The nucleotide-oligomerization domain (NOD) is an important molecule involved in host defense against bacterial infection. To study the role of NODs in the host response to Mycobacterium leprae, we measured the mRNA levels of NODs and related genes in infected mouse tissues. The mRNA expression of NOD1, NOD2, caspase-1 and ASC was increased in mouse footpads. Whereas NOD2 expression in macrophages was increased at 2 and 24 h post-infection with M. leprae, there was no expression of NOD1 at these time points. An increase in caspase-1 expression was observed at 2 h and continued at 24 h. However, the expression of ASC was increased only at the early time point. The expression of caspase-1 is regulated by NOD2-dependent pathway in established HEK 293. Our results suggest NOD2, rather than NOD1, may be associated with the host response to M. leprae and that caspase-1 activation is essential for the host response.

BACKGROUND:Endoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells.METHODS:RAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR.RESULTS:Palmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα.CONCLUSIONS:The findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

Adipose-derived stem cell conditioned medium accelerates keratinocyte differentiation via the upregulation of miR-24 Gimoon Seo, Eunhye Oh, Mihee Yun, Ji-Young Lee, Joon Sung Bae, Kwon Joo, Gue Tae Chae and Seong-Beom Lee Institute of Hansen’s Disease, College of Medicine, The Catholic University of Korea, Seoul, Korea; Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea; JK Plastic Surgery Center, Gangnam-gu, Seoul, Korea Correspondence: Seong-Beom Lee, Institute of Hansen’s Disease, Department of Pathology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea, Tel.: +82-2-2258-7313, Fax: +82-2-595-2241, e-mail: sblee@catholic.ac.kr

The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis of psoriasis. Keratinocytes are a major source of IL-1β and express absent in melanoma 2 (AIM2). AIM2 recognizes a double-stranded DNA and initiates the IL-1β-processing of inflammasome. The AIM2 inflammasome is a cytosolic multiprotein complex composed of AIM2, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Epigallocatechin-3-Gallate (EGCG), a major polyphenolic component of green tea, has anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG suppresses AIM2 inflammasome in human epidermal keratinocytes, neonatal (HEKn). Treatment with EGCG, before or after IFN-γ priming, attenuated poly(dA:dT)-induced IL-1β secretion in HEKn cells. Pre-treatment with EGCG reduced the level of IFN-γ-induced priming signal via the down-regulation of pro-IL-1β and pro-capspase-1 in HEKn cells. Furthermore, treatment with EGCG attenuated poly(dA:dT)-induced ASC oligomerization and caspase-1 activation in IFN-γ-primed HEKn cells. These results suggest that EGCG attenuates AIM2-induced IL-1β secretion by suppressing both IFN-γ-mediated priming and poly(dA:dT)-induced ASC oligomerization of inflammasomes in human epidermal keratinocytes.

It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E2 (PGE2) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE2 release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE2 release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function.

In the present study, we examined the effect of palmitate on adipocyte differentiation in mouse bone marrow-derived mesenchymal stem cells (MSCs). Obesity is associated with increased adipocyte differentiation of preadipocytes or MSCs. Free fatty acids (FFAs) have been reported to have an influence on adipogenesis. Thus, the elevated level of FFAs in obese individuals may set off a vicious cycle of events leading to further increased adipogenesis. Palmitate is one of the most abundant FFAs in plasma. The results in this study showed that treatment with palmitate for 3 days, followed by maintenance with insulin for 4 days (palmitate 3d/insulin 4d), induced lipid accumulation and adipocyte protein 2 (aP2) expression in bone marrow-derived MSCs. Our results also showed that treatment with palmitate 3d/insulin 4d induced expressions of adipogenic transcription factors, namely CCAAT enhancer-binding protein (C/EBP) β, C/EBPα, and PPARΓ in bone marrow-derived MSCs. In comparison of palmitate with stearate, the levels of lipid accumulation, aP2, and adipogenic transcription factors in cells treated with palmitate 3d/insulin 4d were generally higher than those of cells treated with same concentration of stearate 3d/insulin 4d. Taken together, our results suggest that palmitate is a potent adipogenic FFA that triggers adipocyte differentiation of bone marrow-derived MSCs.

