Investigation for immunomodulatory and anti-inflammatory mechanisms of mesenchymal stem cells on T cells and dendritic cells in CIA mice

의과대학/박사Background Mesenchymal stem cells (MSCs) have profound immunomodulatory properties. Using their properties, MSCs-based therapies have been applied in several inflammatory diseases. The immune modulation of MSCs is related to inhibition of immune cell proliferation and activation. Dendritic cells (DCs) play pivotal roles in initiating immune response as DCs are important antigen-presenting cells. DCs are able to mature into inflammatory DCs, sustaining a continuous activation of adaptive immune system. T cells critical effector immune cells in affecting and regulating immune response and can differentiate into one of several subtypes, including TH1, TH2, TH17 or regulatory T cells (Tregs). As the effects of MSCs on DCs and T cells have not been fully understood, the immunomodulatory mechanisms of MSCs on DCs and T cells were investigated. Methods Bone marrow-derived CD11c+ mononuclear cells of DBA/1 mice were differentiated cultured with GM-CSF and IL-4, to immature DCs (iDCs) for 7 days. CD4+ T cells were isolated from spleen of DBA/1 mice by negative selection using CD4+ T cell isolation kit. The iDCs were maturated using lipopolysaccharides (LPS). We co-cultured human bone marrow-derived MSCs (BM-MSCs) and mouse DCs and CD4+ T cells directly with different ratios. The effect of BM-MSCs on DC maturation and T cell differentiation was assessed by maturation markers by flow cytometry, and supernatants for induced production of cytokines. Gene expressions were analyzed by quantitative RT-PCR and microarray. In addition, immunohistochemistry was performed in inflammatory tissues in collagen-induced arthritis (CIA) mice. Results Human BM-MSCs significantly inhibited the DCs maturation by decreasing CD86 and major histocompatibility complex (MHC)Ⅱ expressions in both 1:1 and 1:10 co-culture ratio. And, human BM-MSCs decreased the levels of interleukin (IL)-12p70, tumor necrosis factor (TNF)-α, and IL-6 effectively, and increased the levels of IL-10 and transforming growth factor (TGF)-β. Also, the relative gene expressions of indoleamine 2,3-dioxygenase (IDO) and heme oxygenase (HO)-1 were increased in 1:1 co-culture ratio at 6 hr. The expression of CD4+CD25+FoxP3+ Tregs were highly induced in co-culture condition with hBM-MSCs. Human BM-MSCs significantly induced the Tregs by increasing programmed death-1 (PD-1) and neuropilin-1 (Nrp-1) expressions in different co-culture ratio. And, human BM-MSCs increased the levels of IL-10 and TGF-β1. Moreover, immunohistochemical analysis of inflamed tissues in BM-MSC treated CIA mice showed significant immunopositive staining for PD-1 on CD4+ T cells. Conclusion The results demonstrated that human BM-MSCs inhibited the maturation of DCs and induced anti-inflammatory signals of DCs in in vitro. Moreover, these data showed that human BM-MSCs highly induced the CD4+CD25+Foxp3+ Tregs in both in vitro and in vivo.ope

Wound Healing Potential of Low Temperature Plasma in Human Primary Epidermal Keratinocytes

Background: Low temperature plasma (LTP) was recently shown to be potentially useful for biomedical applications such as bleeding cessation, cancer treatment, and wound healing, among others. Keratinocytes are a major cell type that migrates directionally into the wound bed, and their proliferation leads to complete wound closure during the cutaneous repair/regeneration process. However, the beneficial effects of LTP on human keratinocytes have not been well studied. Therefore, we investigated migration, growth factor production, and cytokine secretion in primary human keratinocytes after LTP treatment. Methods: Primary cultured keratinocytes were obtained from human skin biopsies. Cell viability was measured with the EZ-Cytox cell viability assay, cell migration was evaluated by an in vitro wound healing assay, gene expression was analyzed by quantitative real-time polymerase chain reaction, and protein expression was measured by enzyme-linked immunosorbent assays and western blotting after LTP treatment. Results: Cell migration, the secretion of several cytokines, and gene and protein levels of angiogenic growth factors increased in LTP-treated human keratinocytes without associated cell toxicity. LTP treatment also significantly induced the expression of hypoxia inducible factor-1α (HIF-1α), an upstream regulator of angiogenesis. Further, the inhibition of HIF-1α expression blocked the production of angiogenic growth factors induced by LTP in human keratinocytes. Conclusion: Our results suggest that LTP treatment is an effective approach to modulate wound healing-related molecules in epidermal keratinocytes and might promote angiogenesis, leading to improved wound healing.ope

