Eun-Kyeong Jo

Vitamin D3 Induces Autophagy in Human Monocytes/Macrophages via Cathelicidin

Autophagy and vitamin D3-mediated innate immunity have been shown to confer protection against infection with intracellular Mycobacterium tuberculosis. Here, we show that these two antimycobacterial defenses are physiologically linked via a regulatory function of human cathelicidin (hCAP-18/LL-37), a member of the cathelicidin family of antimicrobial proteins. We show that 1,25-dihydroxyvitamin D3 (1,25D3), the active form of vitamin D, induced autophagy in human monocytes via cathelicidin, which activated transcription of the autophagy-related genes Beclin-1 and Atg5. 1,25D3 also induced the colocalization of mycobacterial phagosomes with autophagosomes in human macrophages in a cathelicidin-dependent manner. Furthermore, the antimycobacterial activity in human macrophages mediated by physiological levels of 1,25D3 required autophagy and cathelicidin. These results indicate that human cathelicidin, a protein that has direct antimicrobial activity, also serves as a mediator of vitamin D3-induced autophagy.

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.

A Critical Role of Toll-like Receptor 2 in Nerve Injury-induced Spinal Cord Glial Cell Activation and Pain Hypersensitivity

The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular mechanisms underlying glial cell activation, however, have not been clearly elucidated. In this study, we found that damaged sensory neurons induce the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and inducible nitric-oxide synthase genes in spinal cord glial cells, which is implicated in the development of neuropathic pain. Studies using primary glial cells isolated from toll-like receptor 2 knock-out mice indicate that damaged sensory neurons activate glial cells via toll-like receptor 2. In addition, behavioral studies using toll-like receptor 2 knock-out mice demonstrate that the expression of toll-like receptor 2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the toll-like receptor 2 knock-out mice. Similarly, the nerve injury-induced pro-inflammatory gene expression in the spinal cord is also reduced in the toll-like receptor 2 knock-out mice. These data demonstrate that toll-like receptor 2 contributes to the nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity.

A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis.

We examined the distribution of single nucleotide polymorphisms (SNPs) in nitric oxide synthase 2A, monocyte chemoattractant protein–1 (MCP-1), regulated on activation, normal T cell expressed and secreted, and macrophage inflammatory protein–1 α genes in tuberculosis patients and healthy controls from Mexico. The odds of developing tuberculosis were 2.3- and 5.4-fold higher in carriers of MCP-1 genotypes AG and GG than in homozygous AA. Cases of homozygous GG had the highest plasma levels of MCP-1 and the lowest plasma levels of IL-12p40, and these values were negatively correlated. Furthermore, stimulation of monocytes from healthy carriers of the genotype GG with Mycobacterium tuberculosis antigens yielded higher MCP-1 and lower IL-12p40 concentrations than parallel experiments with monocytes from homozygous AA. Addition of anti–MCP-1 increased IL-12p40 levels in cultures of M. tuberculosis–stimulated monocytes from homozygous GG, and addition of exogenous MCP-1 reduced IL-12p40 production by M. tuberculosis–stimulated monocytes from homozygous AA. Furthermore, we could replicate our results in Korean subjects, in whom the odds of developing tuberculosis were 2.8- and 6.9-fold higher in carriers of MCP-1 genotypes AG and GG than in homozygous AA. Our findings suggest that persons bearing the MCP-1 genotype GG produce high concentrations of MCP-1, which inhibits production of IL-12p40 in response to M. tuberculosis and increases the likelihood that M. tuberculosis infection will progress to active pulmonary tuberculosis.

Molecular mechanisms regulating NLRP3 inflammasome activation

Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inflammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome.

Mycobacterial interaction with innate receptors: TLRs, C-type lectins, and NLRs.

