Jwa-Jin Kim

Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

Despite the recent attention focused on the roles of the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in the pathogenesis of type 2 diabetes, little is known about the ex vivo profile of inflammasome activation in type 2 diabetic patients. In this study, we investigated patterns of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) from drug-naïve patients with newly diagnosed type 2 diabetes. Type 2 diabetic subjects had significantly increased mRNA and protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and proinflammatory cytokines in MDMs cultured with autologous sera compared with healthy controls. Upregulated interleukin (IL)-1β maturation, IL-18 secretion, and caspase-1 cleavage were observed in MDMs from type 2 diabetic patients after stimulation with various danger molecules (ATP, high-mobility group protein B1, free fatty acids, islet amyloid polypeptide, and monosodium uric acid crystals). Mitochondrial reactive oxygen species and NLRP3 were required for IL-1β synthesis in MDMs. Finally, 2 months of therapy with the antidiabetic drug metformin significantly inhibited the maturation of IL-1β in MDMs from patients with type 2 diabetes through AMP-activated protein kinase (AMPK) activation. Taken together, these data suggest that NLRP3 inflammasome activation is elevated in myeloid cells from type 2 diabetic patients and that antidiabetic treatment with metformin contributes to modulation of inflammasome activation in type 2 diabetes.

The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors

The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-κB signaling pathway, acting as both a repressor of transactivation of the NF-κB subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase–dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), β (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism.

NLRP3 Inflammasome and Host Protection against Bacterial Infection

The inflammasome is a multi-protein complex that induces maturation of inflammatory cytokines interleukin (IL)-1β and IL-18 through activation of caspase-1. Several nucleotide binding oligomerization domain-like receptor family members, including NLRP3, recognize unique microbial and danger components and play a central role in inflammasome activation. The NLRP3 inflammasome is critical for maintenance of homeostasis against pathogenic infections. However, inflammasome activation acts as a double-edged sword for various bacterial infections. When the IL-1 family of cytokines is secreted excessively, they cause tissue damage and extensive inflammatory responses that are potentially hazardous for the host. Emerging evidence has shown that diverse bacterial pathogens or their components negatively regulate inflammasome activation to escape the immune response. In this review, we discuss the current knowledge of the roles and regulation of the NLRP3 inflammasome during bacterial infections. Activation and regulation of the NLRP3 inflammasome should be tightly controlled to prevent virulence and pathology during infections. Understanding the roles and regulatory mechanisms of the NLRP3 inflammasome is essential for developing potential treatment approaches against pathogenic infections.

Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome

Excessive activation of the NLRP3 inflammasome results in damaging inflammation, yet the regulators of this process remain poorly defined. Herein, we show that the orphan nuclear receptor small heterodimer partner (SHP) is a negative regulator of NLRP3 inflammasome activation. NLRP3 inflammasome activation leads to an interaction between SHP and NLRP3, proteins that are both recruited to mitochondria. Overexpression of SHP competitively inhibits binding of NLRP3 to apoptosis-associated speck-like protein containing a CARD (ASC). SHP deficiency results in increased secretion of proinflammatory cytokines IL-1β and IL-18, and excessive pathologic responses typically observed in mouse models of kidney tubular necrosis and peritoneal gout. Notably, the loss of SHP results in accumulation of damaged mitochondria and a sustained interaction between NLRP3 and ASC in the endoplasmic reticulum. These data are suggestive of a role for SHP in controlling NLRP3 inflammasome activation through a mechanism involving interaction with NLRP3 and maintenance of mitochondrial homeostasis.

Controlled delivery of a hydrophilic drug from a biodegradable microsphere system by supercritical anti-solvent precipitation technique.

The purpose of this study was to prepare microspheres loaded with hydrophilic drug, bupivacaine HCl using poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA). Microspheres were prepared with varying the PLGA/PLLA ratio with two different levels of bupivacaine HCl (5 and 10%) using a supercritical anti-solvent (SAS) technique. Microspheres ranging from 4–10 µm in geometric mean diameter could be prepared, with high loading efficiency. Powder X-ray diffraction (PXRD) revealed that bupivacaine HCl retained its crystalline state within the polymer and was present as a dispersion within the polymer phase after SAS processing. The release of bupivacaine HCl from biodegradable polymer microspheres was rapid up to 4 h, thereafter bupivacaine HCl was continuously and slowly released for at least 7 days according to the PLGA/PLLA ratio and the molecular weight of PLLA.

