[보고서]통합적 오믹스 연구를 통한 베체트 장염의 진단, 예후 예측의 바이오마커 및 치료 표적의 발굴

천재희

보고서 정보

주관연구기관

연세대학교
Yonsei University

연구책임자

천재희

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2016-11

과제시작연도

2015

주관부처

보건복지부
[Ministry of Health & Welfare(MW)(MW)

연구관리전문기관

한국보건산업진흥원
Korea Health Industry Development Institute

등록번호

TRKO201800042822

과제고유번호

1465019014

사업명

질환극복기술개발

DB 구축일자

2018-11-24

키워드

베체트 장염.통합적 오믹스.바이오마커.치료 표적.베체트 장염 질병 모델.Behcet's disease.integrated omics.biomarker.therapeutic target.Behcet's disease model.

표 Baseline clinical characteristics of patients with Behcet’s disease
표 Baseline clinical characteristics of patients with intestinal BD
표 Sequence quality
표 q-q plot
표 2-dimensional electrophoresis
표 BD targets
표 SAA expression in BD patients
표 2 dimensional electrophoresis
표 Immunostaining against galectin-3
표 환자군의 기초 정보
표 베체트 궤양의 형태에 따른 임상 양상 및 분변 마커의 변화
표 베체트 궤양의 형태에 따른 분변 calprotectin 값의 차이
표 Halplotype analysis of susceptible genes for intestinal Behcet disease and
표 Pathway analyses of genes susceptible to intestinal Behçet’s disease, Behçet’s disease without intestinal involvement, and inflammatory bowel disease (IBD)
표 LGALS3, IL1B, NLRC4 mRNA의 베체트 장염이 있는 환자의 대장에서의 발현비교
표 Galectin 3 deficient BMDM에서의 inflammasome 자극(LPS+ATP)에 의한 cytokine 발현의 변화
표 BMDM에서의 inflammasome 자극(LPS+ATP)에 의한 inflammasome 활성(caspase, IL1-b)의 변화
표 Galectin 3 deficienty BMDM에서의 inflammasome 자극(almonella (Sal) infection)에 의한 inflammasome 활성(caspase, IL1-b)의 변화
표 control cell line (CTL 4, 5, 9) 및 LGALS3 shRNA stable cell (G3 sh 5, 8,30)에서의 Lgals3의 유전자 발현확인
표 control cell line (CTL 4, 5, 9) 및 LGALS3 shRNA stable cell (G3 sh5, 8,30)에서의 Lgals3의 및 Illb 유전자 발현의 상관관계
표 IL10/MyD88 double knockout mouse의 genotyping 결과
표 Lgals3, Nlrc4 mRNA의 마우스 대장에서의 발현비교
표 UPGMA Dendrogram
표 Taxonomic composition of Control (1%)
표 Taxonomic composition of UC (1%)
표 Taxonomic composition of CD (1%)
표 Taxonomic composition of BD (1%)
표 Taxonomic composition of Control
표 Taxonomic composition of BD
표 Taxonomic composition of IBD (Phylum level)
표 Taxonomic composition of IBD (Class level)
표 Taxon XOR analysis of Control and BD
표 Taxon XOR analysis of BD and Control
표 Taxon XOR analysis of UC and BD
표 Taxon XOR analysis of UC and BD
표 Taxon XOR analysis of CD and BD
표 Source tracking of IBD
표 Source tracking of IBD
표 Diversity indices of BD and CD
표 Clinical Characteristics of Patients according to the Disease Type
표 Categories of Infectious Complications in Patients with Inflammatory Bowel Disease and Intestinal Behçet’′s Disease
표 Relationship between the Procalcitonin Level and Disease Activity in Patients with Inflammatory Bowel Disease and Intestinal Behçet′s Disease
표 Sensitivity, Specificity and Positive and Negative Predictive Values of Procalcitonin and C-reactive Protein
표 Selection of the study subjects
표 Fecal calprotectin measured by the three fecal calprotectin kits in three groups of patients. Fecal calprotectin according to the three different kits was significantly elevated in IBD compared to IBS. Fecal calprotectin determined by the three kits was not different between IBS and the “other colitis” group (see the METHODS section for the definition)
표 Diagnostic flow of the study population