Methotrexate-loaded multifunctional nanoparticles with near-infrared irradiation for the treatment of rheumatoid arthritis

Backgrounds: Despite the advances of rheumatoid arthritis (RA) therapeutics, several patients do not receive adequate treatment due to the toxicity and/or insufficient response of drugs. The aim of this study is to design photothermally controlled drug release from multifunctional nanoparticles (MNPs) at a near-infrared (NIR) irradiated site to improve therapeutic efficacy for RA and reduce side effects. Methods: Au film was deposited onto methotrexate (MTX)-loaded poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles, resulting in MTX-loaded MNPs. The synergistic effects of MTX-loaded MNPs with NIR irradiation were investigated using RA fibroblast-like synoviocytes (FLSs) and collagen-induced arthritis (CIA) mice. Results: Upon NIR irradiation, NIR resonance of the Au half-shell generated heat locally, accelerating MTX release from PLGA nanoparticles. In vivo NIR images of MTX-loaded MNPs indicated effective delivery of the MNPs to the inflamed joints. Moreover, in collagen-induced arthritis mice, MTX-loaded MNPs containing 1/1400 of MTX solution (repeated-dose administration) had therapeutic effects comparable to conventional treatment with MTX solution. In vitro experiments showed higher therapeutic efficacy of MTX-loaded MNPs with NIR irradiation than that of chemotherapy alone. Conclusions: A combination therapy of MTX-loaded MNP and NIR irradiation showed durable and good treatment efficacy for the suppression of arthritis in a single administration of small dose of MTX. Our results demonstrate that the treatment modality using drug-loaded MNP with NIR irradiation may be a promising therapeutic strategy for the treatment of RA and allow in vivo NIR optical imaging.ope

Transcriptional Interactomic Inhibition of RORα Suppresses Th17-Related Inflammation

Purpose: Th17 cells and their cytokines are implicated in the pathogenesis of various autoimmune diseases. Retinoic acid-related orphan receptor alpha (RORα) is a transcription factor for the differentiation and the inflammatory functions of Th17 cells. In this study, we generated the nucleus-transducible form of transcription modulation domain of RORα (nt-RORα-TMD) to investigate the functional roles of RORα in vitro and in vivo under normal physiological condition without genetic alteration. Methods: The functions of nt-RORα-TMD were analyzed in vitro through flow cytometry, luciferase assay, ELISA, and transcriptome sequencing. Finally, the in vivo therapeutic effects of nt-RORα-TMD were verified in dextran sulfate sodium (DSS)-induced colitis mice. Results: nt-RORα-TMD was effectively delivered into the cell nucleus in a dose- and time-dependent manner without any cellular toxicity. nt-RORα-TMD competitively inhibited the RORα-mediated transcription but not RORγt-mediated transcription. Secretion of IL-17A from the splenocytes was suppressed by nt-RORα-TMD without affecting the secretion of Th1- or Th2-type cytokine and T cell activation events such as induction of CD69 and CD25. The differentiation potential of naïve T cells into Th17 cells, not into Th1, Th2, or Treg cells, was significantly blocked by nt-RORα-TMD. Consistently, mRNA sequencing analysis showed that nt-RORα-TMD treatment down-regulated the expression of the genes related to the differentiation and functions of Th17 cells. Treatment of DSS-induced colitis mice with nt-RORα-TMD improved the overall symptoms of colitis, such as body weight change, colon length, infiltration of inflammatory cells, and the level of inflammatory cytokines in the serum. In the mesenteric lymph node (MLN) of the nt-RORα-TMD-treated mice, the population of CD4+IL-17A+ Th17 cells was reduced, and the population of CD4+Foxp3+ Treg cells increased. Conclusion: nt-RORα-TMD has a potential to be developed as a novel therapeutic reagent for treating various inflammatory diseases in which Th17 cells are the leading pathological player.ope

