Mitochondrial dsRNA and TLR3 in hepatic inflammation

Won-Il Jeong 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Previously, mitochondrial double-stranded RNA (mtdsRNA) and its immune responses have been reported. However, the effects of generated mtdsRNA on alcoholic liver disease (ALD) remain unclear. Therefore, we report that mtdsRNA originated from hepatocytes stimulates toll-like receptor 3 (TLR3) in Kupffer cells through the exosome, subsequently enhancing IL-17A production in ALD. Following binge ethanol drinking, IL-17A production primarily increased in γδ T cells of wild type (WT) mice, whereas the production of IL-17A was not observed in Kupffer cell-depleted WT and TLR3 KO mice. In addition, the expression of PNPase, an mtdsRNA restricting enzyme, was significantly decreased in ethanol-exposed livers and hepatocytes of WT mice. Immunostaining revealed that mtdsRNA colocalized with the mitochondria in ethanol-treated hepatocytes from WT mice and healthy humans. Bioanalyzer analysis revealed that small-sized RNAs were enriched in ethanol-treated exosomes (EtOH-Exo) rather than EtOH-treated microvesicles in hepatocytes of WT mice and humans. qPCR and RNA sequencing analyses indicated that mRNA expression of mitochondrial genes encoded by heavy and light strands was robustly increased in EtOH-Exo from mice and humans. After direct treatment with EtOH-Exo, IL-1β expression was significantly increased in WT Kupffer cells but not in TLR3 KO Kupffer cells, augmenting IL-17A production of γδ T cells in mice and humans. In conclusion, ethanol-mediated generation of mtdsRNA contributes to TLR3 activation in Kupffer cells through exosomal delivery. Consequently, increased IL-1β expression in Kupffer cells triggers IL-17A production in γδ T cells at the early stage that may accelerate IL-17A expression in CD4+ T cells in the later stage of ALD. Therefore, mtdsRNA and TLR3 may function as novel therapeutic targets in ALD.

Association of DOCK8, IL17RA, and KLK12 Polymorphisms with Atopic Dermatitis in Koreans

( Won Il Heo ),( Kui Young Park ),( Mi-kyung Lee ),( Yu Jeong Bae ),( Nam Ju Moon ),( Seong Jun Seo ) 대한피부과학회 2020 Annals of Dermatology Vol.32 No.3

Background: Early-onset and severe atopic dermatitis (AD) in patients increase the probability of the development of allergic rhinitis or asthma. Treatment and prevention strategies in infants and young children with AD are targeted toward treating the symptoms, restoring skin barrier functions, and reducing the absorption of environmental allergens in an attempt to attenuate or block the onset of asthma and food allergy. Objective: Given that the initiating events in AD remain poorly understood, identifying those at risk and implementing strategies to prevent AD is necessary. Methods: Whole-exome sequencing (WES) was performed in a 43 control group and a disease group with 20 AD patients without atopic march (AM) and 20 with AM. Sanger sequencing was carried out to validate found variants in cohorts. Results: DOCK8, IL17RA, and KLK12 single-nucleotide polymorphisms were identified by WES as missense mutations: c.1289C>A, p.P97T (rs529208); c.1685C>A, p.P562G (rs12484684); and c.457+27>C, rs3745540, respectively. A case-control study show that total immunoglobulin E (IgE) level was significantly increased in the AA genotype of DOCK8 compared to the CA genotype in allergic patients. The rs12484684 of IL17RA increased risk of adult-onset AD (odds ratio: 1.63) compared to the control for (A) allele frequency. AD and AM Patients with the IL17RA CA genotype also had elevated IgE levels. rs3745540 of KLK12 was associated with AD in dominant model (odds ratio: 2.86). Conclusion: DOCK8 (rs529208), IL17RA (rs12484684), and KLK12 (rs3745540), were identified using a new WES filtering method. the result suggests that polymorphism of DOCK8 and IL17RA might be related to increase the total IgE level. (Ann Dermatol 32(3) 197∼205, 2020)

Evaluation of ‘TNF-α, IL-6, and MMP-9’ Test Kit for Screening of Meibomian Dysfunction in Patients with Inflammatory Dry Eye Syndrome