표 Correlation between alterations in fecal calprotectin levels and DAIBD from diagnosis until 3 months of follow-up. rho=0.470, P=0.027, DAIBD, disease activity index for intestinal Behçt’s disease
표 Micorobiome changes between wild type mice and TLR4-deficient mice
표 Effects of IL-33 on dextran sodium sulfate (DSS)-induced colitis in mice.(A) Body weight changes. The weight of each mouse was followed daily. (B) Effects of IL-33 on colon length. (C) Effects of IL-33 on the disease activity index (DAI). Disease activity at day 7 was scored according to the criteria outlined in the Materials and Methods. (D) Representative image of periodic acid-Schiff (PAS)-stained goblet cells in the colon.(E,H,I)mRNA expression in the mouse colon. mRNA expression of Muc2 (E), M2 markers (Cd206, Cd163, and Arg1) (H), and Tnfa (I) were evaluated by quantitative RT-PCR. (F) Flow cytometry analysis of the M2 macrophage (CD206+) populations in peritoneal cavity cells (PCCs) from treated mice.(G) Flow cytometry analysis of the Treg (Foxp3+) and Th1 (Gata3+) populations in the spleens of treated mice. Data are presented as means ±SE(n=5 mice/group). Recombinant IL-33 (mrIL-33) was i.p. injected daily (0.2 μg/mouse) for 5 days after treatment with 3% (w/v) DSS. #P < 0.05 vs. Sham control, *P < 0.05 vs. DSS as assessed by ANOVA
표 MyD88 independent effects of IL-33 on goblet cell restoration in inflammation. (A, B, E) Effects of IL-33 on colitis and goblet cells in the colon of wild-type (WT) and MyD88 deficient (MyD88-/-)mice after low-dose 2,4,6-trinitrobenzenesulfonic acid (TNBS) administration (n = 5 mice/group). To assess the effects of IL-33 on enterocyte early in colitis, 100 μl TNBS solution (1.8% (w/v)) in 40% ethanol was injected into the colon of each mouse. (A) Changes in body weight. The weight of each mouse was followed daily. (B) Representative image of periodic acid-Schiff (PAS) staining of the colonic mucosa for goblet cells. Original magnification: ×200. (C, D) Effects of IL-33 on MUC2 (C) and KLF4 (D) mRNA expression in human colonic epithelial cells. HT-29 cells were treated with vehicle (Veh) or lipopolysaccharide (LPS, 100 ng/ml), with or without human recombinant IL-33 (hrIL-33), for 4 hours. Total RNA was extracted and analysed by quantitative RT-PCR. Target mRNA expression levels were normalised to that of β-actin. Results are representative of two independent experiments. (E) Effects of IL-33 on Klf4 mRNA expression in the mouse colon. Values are expressed as means; error bars represent ±SD. #P < 0.05 vs. Sham control, *P < 0.05 vs. TNBS or LPS, **P < 0.01 vs. TNBS or LPS, ***P < 0.005 vs.LPS as assessed by ANOVA. Veh, injected with vehicle; IL-33, injected with mrIL-33 (1μg /mouse) or hrIL-33 (20 ng/ml); TNBS, injected with TNBS
표 IL-33-treated peritoneal cavity cells ameliorate mouse colitis. Male C57BL/6 mice were inoculated i.p. with IL-33 (mrIL-33, 1 μg/mouse) or PBS for 3 consecutive days. Next, peritoneal cavity cells (PCCs) were extracted from the peritoneum of each mouse by washing the peritoneal cavity with cold PBS (A, B). F4/80 positive cells were sorted from the harvested PCCs and i.p. injected (1×106 cells) into recipient mice. After 2days, 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution (2.2%(w/v)) was injected into the colon of each