Punicalagin Ameliorates Lupus Nephritis via Inhibition of PAR2

Lupus nephritis (LN) is the most frequent phenotype in patients with systemic lupus erythematosus (SLE) and has a high rate of progression to end-stage renal disease, in spite of intensive treatment and maintenance therapies. Recent evidence suggests that protease-activated receptor-2 (PAR2) is a therapeutic target for glomerulonephritis. In this study, we performed a cell-based high-throughput screening and identified a novel potent PAR2 antagonist, punicalagin (PCG, a major polyphenol enriched in pomegranate), and evaluated the effects of PCG on LN. The effect of PCG on PAR2 inhibition was observed in the human podocyte cell line and its effect on LN was evaluated in NZB/W F1 mice. In the human podocyte cell line, PCG potently inhibited PAR2 (IC50 = 1.5 ± 0.03 µM) and significantly reduced the PAR2-mediated activation of ERK1/2 and NF-κB signaling pathway. In addition, PCG significantly decreased PAR2-induced increases in ICAM-1 and VCAM-1 as well as in IL-8, IFN-γ, and TNF-α expression. Notably, the intraperitoneal administration of PCG significantly alleviated kidney injury and splenomegaly and reduced proteinuria and renal ICAM-1 and VCAM-1 expression in NZB/W F1 mice. Our results suggest that PCG has beneficial effects on LN via inhibition of PAR2, and PCG is a potential therapeutic agent for LN.ope

O-linked N-acetylglucosamine glycosylation of p65 aggravated the inflammation in both fibroblast-like synoviocytes stimulated by tumor necrosis factor-α and mice with collagen induced arthritis

INTRODUCTION: We investigated the inflammatory potential of O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation) of p65 in rheumatoid arthritis (RA). METHODS: Fibroblast-like synoviocytes (FLS) and MH7A cells were treated with synthetic ThiaMet-G (200 μM), an O-GlcNAcase (OGA) inhibitor, followed by tumor necrosis factor (TNF)-α (10 μg/mL). Proliferation of synovial cells was measured by MTT assay, and the levels of mRNAs encoding pro-inflammatory molecules were quantitated by RT-PCR. The nuclear localization of O-GlcNAcylated of p65 and its DNA binding affinity and transcriptional activity were assessed. The severity assessment of arthritis and a histopathological examination were done in mice with collagen induced arthritis (CIA). ThiaMet-G (20 mg/kg) intraperitoneal injection was done every other day for 26 days. Fluorescence-activated cell sorting (FACS) analysis of T cells was performed. RESULTS: Hyper-O-GlcNAcylation increased the proliferation and mRNA expression of pro-inflammatory genes in synoviocytes stimulated by TNF-α. Moreover, O-GlcNAcylation of p65 enhanced its proportion of nuclear localization, DNA binding affinity and transcriptional activity. In CIA mice, ThiaMet-G significantly aggravated the severity of arthritis clinically and histologically, and it also increased CD4 + IFN-γ + T cells and CD4 + IL-17+ T cells. CONCLUSIONS: O-GlcNAcylation of p65 increased the effects of TNF-α-mediated inflammation both in vitro (in synovial cells) and in vivo (in mice with CIA).ope

Agmatine Attenuates Nitric Oxide Synthesis and Protects ER-structure from Global Cerebral Ischemia in Rats

In ischemic strokes, apoptosis is caused by excitotoxicity, ionic imbalance, oxidative/nitrosative stress, and apoptotic-like pathways. Nitric oxide (NO), a free radical, is elevated after ischemic insult. NO, which is generated primarily by neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS), promotes neuronal damage following ischemia. Evidence obtained in recent years has demonstrated that endoplasmic reticulum (ER)-mediated cell death plays an important role in cerebral ischemia. Agmatine is an endogenous substance synthesized from L-arginine by arginine decarboxylase (ADC) and is present in mammalian brain. We had previously reported that agmatine contributes to neuroprotection against ischemic injury. In continuation of our earlier work, we intended to investigate whether agmatine protects brain from transient global ischemia, and also tried to determine the neuroprotective mechanism of agmatine. Twenty minutes of transient global ischemia was induced by 4 vessel occlusion (4-VO). Agmatine (100 mg/kg, IP) was administered simultaneously with reperfusion. Samplings of brain were done at 6, 24, 48, and 72 h after reperfusion to determine the effect of agmatine on ischemic injured hippocampus. ER-damage was also investigated using electron microscope. Results showed that agmatine treatment prevented delayed neuronal cell death in hippocampal CA1 neurons after global cerebral ischemia. It also blocked NOS expression in the rat brain. Agmatine induced the increased expression of glucose-regulated protein 78 (Grp78). These results suggest that agmatine inhibits the production of NO by decreasing the expression of nNOS and iNOS on global forebrain ischemia and the neuroprotective effect of agmatine were concerned with the ER stress-mediated conditionope