Min-Hye Park,Jung-Eun Park,Jang-Won Byun,Min-Ji Choi,Il-Hoon Cho,Myeong-Jin Jeong,Yoon-Jung Choy,Koon-Ja Lee 대한시과학회 2020 대한시과학회지 Vol.22 No.1

목적 : 마이봄샘기능저하증(meibomian gland dysfunction, MGD)을 수반하는 염증성 건성안의 감별진단에 대한 ‘TNF-α, IL-6, MMP-9’ 검사키트의 유용성을 평가하였다. 방법 : 건성안 이외의 안질환이 없는 20~30대 중 OSDI 설문 검사에 따른 건성안 총 118안을 대상하였고, 결막낭 메니스커스로부터 소량의 눈물을 채취하여 TNF-α, IL-6 및 MMP-9 검사를 하였다. 각막염색과 결막충혈 이 모두 Grade 1 이상인 경우는 염증성 건성안으로, 마이봄샘폐쇄와 마이봄샘구멍막힘이 모두 grade 1 이상인 경우는 MGD 관련 건성안으로 평가하였다. 염증성 건성안 및 MGD와 TNF-α, IL-6, MMP-9과의 상관성은 카 이제곱검정(Chi-square test)으로 분석하였고, ‘TNF-α, IL-6, MMP-9’ 검사키트의 염증성 건성안과 MGD를 수반하는 염증성 건성안 감별능력은 ROC 커브를 이용하여 민감도, 특이도 및 AUC(Area under the curve)를 구하고 정확도를 평가하였다. 결과 : 염증성 건성안은 TNF-α와 IL-6와 유의한 상관성을 보였고(p<.050), ‘TNF-α, IL-6, MMP-9’ 검사 키트는 MMP-9 검사키트와 80.20%의 높은 일치도를 나타냈으나(p<.050), 염증성 건성안 감별에 대한 민감도, 특이도, 정확도는 MMP-9 검사키트보다 낮았다. MGD는 MMP-9 검사와 상관성을 보이지 않았고, TNF-α와 IL-6 검사와는 유의한 상관성을 보였으며, MGD 감별에 대한 민감도, 특이도, 정확도는 각각 85.50%, 34.70%, 0.601, 85.50%, 32.70%, 0.591로 나타났다. MGD 수반한 염증성 건성안 감별에 대한 ‘TNF-α, IL-6, MMP-9’ 검사키트의 민감도, 특이도 및 정확도는 100.00%, 34.10%, 0.670로 MMP-9 검사키트보다 더 높았다. 결론 : MGD 진단에는 TNF-α, IL-6 검사가 유용하며, ‘TNF-α, IL-6, MMP-9’ 검사키트는 MGD를 수반한 염증성 건성안 평가에 유용할 것으로 사료된다. Purpose : To evaluated the ‘TNF-α, IL-6, MMP-9’ test kit for screening of inflammatory dry eye and IDE (inflammatory dry eye) with MGD (meibomian gland dysfunction). Methods : A total of 118 dry eyes were selected using OSDI (ocular surface disease index) questionnaire among participated 20~30s without ophthalmologic diseases except for dry eye. Small amount of tear obtained from meniscus of the conjunctiva were tested with TNF-α, IL-6, and MMP-9 kit. IDE refers to the criteria which specifies the corneal staining and conjunctival hyperemia more than grade 1 and MGD refers to the criteria which specifies meibomian gland blockage and meibomian orifice obstruction with more than grade 1. Chi-square test was performed to analyze the correlation between the IDE, MGD and the results of ‘TNF-α, IL-6, MMP-9’ tests. and ROC (receiver operate characteristics) curve was used for the sensitivity, specificity and AUC (area under the curve) for the accuracy of ‘TNF-α, IL-6, MMP-9’ tests. Results : TNF-α and IL-6 were significantly correlated with IDE (p<.050) and ‘TNF-α, IL-6, MMP-9’ test kit showed a high agreement of 80.20% with MMP-9 test kit(p<.050) although the accuracy was lower than MMP-9 test kit. The MMP-9 showed no correlation with MGD, however TNF-α, IL-6 were significantly correlated with MGD (p<.050). sensitivity, specificity, and AUC of TNF-α, IL-6 tests for MGD were 85.50%, 34.70%, 0.601, 85.50%, 32.70%, and 0.591. The sensitivity, specificity, and AUC of ‘TNF-α, IL-6, MMP-9’ test kit for IDE with MGD were 100.00%, 34.10%, and 0.670, respectively, which shows higher accuracy than MMP-9. Conclusion : TNF-α and IL-6 tests are useful for the diagnosis of MGD, and ‘TNF-α, IL-6, MMP-9’ test kit is useful for screening IDE with MGD.

