Jae-Min Yuk

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.

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.

Autophagy Negatively Regulates Keratinocyte Inflammatory Responses via Scaffolding Protein p62/SQSTM1

The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.

Mycobacterium abscessus activates the macrophage innate immune response via a physical and functional interaction between TLR2 and dectin-1.

Mycobacterium abscessus (Mab) is an emerging and rapidly growing non‐tuberculous mycobacterium (NTM). Compared with M. tuberculosis, which is responsible for tuberculosis, much less is known about NTM‐induced innate immune mechanisms. Here we investigated the involvement of pattern‐recognition receptors and associated signalling in Mab‐mediated innate immune responses. Mab activated the extracellular signal‐regulated kinase (ERK)1/2 and p38 mitogen‐activated protein kinases (MAPKs), and induced the secretion of tumour necrosis factor‐α, interleukin (IL)‐6 and IL‐12p40 in murine macrophages via Toll‐like receptor (TLR) 2. Notably, the activation of ERK1/2, but not p38, was crucial for Mab‐induced pro‐inflammatory cytokine production. The ITAM‐like motif of dectin‐1 critically contributed to Mab internalization and cytokine secretion by macrophages. In addition, dectin‐1, in cooperation with TLR2, was required for the efficient phagocytosis of Mab, ERK1/2 activation and pro‐inflammatory cytokine secretion. Co‐immunoprecipitation and confocal analysis showed the physical interaction and colocalization of dectin‐1 with TLR2 following Mab stimulation. Moreover, dectin‐1‐induced Syk activation was essential for the production of inflammatory cytokines and the release of reactive oxygen species by Mab‐infected macrophages. Collectively, these data demonstrate that Mab actively internalizes into and robustly activates innate immune responses in macrophages through a physical and functional interaction between TLR2 and dectin‐1.

Roles of Autophagy in Elimination of Intracellular Bacterial Pathogens

As a fundamental intracellular catabolic process, autophagy is important and required for the elimination of protein aggregates and damaged cytosolic organelles during a variety of stress conditions. Autophagy is now being recognized as an essential component of innate immunity; i.e., the recognition, selective targeting, and elimination of microbes. Because of its crucial roles in the innate immune system, therapeutic targeting of bacteria by means of autophagy activation may prove a useful strategy to combat intracellular infections. However, important questions remain, including which molecules are critical in bacterial targeting by autophagy, and which mechanisms are involved in autophagic clearance of intracellular microbes. In this review, we discuss the roles of antibacterial autophagy in intracellular bacterial infections (Mycobacteria, Salmonella, Shigella, Listeria, and Legionella) and present recent evidence in support of molecular mechanisms driving autophagy to target bacteria and eliminate invading pathogens.

Dectin-1 is Inducible and Plays an Essential Role for Mycobacteria-Induced Innate Immune Responses in Airway Epithelial Cells

IntroductionAirway epithelial cells are the first cells to be challenged upon contact with mycobacteria. In response, they express pattern-recognition receptors that play fundamental roles as sentinels and mediators of pulmonary innate immunity. The c-type lectin Dectin-1 is expressed predominantly on the surface of myeloid lineage cells. In this study, we examined the induction, regulation, and functions of Dectin-1 in pulmonary epithelial cells.ResultsMycobacterium tuberculosis (Mtb) actively induced the expression of Dectin-1 mRNA and protein in A549 cells in a toll-like receptor (TLR) 2-dependent manner. In addition, Mtb-mediated generation of reactive oxygen species and Dectin-1 induction were mutually dependent. Moreover, Mtb actively induced the phosphorylation of Src family kinases at Tyr416 via TLR2. Selective inhibition of Src markedly attenuated the induction of Mtb-dependent Dectin-1 expression, indicating that Src kinases are crucial regulators of Dectin-1-dependent signaling. Mtb internalization was partially blocked by silencing Dectin-1 expression, inhibiting Src kinases, or pretreating with antioxidants. Finally, Dectin-1 was required for pro-inflammatory cytokine release and antimicrobial effects on intracellular mycobacterial growth in A549 cells.ConclusionCollectively, our findings demonstrate the novel induction of Dectin-1 in type II airway epithelial cells and its critical role in the innate immune response against Mtb in non-phagocytic cells.