Mesenchymal stem cells (MSCs) are known to be effective in wound healing, but not much has been reported on quantitative correlations between MSCs injected into the wound site and MSCs that actually participate in wound healing. This study traced MSCs participating in wound healing by using small intestinal submucosa (SIS) as a cell carrier, identified their moving path and calculated the number of MSCs involved in wound healing. First, MSCs were isolated from the nude mouse and 1x106 cells were seeded onto the centre of the SIS. MSC-seeded SIS complexes were injected onto full-thickness skin wounds made on the dorsum of nude mice. Tracing of MSC-seeded SIS complex transplanted to the wound site revealed that 27.6% of the MSCs were migrated to the wound site at the first attempt. Second, repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs present in the wound site remains constant in the range 23x105 from day 1 to day 10. The expression of skin regeneration-related growth factors was confirmed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). MSCs participating in wound healing were found not only to suppress inflammation of the wound but also to increase the skin regeneration-related growth factors that enable the recovery of the skin. An optimal number of about 3x105 MSCs injected into the site was found to adapt themselves to the skin wound-healing process effectively. Copyright (c) 2012 John Wiley & Sons, Ltd.

The objective of the present study was to evaluate non-woven chitosan/poly(lactic-co-glycolic acid) fibrous scaffold (chitosan/PLGA FS) prepared by a co-electrospinning process for tissue engineering applications, as compared to chitosan and PLGA FSs. The morphological, structural, and mechanical properties of the FSs were assessed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy-attenuated total reflection mode (FTIR-ATR), and a universal testing machine (UTM). The biocompatibility of the FSs was also evaluated in vitro in cultures of mouse fibroblasts and in vivo by subcutaneous implantation studies in rats. SEM image of the chitosan/PLGA FS showed morphological similarities to the natural ECM, characterized by high surface-to-volume ratio, high porosity, and variable pore size distributions. The FTIR-ATR spectrum of chitosan/PLGA FS revealed incorporation of the characteristic bands of chitosan and PLGA, indicating the co-existence of two fibrous structures. The poor mechanical properties of chitosan FS was improved by co-electrospinning with PLGA. In vitro L929 cell proliferation assay revealed that the cytocompatibility of chitosan/PLGA FS was increased compared to that of PLGA. In addition, chitosan/PLGA FS showed the improved ability to resolve foreign body reactions compared to PLGA FS due to the presence of chitosan.

The purpose of this study is to understand the effect of chitin on macrophage mediated immunity, which is a significant factor to wound healing and tissue regeneration. In this work, water soluble chitin (WSC) was prepared by re-acetylation of chitosan and was treated with the murine RAW 264.7 macrophage cell lines (ATCC TIB-71). WSC induced classical activation in the RAW 264.7 cells, accompanied by the induction of associated genes. The results suggest that WSC is one of the functional chitin molecules that are responsible for the immune response, especially present in macrophage classical activation. (c) 2012 Elsevier B.V. All rights reserved.

OBJECTIVE:The kinase receptor-interacting protein (RIP) 1, a serine/threonine protein kinase, is a key signaling molecule for necrosis. The possible involvement of RIP1 in palmitate-induced macrophage death and its underlying molecular mechanism was investigated in this study.METHODS:Cell viability was measured by an MTT reduction assay. The type of cell death was determined by staining with annexin V, propidium iodide (PI) and the APOPercentage dye, and by examining cell morphology using transmission electron microscopy. The down-regulation of RIP1 was performed by siRNA transfection. Intracellular reactive oxygen species (ROS) were measured by staining with H(2)DCF-DA.RESULTS:Palmitate largely induced necrosis in RAW 264.7 cells, whereas C2-ceramide induced apoptosis. Palmitate-induced necrosis was inhibited by Necrostatin-1, an inhibitor of RIP1, and by RIP1 siRNA transfection, whereas ordinary cell death was not inhibited by z-VAD-fmk. In addition, the presence of palmitate caused a significant increase in intracellular ROS levels compared to control cells. Pre-treatment with Tempol, a cell permeable ROS scavenger, and MnTBAP, an inhibitor of mitochondrial oxidative stress, protected cells from palmitate-induced cell death. Furthermore, the down-regulation of RIP1 by siRNA transfection significantly decreased palmitate-induced ROS generation compared to control cells.CONCLUSION:The findings reported herein indicate that palmitate induces necrotic cell death via RIP1-dependent ROS generation in RAW 264.7 cells. These findings may provide a new mechanism that explains the link between elevated levels of free fatty acids (FFAs) and macrophage death.