recipient mouse (C-F). (A, B) Representative flow cytometry plots of PCCs and spleen cells from IL-33-treated naïve mice. (A) Dot plot of the M2 (CD206+) macrophage (F4/80+) populations in the PCCs and percentages of the M2 macrophage populations in the PCCs. (B) Treg (Foxp3+) and Th1 (Gata3+) populations in thespleensofnaïvemice. (C) Body weight changes of PCC-transplanted mice. (D) Colon lengths of PCC-transplanted mice. (E) Disease activity index (DAI) scores of PCC-transplanted mice. (F) Flow cytometry analysis of Treg (Foxp3+) and Th1 (Gata3+) populations isolated from the spleen of PCC-transplanted mice. Values are expressed as means (n = 5); error bars represent ±SD. #P < 0.05 vs. TNBS, ##P < 0.01 vs. TNBS, *P < 0.05 vs. Veh or PBS PCC, **P < 0.01 vs. Veh or PBS PCC as assessed by ANOVA. Veh, injected with vehicle; IL-33, injected with mrIL-33; PCC, injected with F4/80+PCCs;TNBS, injected with TNBS
표 IL-33-treated peritoneal cavity cells rescue M2 macrophages and the goblet cells in colon tissues with colitis. (A) Representative flow cytometry plots of M2 (CD206+) macrophage (F4/80+) populations in colon samples. (B-D) Effects of IL-33 on the mRNA expression levels of M2 (Cd206 and Cd163, B), M1 marker (Tnfa, C), and goblet cell marker (Klf4, D) in the colons. (E) Representative periodic acid-Schiff (PAS) staining of colonic mucosal samples. Original magnification: ×200. Values are expressed as means (n = 5); error bars represent ±SDs. *P < 0.05 vs. PBS PCC as assessed by ANOVA. PCC, peritoneal cavity cell; Veh, injected with vehicle; IL-33, injected with mrIL-33; TNBS, injected with TNBS
표 Primers for qRT-PCR
표 Levels of circulating IL-33 and soluble ST2 (sST2). Serum IL-33 and sST2 concentrations were determined in duplicate by enzyme-linked immunosorbent assay (ELISA). Two different anti-IL-33 antibodies were used, each of which recognized a different IL-33 epitope. The levels of IL-33 (A) and sST2 (B) were assessed by ELISA in serum samples from normal controls (NC) and patients with Crohn’s disease (CD),ulcerative colitis (UC), and Behçet’s disease (BD). (C) Correlations between serum IL-33 and sST2 levels and clinical disease activity of inflammatory bowel disease (IBD). Solid line, references on the X and Y axes; dotted line, fit for total variables. **P < 0.01 vs.NC, ***P < 0.005 vs. NC as assessed by ANOVA. NC, normal control; CDAI, Crohn
표 Ratios of circulating IL-33/ST2 and its correlation with disease activity. (A) Serum IL-33/sST2 ratios in patients with inflammatory bowel disease (IBD). (B) Correlation between serum IL-33/sST2 ratio and IBD clinical disease activity. Solid line, references on the X and Y axes; dotted line, fit for total variables. Results are shown as individual values (symbols) and SDs (lines) for each group of patients. ***P < 0.005 vs. NC as assessed by ANOVA. NC, normal control; CD, Crohn