전뇌 허혈 동물 모델에서 아그마틴의 신경세포 보호 효과

Dept. of Medical Science/석사[한글] 허혈성 뇌질환에서 세포사멸은 과독성, 이온 불균형, 산화/질산화 스트레스와 세포사멸 관련 기전에 의해 일어난다. 허혈성 질환이 발생한 후 유도되는 자유이온의 하나가 일산화질소이다. 일산화생성효소에 의해 생산된 일산화질소는 허혈에 따른 신경세포 저해를 일으킨다. 최근 몇 년 동안 소포체 관련 세포사가 전뇌허혈에 중요한 역할을 할 것이라고 보고되어 왔다.아그마틴은 알기닌 탈탄산효소에 의해 L-arginine으로부터 합성되는 내재성 물질로, 포유류의 뇌 속에도 존재한다. 이전 연구들은 아그마틴의 신경세포보호작용을 세포성 모델과 허혈성 동물 모델로 제시해 오고 있다. 또한, 우리는 중간대뇌동맥결찰 마우스 모델을 제작, 그것을 통해 아그마틴이 허혈성 저해로부터 신경세포를 보호하고 있음을 이미 보고하였다.4-VO 전뇌 허혈성 동물 모델은 1979년 Pulsinelli et al.의 모델을 참조하여 제작하였다. 동물은 이소푸르란을 이용하여 마취하였고, 아그마틴은 100 mg/kg 양을 재관류 시작과 동시에 복강주사하였다. 일산화질소생성효소의 발현에 아그마틴이 미치는 영향을 알아내기 위해 재관류 시작 후 6, 24, 48, 72 시간이 경과하면 뇌를 적출하였다. 또한 소포체 관련 칼슘 분비와 아그마틴의 조절 관계를 확인하기 위해 조직면역화학염색법을 실시하였고, 일산화질소생성효소의 발현 정도를 알기 위해 immunoblotting을 수행하였다. 아그마틴 처리는 전뇌허혈성 저해로부터 해마 CA1 구역의 신경세포들을 보호하였고, 일산화질소생성효소의 발현도 저해시켰다. 한편, 아그마틴에 의한 일산화질소생성효소의 발현 조절은 뇌 지역에 따라 차이를 보였다. 아그마틴 처리는 NF-kB, Hsp70와 Grp78의 발현을 유도하는 반면, MMP-9과 MMP-2의 발현은 감소시켰다. 또한, 전자현미경적 분석을 통해 아그마틴 처리군은 실험 대조군보다 소포체와 골지체를 비롯한 세포소기관의 형태가 정상군에 보다 가까움을 알 수 있었다.이상의 결과들은 아그마틴이 전뇌허혈성 동물 모델에서 3가지 일산화질소생성효소의 발현을 다르게 조절 및 감소시킴으로써 일산화질소의 생성을 억제함을 보여준다. 아그마틴은 전뇌허혈성 저해로부터 신경세포를 보호하기 위해 여러 경로를 통해 MMPs, Hsp70, NF-kB, Grp78 등 anti-apoptotic 단백질과 pro-apoptotic 단백질들의 발현을 조절한다. 또한, 아그마틴은 소포체성 세포사멸과 연관된 허혈성 저해로부터 신경세포를 보호한다. [영문]In ischemic strokes, apoptosis is caused by excitotoxicity, ionic imbalance, oxidative/nitrosative stress and apoptotic-like pathways. One of free radicals which are elevated after the ischemic insult is nitric oxide (NO). NO generated primarily by neuronal and inducible NO synthases (NOS) promotes neuronal damage following ischemia. Evidence obtained in recent years has demonstrated that endoplasmic reticulum（ER)-mediated cell death plays an important role in cerebral ischemia.Agmatine is an endogenous substance synthesized from L-arginine by arginine decarboxylase (ADC) and is present in the mammalian brains. Previous studies have shown that agmatine may be neuroprotective in in vitro and in vivo ischemia models. Also, we have previously studied and reported the effects of agmatine on the middle cerebral artery occlusion (MCAO) model in mice and shown that agmatine contributes to neuroprotection from ischemic injury.Transient global ischemia for 20 minutes was induced by 4 vessel occlusion (4-VO) using the method described originally by Pulsinelli et al. (1979). Animals were anesthetized with isoflurane and agmatine was administered by IP injection in dose of 100 mg/kg at the same time of reperfusion. The rats were sampled at 6, 24, 48, and 72h after reperfusion to determine the effect of agmatine on NOS expression. We also checked the relation of agmatine and calcium influx with ER by using immunohistochemistry. We detected the expression of NOS by using immunoblotting. Agmatine treatment prevented the delayed neuronal cell death in the hippocampal CA1 neurons in global cerebral ischemia. It also blocked NOS expression and calcium influx in the rat brain. On the other hand, the expression of NOS was different according to the area of the rat brain. Agmatine induced the level of NF-kB, Hsp70 and Grp 78, and so on. Also, agmatine reduced the level of MMP-9 and MMP-2 slightly. Agmatine has shown different neuroprotective effects according to various brain regions.These results show that agmatine inhibits the production of nitric oxide by decreasing the expression of three isoforms of NOS differently on global forebrain ischemia. It regulates anti-apoptotic and pro-apoptotic proteins through different routes to protect neurons from transient global ischemia. Moreover, neuroprotective effects of agmatine were concerned with ER stress-mediated condition.ope