Purpose of review The recent discovery of novel classes of receptors, including toll-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors is challenging the crucial role of the innate immune system in the recognition of Mycobacterium tuberculosis. The present review is to focus on the roles and mechanisms of specific pattern-recognition receptor–microbial interaction for the host defense against mycobacterial infections. Recent findings Toll-like receptors, key players in innate immunity, are now known to be important for the initiation and coordination of host responses to Mycobacterium tuberculosis. The interaction of Mycobacterium tuberculosis with toll-like receptors triggers intracellular signaling cascades that culminate in a proinflammatory response, but can also trigger signals that dampen the innate immune response. Other membrane-bound pattern-recognition receptors, including the mannose receptor, DC-SIGN, and Dectin-1, contribute to the propagation of Mycobacterium tuberculosis inflammatory signals, and Nod-like receptors (cytosolic pattern-recognition receptors) also act in modulating host recognition of Mycobacterium tuberculosis. Interactions between toll-like receptors and other pattern-recognition receptors are also evident in responses to Mycobacterium tuberculosis, as are possible mechanisms for coordination of innate and adaptive immunity. Summary The complexity of Mycobacterium tuberculosis–pattern-recognition receptor interactions and their effects on host cell responses suggest key roles for innate immunity in controlling Mycobacterium tuberculosis, and the possibility of developing novel therapeutics for tuberculosis that target Mycobacterium tuberculosis-regulated innate immunity pathways.

NADPH oxidase 2-derived reactive oxygen species in spinal cord microglia contribute to peripheral nerve injury-induced neuropathic pain

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain.

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.

NADPH oxidase 2 interaction with TLR2 is required for efficient innate immune responses to mycobacteria via cathelicidin expression.

Gp91phox/NADPH oxidase (NOX) 2 is the main catalytic component of NOX, which mediates the phagocytic killing of ingested pathogens via the production of reactive oxygen species (ROS). However, Mycobacterium tuberculosis (Mtb) is relatively resistant to the microbicidal effects of ROS. Thus, the exact roles of NOX2 in the innate immune control against Mtb infection are not fully resolved. In this study, we show that NOX2 is essential for TLR2-dependent inflammatory responses and 1,25-dihydroxyvitamin D3 (1,25D3)-mediated antimicrobial activity against Mtb via cathelicidin expression. NOX2-null macrophages prominently abrogated Mtb-induced ROS production and inflammatory signaling activation in a TLR2-dependent manner. Mtb triggered a physical association between NOX2 and TLR2. In addition, the knockdown of NOX2 inhibited 1,25D3-triggered antimicrobial activity against viable Mtb through the modulation of cathelicidin expression in human macrophages. Treatment of NOX2 knocked down cells with cathelicidin restored the 1,25D3-induced antimicrobial effect, suggesting that the NOX2-dependent induction of cathelicidin in macrophages is part of a defensive strategy against Mtb. Furthermore, cathelicidin expression was required for the Mtb-induced release of ROS and the production of proinflammatory cytokines/chemokines, indicating a positive circuit of inflammation in response to Mtb. Our data collectively demonstrate a novel regulatory mechanism for TLR2-dependent innate responses to Mtb involving crosstalk between NOX2 and TLR2 and the expression of cathelicidin.

Role of microglial IKKβ in kainic acid-induced hippocampal neuronal cell death

Microglial cells are activated during excitotoxin-induced neurodegeneration. However, the in vivo role of microglia activation in neurodegeneration has not yet been fully elucidated. To this end, we used Ikkbeta conditional knockout mice (LysM-Cre/Ikkbeta(F/F)) in which the Ikkbeta gene is specifically deleted in cells of myeloid lineage, including microglia, in the CNS. This deletion reduced IkappaB kinase (IKK) activity in cultured primary microglia by up to 40% compared with wild-type (Ikkbeta(F/F)), and lipopolysaccharide-induced proinflammatory gene expression was also compromised. Kainic acid (KA)-induced hippocampal neuronal cell death was reduced by 30% in LysM-Cre/Ikkbeta(F/F) mice compared with wild-type mice. Reduced neuronal cell death was accompanied by decreased KA-induced glial cell activation and subsequent expression of proinflammatory genes such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Similarly, neurons in organotypic hippocampal slice cultures (OHSCs) from LysM-Cre/Ikkbeta(F/F) mouse brain were less susceptible to KA-induced excitotoxicity compared with wild-type OHSCs, due in part to decreased TNF-alpha and IL-1beta expression. Based on these data, we concluded that IKK/nuclear factor-kappaB dependent microglia activation contributes to KA-induced hippocampal neuronal cell death in vivo through induction of inflammatory mediators.