표 IL-33 mRNA expression in inflamed mucosal tissue samples. (A) Colonic biopsy samples were harvested from noninflamed areas of normal control (NC, n = 3) patients and from inflamed areas of patients with UC (n = 9), BD (n = 7), and CD (n =6). Total RNA was extracted and processed for quantitative RT-PCR. IL-33 expression levels were normalised to that of β-actin. Relative mRNA levels were calculated using the 2DCt formula. The significance of differences between groups was analysed using Student’s t-test. (B) Effects of IL-33 on dextran sodium sulfate (DSS)-induced colitis in mice (n = 5 mice/group). *P < 0.05 vs. NC, *P < 0.05 vs. Sham. NC, normal control; UC, ulcerative colitis; BD, Behçet’s disease; CD, Crohn’s disease; Veh, injected with vehicle; IL-33, injected with mrIL-33; TNBS, injected with 2,4,6-trinitrobenzenesulfonic acid
표 Histological score of dextran sodium sulfate (DSS)-induced colitis in mice (n = 5). **P < 0.01 vs. DSS. IL-33, 5 daily injections of IL-33 (mrIL-33, 0.2 μg/mouse); DSS, treated with 3% (w/v) DSS
표 Effects of IL-33 on the survival of mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Survival (n = 7) was analysed by generating a Kaplan-Meier plot. Veh, treated with vehicle; IL-33, treated with mrIL-33; TNBS,injected with 2,4,6-trinitrobenzenesulfonic acid
표 Effects of IL-33 on disease activity index (DAI, A), histological score (B),and Muc2 expression in colon tissue (C) of wild-type (WT) and Myd88 deficient (Myd88-/-)micewith2,4,6-trinitrobenzenesulfonicacid(TNBS)-inducedcolitis(n=5).Toassessthe effectsofIL-33onenterocyteandmacrophageearlyindisease,100μlTNBSsolution(1.8%(w/v))in40 %ethanolwasinjectedintothecolonofeachmouseandDAIscoreatday6(D6)wasobtained.##P < 0.01 vs. Sham, ###P < 0.005 vs. Sham, **P < 0.01 vs. TNBS, ***P < 0.005 vs. TNBS. Veh, treated with vehicle; IL-33, treated with mrIL-33; TNBS, injected with 2,4,6-trinitrobenzenesulfonic acid
표 Male C57BL/6 mice were inoculated i.p. with IL-33 (1 μg/mouse) or PBS for 3 consecutive days (A). Next, peritoneal cavity cells (PCCs) were extracted from the peritoneum of each mouse by washing the peritoneal cavity with cold PBS. F4/80 positive cells were sorted from the extracted PCCs and i.p. injected (1 ´106cells)intotherecipientmice.After2days,2,4,6-trinitrobenzenesulfonicacid(TNBS)solutionwasin jectedintothecolonofeachrecipientmouse(B, C). (A) Representative flow cytometry gating strategies of peritoneal cavity cells (PCCs) isolated from PCC-treated mice (n = 5). (B) Histological score of the colon from TNBS-treated mice. Values are expressed as means (n = 5); error bars represent ±SDs. (C) Representative image of mouse colon immunostaining (n = 3-4). CD206 is visualized in green using fluorescent conjugated antibodies. Nuclei were counterstained with DAPI (blue). ###P < 0.05 vs. TNBS, *P < 0.05 vs. PBS PCC as assessed by ANOVA. Veh, treated with vehicle; IL-33, treated with mrIL-33; TNBS, injected with 2,4,6-trinitrobenzenesulfonic acid
표 Effects of IL-33 on the mRNA expression levels of M2 and goblet cell markers in the colons from peritoneal cavity cell (PCC)-treated mice. (A) mRNA expression of Il10 and Tgfb. (B) mRNA expression of Muc2. Male C57BL/6 mice (n = 5) were inoculated i.p. with IL-33 (1 μg/mouse) or PBS for 3 consecutive days. Next, peritoneal cavity cells (PCCs) were extracted from the peritoneum of each mouse by washing the peritoneal cavity with cold PBS. F4/80 positive cells were sorted from the isolated PCCs and i.p. injected (1x 106cells) into the recipient mice. After 2 days, ,4,6-trinitrobenzene sulfonic acid (TNBS) solution was injected into the colon of each recipient mouse.***P < 0.005 vs. PBS PCCs as assessed by ANOVA. Veh, treated with vehicle; IL-33, treated with mrIL-33; TNBS, injected with 2,4,6-trinitrobenzenesulfonic acid