코디세핀이 마우스 복강 대식세포에서 전염증성 사이토카인의 생성에 미치는 영향

서민정(Min-Jeong Seo),강병원(Byoung-Won Kang),김민정(Min-Jeong Kim),이혜현(Hye-Hyeon Lee),서권일(Kwon-il Seo),김광혁(Kwang-Hyuk Kim),정영기(Yong-Kee Jeong) 한국식품과학회 2014 한국식품과학회지 Vol.46 No.1

본 연구는 동충하초(Cordyces militaris) 유래의 기능성 물질인 코디세핀의 면역활성을 검증하기 위하여 C57BL6 마우스 복강 대식세포를 이용하여 코디세핀이 대식세포의 활성화에 미치는 영향에 대하여 시험하였다. 그 결과 LPS에 의해 유도된 마우스 복강세포는 코디세핀의 작용에 의해 IL-1β, IL-12, TNF-α의 염증성 사이토카인의 생성이 증대되어 초기 염증매개 반응을 유도하여 선천면역반응의 활성화와 그리고 면역작용에 있어 후기 적응면역의 전환으로의 T 림프구의 활성화가 예상된다. 또한 IL-6의 생성증대로 활성화된 T 림프구에 의해 B 림프구의 항체생성반응을 매개하는 면역반응도 상승할 것으로 사료된다. 그리고 대식세포에 의한 염증반응에서 염증매개인자인 NO와 H₂O₂의 생성을 증대시킴에 따라 대식세포의 독성작용을 활성화시켜 염증반응을 효과적으로 유도할 것으로 보이며, 또한 H₂O₂의 후기 생성을 저해하였는데 이는 염증반응에 유도될 수 있는 세포의 손상으로부터 세포를 보호할 수 있을 것으로 사료된다. 따라서 코디세핀은 외부인자로부터 염증매개성 면역반응의 증강작용을 나타내는 것으로 사료된다. The effect of cordycepin purified from Cordyceps militaris on macrophage activation was investigated in peritoneal macrophages isolated from C57BL6 mice. Lipopolysaccharide-induced mouse peritoneal cells showed that cordycepin treatment increased the expression of the inflammatory cytokines interleukin (IL)-1β, IL-12, and tumor necrosis factor-α (TNF-α), leading to early inflammation-mediated reactions, the activation of immunological responses, and T lymphocyte activation. T lymphocytes, activated by a greater production of IL-6, resulted in antibody-generating immune reactions, suggesting that cordycepin was effective at inducing immunological responses. Consistent with the increase in the inflammation-mediating factors including nitric oxide (NO) and hydrogen peroxide (H₂O₂), the toxic response of macrophages was activated and effectively induced inflammation. These findings demonstrate that cordycepin is involved in reducing cell injury provoked by inflammatory reactions. Therefore, these results suggest that cordycepin treatment of mouse peritoneal cells induces inflammation-mediated immunological responses and immunostimulation.

Blockade of indoleamine 2,3-dioxygenase protects mice against lipopolysaccharide-induced endotoxin shock.