The utility of a biodegradable hyaluronic acid (HyA)-based hydrogel as a tissue scaffold for intravitreal carriage of mesenchymal stem cells (MSCs) into the retina was tested. A rat model of retinal ischemia-reperfusion (IR) injury was used. DiI-labeled MSCs from three passages were loaded onto hydrogel and injected into the vitreous body of experimental and control eyeballs 1 week after IR. The neutral hydrogel was also modified to a basic pH. A control carrier, 0.01 M phosphate-buffered saline (pH 7.2; PBS) was compared. Retinal tissues were prepared 1 week and 2 weeks after MSCs application. MSCs localization and effects were evaluated on immunostained retinal preparations with confocal microscopy. MSCs localization was apparent in the retinas loaded onto hydrogels, adhering tightly to the inner limiting membrane, whereas those in PBS floated in the vitreous body. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were expressed in the end feet of Müller cells in the normal retina. NGF expression was slightly reduced 2 weeks IR, had expanded into the proximal processes 3 weeks IR, but it appeared reversely 1 week and 2 weeks after MSCs application, respectively. BDNF expression was higher in the ischemic and MSCs-treated ischemic retinas than in the normal and MSCs-treated control retinas, respectively. These findings demonstrate that HyA-based hydrogel is an efficient vehicle for intravitreal MSC transplantation into the retina, and that MSCs thus transplanted induce Müller glial cells to produce growth factors concerned with the survival of retinal ganglion cells.

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1β and TNF-α was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-κB activation and cytokine expression. Treatment with M. leprae significantly increased NF-κB activation and expression of TNF-α and IL-1β in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.

A novel mechanism for H2O2-induced autophagic cell death in GSH-depleted RAW 264.7 cells, a murine macrophage cell line, is proposed. Under GSH-depleted conditions, H2O2-induced autophagic cell, characterized by an increased LC3-II/I ratio, a decreased level of p62 and the formation of autophagic vacuoles, was inhibited by bafilomycin A1 and by Atg5 siRNA transfection, whereas the cell death was not inhibited by zVAD-fmk, by PI3K inhibitors or by Beclin 1 siRNA transfection. In addition, H2O2 treatment reduced the activity of mTOR and promoted the ubiquitination and degradation of Rheb, a key upstream activator of mTOR. Furthermore, proteasome inhibition with MG132 restored the expression of Rheb and increased mTOR activity, resulting in an increased viability of H2O2-treated cells. Collectively, these findings demonstrate that H2O2 induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.

BACKGROUND:The active metabolite (1, 25-dihydroxycholecalciferol) of vitamin D (25-hydroxycholecalciferol) leads to activation of macrophages and deficiency of vitamin D seems to be involved in the risk of tuberculosis. The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and may be influenced by polymorphism in the VDR gene. In this study, variation in the VDR gene was investigated in Korean population with tuberculosis.METHODS:We typed three VDR polymorphisms of restriction endonuclease sites for TaqI, BsmI and FokI in 155 patients with tuberculosis and 105 healthy volunteers.RESULTS:The frequencies of FokI genotypes determined from TB patients were 29.13% for FF, 56.31% for Ff, and 14.56% for ff. We observed 1.4-fold increased prevalence of the Ff genotype in TB patients compared with normal healthy groups (p=0.0857). However, there was no significant association between the genotype groups, TB patient and normal control, for FokI polymorphism. There was also no significant association between VDR gene and tuberculosis in another polymorphism (BsmI and TaqI).CONCLUSION:Three polymorphisms (TaqI, BsmI and FokI) in the VDR gene do not appear to be responsible for host susceptibility to human tuberculosis in Korean population.