표 Representative image of CD206 and IL-33 immunostain in human colon (NC, n = 3; UC, n = 4). (A) CD206 immunostaining (green). (B) IL-33 immunostaining (red). Nuclei were counterstained with DAPI (blue). NC, normal control; UC, ulcerative colitis
표 Wound healing assay. Monocytes were isolated from PBMCs and differentiated into macrophages for 7 days. Macrophages were cocultured with the HT-29 cells and wound healing assays were performed. Representative images show increased wound closures by IL-33 treatment. Data are presented as means ±SD (n = 2). *P < 0.05 vs. Veh as assessed by Student’s t-test. NC, not cocultured; Veh, treated with vehicle; IL-33, treated with hrIL-33; LPS, treated with lipopolysaccharide. *P < 0.05 vs. LPS
표 Baseline characterictics of the study population
표 The distribution of serum interleukin (IL)-12B levels according to disease activity.A, IL-12B levels inpatientswithCrohn’ disease (CD). B, IL-12B levels in patientswith ulcerative colitis (UC). C, IL-12B levels in patients with intestinal Behc¸et’ disease (BD)
표 TABLE 3. IL-12B Levels (pg/mL) According to Endoscopic Disease Activity
표 Baseline characteristics of study population
표 Hazard ratio of postoperative thiopurines compared with 5-ASA
표 The effects of guggulsterone on NF-κB p65 and TLR4 signalling and the expression of TREM-1. (a) Effects of guggulsterone on NF-κB p65 nuclear translocation. NF-κB p65 (green colour) nuclear translocation was analysed by immunofluorescent (IF) staining. Figures are representative of 2 independent experiments (original magnification ×400). (b) Levels of TLR4, TREM-1, TNF-α, and β-actin and phosphorylation of NF-κB p65 (p-NF-κB) and IκB-α (p-IκB-α) were analysed using Western blotting. Blots are representative of 3 independent experiments. (c) Secreted levels of TREM-1 protein in culture media were measured by enzyme-linked immunosorbent assay (ELISA). Data represent means ± S.D. values (n = 2). (d-f) mRNA levels of Il10 (d), Trem1 and Tlr4 (e), and Tnfa and Il6 (f). Data represent means ± S.D. values (n = 3-4). RAW264.7macrophage cells were treated with LPS (10 μg/mL) and the indicated concentration or 20 μM of GGS for 24 h for IF staining, Western blot, and ELISA and for 4 h for mRNA measurements. Veh, treated with vehicle; GGS, treated with guggulsterone; LPS, treated with lipopolysaccharide; TREM-1 Fc, treated with TREM-1 Fc chimera antibody (1μg/mL). #p < 0.05 vs. Veh, ##p < 0.01 vs. Veh, ###p < 0.005 vs. Veh, *p < 0.05 vs. LPS,**p < 0.01 vs. LPS, ***p < 0.005 vs. LPS
표 The effects of guggulsterone on amplification of TLR4 signalling by TREM-1 stimulation. (a) Levels of TREM-1, TLR4, IκB-α, TNF-α, IL-6, IL-17, COX-2, MMP-9, and β-actin, and phosphorylation of NF-κB p65 and IκB-α were analysed using Western blotting, which was carried out after 24 h of treatment. Blots are representative of 3 independent experiments. (b) Levels of TNF-α. (c) Levels of MMP-9. Levels of TNF-α and MMP-9 in cultured media were measured by enzyme-linked immunosorbent assay after 12 h or 24 h of treatment. Data represent means ± S.D. values (n = 2). The RAW264.7 cells were pre-treated with GGS (20 μM) and treated with LPS (10 μg/mL) in the presence of IgG or anti-TREM-1 antibody (0.4 μg/mL). α-TREM-1, treated with TREM-1 agonist antibody; GGS, treated with guggulsterone; IgG, immunoglobulin Gisotype control; LPS, treated with lipopolysaccharide. ***p < 0.005 vs. LPS