Nanoparticles up-regulate tumor necrosis factor-α and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation

Evaluating the toxicity of nanoparticles is an integral aspect of basic and applied sciences, because imaging applications using traditional organic fluorophores are limited by properties such as photobleaching, spectral overlaps, and operational difficulties. This study investigated the toxicity of nanoparticles and their biological mechanisms. We found that nanoparticles, quantum dots (QDs), considerably activated the production of tumor necrosis factor (TNF)-alpha and CXC-chemokine ligand (CXCL) 8 through reactive oxygen species (ROS)- and mitogen-activated protein kinases (MAPKs)-dependent mechanisms in human primary monocytes. Nanoparticles elicited a robust activation of intracellular ROS, phosphorylation of p47phox, and nicotinamide adenine dinucleotide phosphate oxidase activities. Blockade of ROS generation with antioxidants significantly abrogated the QD-mediated TNF-alpha and CXCL8 expression in monocytes. The induced ROS generation subsequently led to the activation of MAPKs, which were crucial for mRNA and protein expression of TNF-alpha and CXCL8. Furthermore, confocal and electron microscopy analyses showed that internalized QDs were trapped in cytoplasmic vesicles and compartmentalized inside lysosomes. Finally, several repeated intravenous injections of QDs caused an increase in neutrophil infiltration in the lung tissues in vivo. These results provide novel insights into the QD-mediated chemokine induction and inflammatory toxic responses in vitro and in vivo.

The role of nitric oxide in mycobacterial infections.

Although tuberculosis poses a significant health threat to the global population, it is a challenge to develop new and effective therapeutic strategies. Nitric oxide (NO) and inducible NO synthase (iNOS) are important in innate immune responses to various intracellular bacterial infections, including mycobacterial infections. It is generally recognized that reactive nitrogen intermediates play an effective role in host defense mechanisms against tuberculosis. In a murine model of tuberculosis, NO plays a crucial role in antimycobacterial activity; however, it is controversial whether NO is critically involved in host defense against Mycobacterium tuberculosis in humans. Here, we review the roles of NO in host defense against murine and human tuberculosis. We also discuss the specific roles of NO in the central nervous system and lung epithelial cells during mycobacterial infection. A greater understanding of these defense mechanisms in human tuberculosis will aid in the development of new strategies for the treatment of disease.

Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern‐recognition receptors expressed in keratinocytes. We found that keratinocyte Toll‐like receptors (TLRs) 2 and 4 and Dectin‐1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL‐37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin‐1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL‐37. In addition, TLR2, Dectin‐1 and, to an extent, TLR4 are essential for the MU‐mediated expression of CXCL8, CCL2 and LL‐37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin‐1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL‐37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin‐1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

MicroRNA-125a Inhibits Autophagy Activation and Antimicrobial Responses during Mycobacterial Infection

MicroRNAs (miRNAs) are small noncoding nucleotides that play critical roles in the regulation of diverse biological functions, including the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against Mycobacterium tuberculosis. Although the pathways associated with autophagy must be tightly regulated at a posttranscriptional level, the contribution of miRNAs and whether they specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that M. tuberculosis infection of macrophages leads to increased expression of miRNA-125a-3p (miR-125a), which targets UV radiation resistance-associated gene (UVRAG), to inhibit autophagy activation and antimicrobial responses to M. tuberculosis. Forced expression of miR-125a significantly blocked M. tuberculosis–induced activation of autophagy and phagosomal maturation in macrophages, and inhibitors of miR-125a counteracted these effects. Both TLR2 and MyD88 were required for biogenesis of miR-125a during M. tuberculosis infection. Notably, activation of the AMP-activated protein kinase significantly inhibited the expression of miR-125a in M. tuberculosis–infected macrophages. Moreover, either overexpression of miR-125a or silencing of UVRAG significantly attenuated the antimicrobial effects of macrophages against M. tuberculosis. Taken together, these data indicate that miR-125a regulates the innate host defense by inhibiting the activation of autophagy and antimicrobial effects against M. tuberculosis through targeting UVRAG.