Jung, In Duk,Lee, Min-Goo,Chang, Jeong Hyun,Lee, Jun Sik,Jeong, Young-Il,Lee, Chang-Min,Park, Won Sun,Han, Jin,Seo, Su-Kil,Lee, Sang Yong,Park, Yeong-Min Williams Wilkins 2009 JOURNAL OF IMMUNOLOGY Vol.182 No.5

<P>Suppression of an excessive systemic inflammatory response is a promising and potent strategy for treating endotoxic sepsis. Indoleamine 2,3-dioxygenase (IDO), which is the rate-limiting enzyme for tryptophan catabolism, may play a critical role in various inflammatory disorders. In this study, we report a critical role for IDO in the dysregulated immune response associated with endotoxin shock. We found that IDO knockout (IDO(-/-)) mice and 1-methyl-D-tryptophan-treated, endotoxin-shocked mice had decreased levels of the cytokines, TNF-alpha, IL-6, and IL-12, and enhanced levels of IL-10. Blockade of IDO is thought to promote host survival in LPS-induced endotoxin shock, yet little is known about the molecular mechanisms that regulate IDO expression during endotoxin shock. In vitro and in vivo, IDO expression was increased by exogenous IL-12, but decreased by exogenous IL-10 in dendritic cells and splenic dendritic cells. Interestingly, whereas LPS-induced IL-12 levels in serum were higher than those of IL-10, the balance between serum IL-12 and IL-10 following challenge became reversed in IDO(-/-)- or 1-methyl-D-tryptophan-treated mice. Our findings demonstrate that the detrimental immune response to endotoxin shock may occur via IDO modulation. Restoring the IL-12 and IL-10 balance by blocking IDO represents a potential strategy for sepsis treatment.</P>

위암에서 Helicobacter pylori cagA, vacA, iceA 유전자와 숙주 Interleukin-1β및 Interleukin-1 수용체 길항제 유전자 다형성

이성훈 ( Seong Hun Lee ),김태오 ( Tae Oh Kim ),이동현 ( Dong Hyun Lee ),박원일 ( Won Il Park ),김광하 ( Gwang Ha Kim ),허정 ( Jeong Heo ),강대환 ( Dae Hwan Kang ),송근암 ( Geun Am Song ),조몽 ( Mong Cho ) 대한내과학회 2006 대한내과학회지 Vol.71 No.1

Background: Both Helicobacter pylori (H. pylori) cagA, vacA, iceA genotype and host IL-1B/IL-1RN polymorphisms play a role in determining the clinical consequences of H. pylori infection. This study aimed to investigate whether there might be any combinations of H. pylori cagA, vacA, iceA genotype and host IL-1B/IL-1RN polymorphisms that are particularly associated with the occurrence of gastric carcinoma in Korean patients. Methods: This study population was comprised of 239 patients with H. pylori infection: 122 with gastric carcinoma and 117 with gastritis only. DNA was isolated from gastric biopsy sample and H. pylori cagA, vacA and iceA genotype were determined by PCR. IL-1B-511 polymorphisms were genotyped by PCR-RFLP and IL-1RN polymorphisms were analyzed with variable number of tandom repeat after PCR. Results: H. pylori cagA, vacA, and iceA genotype were not associated with an increased risk for gastric carcinoma. IL-1B-511*T carriers and IL-1RN*2 carriers did not show increased risk for gastric carcinoma. On combination of bacterial/host genotypes, cagA+/IL-1B-511*T carriers and cagA+/IL-1RN*2 carriers, vacA s1/IL-1B-511*T carriers, vacA s1/IL-1RN*2 carriers, vacA m1/IL-1B-511*T carriers, vacA m1/IL-1RN*2 carriers, iceA1/IL-1B-511*T carriers, iceA1/IL-1RN*2 carriers showed no increased risk of gastric carcinoma. Conclusions: Combined H. pylori cagA, vacA, iceA genotype and host IL-1B/IL-1RN polymorphisms shows no increased risk of gastric carcinoma. Therefore, it seems other endogenous or exogenous factors may play more important role in the development of gastric carcinoma in Korean.(Korean J Med 71:24-37, 2006)

Gene therapy of intracranial glioma using interleukin 12-secreting human umbilical cord blood-derived mesenchymal stem cells.

