic DENV-infected mice exhibited progressive hunchback posture, limbic seizures, limbic weakness, paralysis, and lethality 7 days post-infection. These symptoms were accompanied by CNS inflammation, neurotoxicity, and blood-brain barrier destruction. Microglial cells surrounding the blood vessels and injured hippocampus regions were activated by DENV infection. Pharmacologically depleting microglia unexpectedly increased viral replication, neuropathy, and mortality in DENV-infected mice. In microglia-depleted mice, the DENV infection-mediated expression of antiviral cytokines and the infiltration of CD8-positive cytotoxic T lymphocytes (CTLs) was abolished. DENV infection prompted the antigen-presenting cell-like differentiation of microglia, which in turn stimulated CTL proliferation and activation. These results suggest that microglial cells play a key role in facilitating antiviral immune responses against DENV infection and acute viral encephalitis.

Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-α production by mononuclear phagocytes

Infection by the dengue virus (DENV) threatens global public health due to its high prevalence and the lack of effective treatments. Host factors may contribute to the pathogenesis of DENV; herein, we investigated the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is activated by DENV in mononuclear phagocytes. DENV infection selectively activates Nrf2 following nuclear translocation. Following endoplasmic reticular (ER) stress, protein kinase R-like ER kinase (PERK) facilitated Nrf2-mediated transcriptional activation of C-type lectin domain family 5, member A (CLEC5A) to increase CLEC5A expression. Signaling downstream of the Nrf2-CLEC5A interaction enhances Toll-like receptor 3 (TLR3)-independent tumor necrosis factor (TNF)-α production following DENV infection. Forced expression of the NS2B3 viral protein induces Nrf2 nuclear translocation/activation and CLEC5A expression which increases DENV-induced TNF-α production. Animal studies confirmed Nrf2-induced CLEC5A and TNF-α in brains of DENV-infected mice. These results demonstrate that DENV infection causes Nrf2-regulated TNF-α production by increasing levels of CLEC5A.

Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells

Annexin A2 (ANXA2), a phospholipid-binding protein, has multiple biological functions depending on its cellular localization. We previously demonstrated that IFN-γ-triggered ANXA2 secretion is associated with exosomal release. Here, we show that IFN-γ-induced autophagy is essential for the extracellular secretion of ANXA2 in lung epithelial cells. We observed colocalization of ANXA2-containing autophagosomes with multivesicular bodies (MVBs) after IFN-γ stimulation, followed by exosomal release. IFN-γ-induced exophagic release of ANXA2 could not be observed in ATG5-silenced or mutant RAB11-expressing cells. Furthermore, knockdown of RAB8A and RAB27A, but not RAB27B, reduced IFN-γ-triggered ANXA2 secretion. Surface translocation of ANXA2 enhanced efferocytosis by epithelial cells, and inhibition of different exophagic steps, including autophagosome formation, fusion of autophagosomes with MVBs, and fusion of amphisomes with plasma membrane, reduced ANXA2-mediated efferocytosis. Our data reveal a novel route of IFN-γ-induced exophagy of ANXA2.

Different Types of Cell Death Induced by Enterotoxins

The infection of bacterial organisms generally causes cell death to facilitate microbial invasion and immune escape, both of which are involved in the pathogenesis of infectious diseases. In addition to the intercellular infectious processes, pathogen-produced/secreted enterotoxins (mostly exotoxins) are the major weapons that kill host cells and cause diseases by inducing different types of cell death, particularly apoptosis and necrosis. Blocking these enterotoxins with synthetic drugs and vaccines is important for treating patients with infectious diseases. Studies of enterotoxin-induced apoptotic and necrotic mechanisms have helped us to create efficient strategies to use against these well-characterized cytopathic toxins. In this article, we review the induction of the different types of cell death from various bacterial enterotoxins, such as staphylococcal enterotoxin B, staphylococcal alpha-toxin, Panton-Valentine leukocidin, alpha-hemolysin of Escherichia coli, Shiga toxins, cytotoxic necrotizing factor 1, heat-labile enterotoxins, and the cholera toxin, Vibrio cholerae. In addition, necrosis caused by pore-forming toxins, apoptotic signaling through cross-talk pathways involving mitochondrial damage, endoplasmic reticulum stress, and lysosomal injury is discussed.