TLR3-Triggered Reactive Oxygen Species Contribute to Inflammatory Responses by Activating Signal Transducer and Activator of Transcription-1

Intracellular reactive oxygen species (ROS) are essential secondary messengers in many signaling cascades governing innate immunity and cellular functions. TLR3 signaling is crucially involved in antiviral innate and inflammatory responses; however, the roles of ROS in TLR3 signaling remain largely unknown. In this study, we show that TLR3-induced ROS generation is required for the activation of NF-κB, IFN-regulatory factor 3, and STAT1-mediated innate immune responses in macrophages. TLR3 induction led to a rapid increase in ROS generation and a physical association between components of the NADPH oxidase (NOX) enzyme complex (NOX2 and p47phox) and TLR3 via a Ca2+-c-Src tyrosine kinase–dependent pathway. TLR3-induced ROS generation, NOX2, and p47phox were required for the phosphorylation and nuclear translocation of STAT1 and STAT2. TLR3-induced activation of STAT1 contributed to the generation of inflammatory mediators, which was significantly attenuated in NOX2- and p47phox-deficient macrophages, suggesting a role for ROS-STAT1 in TLR3-mediated innate immune responses. Collectively, these results provide a novel insight into the crucial role that TLR3-ROS signaling plays in innate immune responses by activating STAT1.

Small Heterodimer Partner-Targeting Therapy Inhibits Systemic Inflammatory Responses through Mitochondrial Uncoupling Protein 2

The orphan nuclear receptor, small heterodimer partner (SHP), appears to play a negative regulatory role in innate immune signaling. Emerging evidence warrants further study on the therapeutic targeting of SHP to suppress excessive and deleterious inflammation. Here we show that fenofibrate, which targets SHP, is required for inhibiting systemic inflammation via mitochondrial uncoupling protein 2 (UCP2). In vivo administration of fenofibrate ameliorated systemic inflammatory responses and increased survival upon experimental sepsis through SHP. An abundance of SHP was observed in mice fed fenofibrate and in cultured macrophages through LKB1-dependent activation of the AMP-activated protein kinase pathway. Fenofibrate significantly blocked endotoxin-triggered inflammatory signaling responses via SHP, but not via peroxisome proliferator-activated receptor (PPAR)-α. In addition to the known mechanism by which SHP modulates innate signaling, we identify a new role of fenofibrate-induced SHP on UCP2 induction, which is required for the suppression of inflammatory responses through modulation of mitochondrial ROS production. These data strongly suggest that the SHP-inducing drug fenofibrate paves the way for novel therapies for systemic inflammation by targeting SHP.

The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages

Background: Ca2+ signaling in FcγR-mediated phagocytosis is unclear. Results: We show that FcγR-mediated phagocytosis requires the production of cyclic ADP-ribose by CD38. Conclusion: CD38 plays a crucial role for Ca2+ signaling in FcγR-mediated phagocytosis. Significance: This study provides new perspectives in immune defense and can help shed light on developing novel methods or drugs for manipulating bacterial infections. Phagocytosis is a crucial event in the immune system that allows cells to engulf and eliminate pathogens. This is mediated through the action of immunoglobulin (IgG)-opsonized microbes acting on Fcγ receptors (FcγR) on macrophages, which results in sustained levels of intracellular Ca2+ through the mobilization of Ca2+ second messengers. It is known that the ADP-ribosyl cyclase is responsible for the rise in Ca2+ levels after FcγR activation. However, it is unclear whether and how CD38 is involved in FcγR-mediated phagocytosis. Here we show that CD38 is recruited to the forming phagosomes during phagocytosis of IgG-opsonized particles and produces cyclic-ADP-ribose, which acts on ER Ca2+ stores, thus allowing an increase in FcγR activation-mediated phagocytosis. Ca2+ data show that pretreatment of J774A.1 macrophages with 8-bromo-cADPR, ryanodine, blebbistatin, and various store-operated Ca2+ inhibitors prevented the long-lasting Ca2+ signal, which significantly reduced the number of ingested opsonized particles. Ex vivo data with macrophages extracted from CD38−/− mice also shows a reduced Ca2+ signaling and phagocytic index. Furthermore, a significantly reduced phagocytic index of Mycobacterium bovis BCG was shown in macrophages from CD38−/− mice in vivo. This study suggests a crucial role of CD38 in FcγR-mediated phagocytosis through its recruitment to the phagosome and mobilization of cADPR-induced intracellular Ca2+ and store-operated extracellular Ca2+ influx.

The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells.

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G2/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G2/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G2 phase, but not at S phase, were ∼2–3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G2 phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G2/M transition.