Ryu, Chung Heon,Park, Sang-Hoon,Park, Soon A,Kim, Seong Muk,Lim, Jung Yeon,Jeong, Chang Hyun,Yoon, Wan-Soo,Oh, Won-il,Sung, Young Chul,Jeun, Sin-Soo Mary Ann Liebert 2011 Human gene therapy Vol.22 No.6

<P>Clinical trials of gene therapy using a viral delivery system for glioma have been limited. Recently, gene therapy using stem cells as the vehicles for delivery of therapeutic agents has emerged as a new treatment strategy for malignant brain tumors. In this study, we used human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) as delivery vehicles with glioma-targeting capabilities, and modified interleukin-12 (IL-12p40N220Q; IL-12M) as a novel therapeutic gene. We also engineered UCB-MSCs to secret IL-12M (UCB-MSC-IL12M) via tetrameric cell-permeable peptide (4HP4)-mediated adenoviral transduction. We confirmed the migratory capacity of UCB-MSC-IL12M toward GL26 mouse glioma cells by an in vitro migration assay and in vivo injection of UCB-MSC-IL12M into the ipsilateral hemisphere of implanted gliomas in C57BL/6 mice. In vivo efficacy experiments showed that intratumoral injection of UCB-MSC-IL12M significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with control mice. Antitumor effects were associated with increased local IL-12M levels, followed by interferon-γ secretion and T-cell infiltration in intracranial gliomas, as well as antiangiogenesis. Interestingly, tumor-free mice after UCB-MSC-IL12M treatment were resistant to ipsilateral and contralateral tumor rechallenge, which was closely associated with tumor-specific long-term T-cell immunity. Thus, our results provide the rationale for designing novel experimental protocols to induce long-term antitumor immunity against intracranial gliomas using UCB-MSCs as an effective delivery vehicle for therapeutic cytokines including IL-12M.</P>

The Effect of Human Placental Extract on Rheumatoid Arthritis in an Animal Model

Jeong Dong Park,신희석,Sang-Il Lee,A Ram Kim,Jong Moon Park,Sang-Yeop Shin,Jun Hwa Shin,Seung Won Moon,Hyun Park,오민균 대한재활의학회 2012 Annals of Rehabilitation Medicine Vol.36 No.2

Objective To assess the effi cacy of human placental extract (HPE) in an animal model of rheumatoid arthritis (RA). Method We used (i) KRN C57BL/6 TCR transgenic x NOD mice (KBx/N) serum transfer arthritis and (ii) collageninduced arthritis (CIA) mice to evaluate the effi cacy of HPE (1 ul or 100 ul, intra-peritoneal, three times per week)on RA. Incidence, severity of arthritis, and hind-paw thickness were quantifi ed. Joint destruction was analyzed using modifi ed mammographic imaging. Histopathological analysis for infl ammation, cartilage, and osteoclasts was performed using Hematoxylin-eosin (H-E), safranin-O, and tartrate-resistant acidic phosphatase (TRAP). ELISAs were used for detection of various cytokines in serum and joint tissue. Results Th ere were no signifi cant diff erences in incidence of arthritis, clinical scores of arthritis, and hind-paw thickness between HPE-treated and vehicle-treated groups for up to 2 weeks in the KBx/N serum transfer arthritis model. Histopathological analysis also showed no diff erences 2 weeks after treatment. Levels of TNF-α, IL-1β, IL-6, IL-10, and RANKL in serum and joint tissues were similar in all groups. Furthermore, there were no diff erences in clinical, radiological, and histological parameters between HPE-treated and vehicle-treated group for 3 weeks in the CIA model. Conclusion Systemic treatment with HPE has no benefi cial eff ects on arthritis in animal models of RA. Th erefore,indiscreet use of HPE in RA should be forbidden.