Drug-loaded gold/iron/gold plasmonic nanoparticles for magnetic targeted chemo-photothermal treatment of rheumatoid arthritis

We have developed methotrexate (MTX)-loaded poly(lactic-co-glycolic acid, PLGA) gold (Au)/iron (Fe)/gold (Au) half-shell nanoparticles conjugated with arginine-glycine-aspartic acid (RGD), which can be applied for magnetic targeted chemo-photothermal treatment, and in vivo multimodal imaging of rheumatoid arthritis (RA). Upon near-infrared (NIR) irradiation, local heat is generated at the inflammation region due to the NIR resonance of Au half-shells and MTX release from PLGA nanoparticles is accelerated. The Fe half-shell layer embedded between the Au half-shell layers enables in vivo T2-magnetic resonance (MR) imaging in addition to NIR absorbance imaging. Furthermore, the delivery of the nanoparticles to the inflammation region in collagen-induced arthritic (CIA) mice, and their retention can be enhanced under external magnetic field. When combined with consecutive NIR irradiation and external magnetic field application, these nanoparticles provide enhanced therapeutic effects with an MTX dosages of only 0.05% dosage compared to free MTX therapy for the treatment of RA.restrictio

Treatment of Collagen-Induced Arthritis Using Immune Modulatory Properties of Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have immune modulatory properties. We investigated the potential therapeutic effects of human bone marrow (BM)-, adipose tissue (AD)-, and cord blood (CB)-derived MSCs in an experimental animal model of rheumatoid arthritis (RA) and explored the mechanism underlying immune modulation by MSCs. We evaluated the therapeutic effect of clinically available human BM-, AD-, and CB-derived MSCs in DBA/1 mice with collagen-induced arthritis (CIA). CIA mice were injected intraperitoneally with three types of MSCs. Treatment control animals were injected with 35 mg/kg methotrexate (MTX) twice weekly. Clinical activity in CIA mice, degree of inflammation, cytokine expression in the joint, serum cytokine levels, and regulatory T cells (Tregs) were evaluated. Mice treated with human BM-, AD-, and CB-MSCs showed significant improvement in clinical joint score, comparable to MTX-treated mice. Histologic examination showed greatly reduced joint inflammation and damage in MSC-treated mice compared with untreated mice. Microcomputed tomography also showed little joint damage in the MSC-treated group. MSCs significantly decreased serum interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and interferon-γ and increased IL-10 and transforming growth factor-β levels. Tregs were increased in mice treated with MSCs compared to untreated or MTX-treated mice. Human BM-, AD-, and CB-MSCs significantly suppressed joint inflammation in CIA mice. The cells decreased proinflammatory cytokines and upregulated anti-inflammatory cytokines and induced Tregs. Therefore, our study suggests that the use of human BM-, AD-, and CB-MSCs could be an effective therapeutic approach for RA.restrictio