Enzyme Hydrolysates of Ginseng Marc Polysaccharides Promote the Phagocytic Activity of Macrophages Via Activation of TLR2 and Mer Tyrosine Kinase

( Jeong Yeon Seo ),( Ji Won Choi ),( Jae Yeon Lee ),( Young Shik Park ),( Yong Il Park ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.6

Although ginseng marc is a by-product obtained during manufacturing of various commercial ginseng products and has been routinely discarded as a waste, it still contains considerable amounts of potential bioactive compounds, including saponins and polysaccharides. Previously, we reported that ginseng oligosaccharides derived from ginseng marc polysaccharides by enzymatic hydrolysis exert immunostimulatory activities in macrophages and these activated macrophages are in turn able to inhibit the growth of skin melanoma cells by inducing apoptosis. In the present study, a more detailed investigation of the immunostimulatory activity and underlying action mechanisms of an enzymatic hydrolysate (GEH) containing these oligosaccharides derived from ginseng marc polysaccharides was performed. The levels of proinflammatory cytokines and anti-inflammatory cytokines were measured in GEH-stimulated RAW264.7 macrophages using RT-PCR analysis and ELISA. The expression levels of Toll-like receptor 2 (TLR2) and TLR4, Dectin-1, and MerTK were measured by RT-PCR analysis or western blot analysis, and the phagocytic activities of GEH-challenged bone marrow-derived macrophages toward apoptotic Jurkat cells were assayed using fluorescence microscopy. GEH induced the production of both proinflammatory cytokines TNF-α and IL-6, and anti-inflammatory cytokine IL-10 in RAW 264.7 cells. The expression of the TLR2 and MerTK mRNAs was increased upon GEH treatment. Phagocytosis of apoptotic Jurkat cells was enhanced in GEH-treated macrophages. Based on the results, this enzymatic hydrolysate (GEH) containing oligosaccharides exerts immunostimulatory effects by maintaining the balance between M1 and M2 cytokines, facilitating macrophage activation and contributing to the efficient phagocytosis of apoptotic cells. Therefore, the GEH could be developed as value-added, health-beneficial food materials with immunostimulatory effects.

Chronic alcohol consumption induces migration of IL-1R2+ monocytes from the bone marrow into the liver by neuro-immunologic pathway

Young-Ri Shim,Hee-Hoon Kim,Keungmo Yang,Tom Ryu,Kyurae Kim,Sung Eun Choi,Minjeong Kim,Chae-Rin Woo,Young-Sun Lee,Won-Il Jeong 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Liver is challenged by diverse detrimental substances through multiple metabolic processes, but it is less prone to inflammation. In chronic alcohol consumption, although the migration of monocytes from bone marrow (BM) into liver is increased, alcoholic hepatitis rarely occurs. Thus, we investigated the sub-population of liver macrophages showing anti-inflammatory roles through single-cell RNA sequencing (scRNA-Seq) after chronic EtOH-feeding. Interestingly, in scRNA-seq and flow cytometry analyses of hepatic macrophages, the phenotype of Ly6Clow (anti-inflammatory) cells was dramatically altered by ethanol intake. In particular, they were highly expressed interleukin-1 type II receptor (IL-1R2), a decoy receptor of IL-1β. Intriguingly, IL-1R2+ Ly6Clow macrophages showed decreased CX3CR1 expression, which was confirmed not only in the liver, but also BM and blood, suggesting monocytes from BM affected by ethanol might migrate into the liver. We found that the Leptin Receptor+ mesenchymal stromal cells (LepR+ MSCs), which were located around blood vessels expressing CX3CL1 to hold CX3CR1+ macrophages, could express alcohol dehydrogenase to metabolize ethanol in BM. Ethanol metabolism in LepR+ MSCs was induced both production of chemokines (CXCL9 and 10) and the excretion of glutamate via cystine-glutamate anti-porter xCT to recruit and activate the CXCR3+ BM NK cells to produce interferon-γ in a metabotropic glutamate receptor 5 (mGluR5)-dependent manner. Indeed, IFN-γ production was significantly decreased in EtOH-fed mice when we depleted mGluR5 in NK cells. In turn, NK cell-derived IFN-γ down-regulated CX3CR1 expression in BM Ly6Clow monocytes, consequently induced egress of Ly6Clow monocytes into the blood and migration into the liver to suppress alcoholic inflammation. In conclusion, glutamate of LepR+ MSCs imposed egress license on anti-inflammatory IL-1R2+ Ly6Clow monocytes through NK cell-derived IFN-γ-mediated suppression of CX3CR1, suggesting a potential therapeutic inter-organ crosstalk between BM and liver in alcoholic liver disease.