표 The effects of NF-κB and proteasome inhibition on amplification of TLR4 signalling by TREM-1 stimulation. (a,b) RAW264.7 cells were pre-treated with an NF-κB inhibitor (MG132 treatment, a) and a proteasome inhibitor (Bay 11-708, b) and were then treated with or without LPS (10 μg/mL) in the presence of IgG antibody or anti-TREM-1 antibody (1 μg/mL) for 24 h. Levels of TREM-1, TLR4, IκB-α, TNF-α, IL-17, IL-6, MMP-9, and β-actin and phosphorylation of IκB-α and Akt were analysed using Western blotting. Blots are representative of 3 independent experiments. (c,d) Measurements of NF-κB and AP-1 transactivation. RAW264.7 cells were transfected with reporter vectors, pNF-κB-Luc (c) or pAP-1-Luc (d), pre-treated with GGS, and treated with or without LPS (10 μg/mL) in the presence of IgG antibody or anti-TREM-1 antibody (0.4 μg/mL). Luciferase activity was normalized based on protein amount and the values are indicated by fold change. α-TREM-1, treated with TREM-1 agonist antibody; Bay, treated with Bay 11-7082 (15 μM); GGS, treated with guggulsterone (20 μM); MG132, treated with MG132 (10 μM); IgG, immunoglobulin Gisotype control; LPS, treated with lipopolysaccharide; ns, not significant. Data represent means ± S.D. values (n = 3). *p < 0.05, ***p < 0.005
표 Anti-inflammatory effects of guggulsterone disappeared in in IL-10-,TLR4-, and MyD88-deficient mice. The ability of guggulsterone (GGS) to inhibit colitis induced by intrarectal injection of TNBS was evaluated in wild-type and TLR4-,MyD88-, and IL-10-deficient mice. (a) Body weight changes. (b) Disease activity index (DAI). DAI at day 3 was scored according to the criteria outlined in the Materials and Methods. (c) Hematoxylin and eosin (H GGS, administered with guggulsterone; TNBS, injected with TNBS. Data are expressed as mean ± S.E.M. (n =4). ##p < 0.01 vs. Veh, ###p < 0.005 vs. Veh, *p < 0.05 vs. TNBS+Veh, **p < 0.01 vs.TNBS+Veh, ***p < 0.005 vs. TNBS+Veh
표 Guggulsterone improves histopathology of TNBS-induced colitis by suppressing TREM-1+ M1 macrophages and increasing M2 macrophages. (a) H GGS, administered with guggulsterone; TNBS, injected with TNBS. #p < 0.05 vs. Veh, ##p < 0.01 vs. Veh, *p < 0.05 vs. TNBS+Veh, **p < 0.01 vs. TNBS+Veh
표 M2 polarisation of mouse bone marrow-derived macrophages by guggulsterone requires IL10 and partly TLR4 signalling. Bone marrow-derived macrophages (BMDMs) were isolated from wild-type (WT), Ill0-/-,Tlr4-/-, and MyD88-/- mice, and were differentiated into BMDMs for 1 week. BMDMs were pretreated with GGS and treated with LPS in the presence or absence of anti-TREM-1 antibody for 4h (a-c) or 24 h (d). Total RNA was isolated, and quantitative real-time PCR was performed using M1 markers (a: Tnfa, Trem1) and M2 markers (b: Cd206; c: Il10). Data represent means ± S.D. (n = 3–4). (d) Immunofluorescent staining of IL-10 (red) in BMDMs isolated from wild-type mice (left) and Myd88-deficient mice (right). DAPI (blue) staining shows the nuclei. Figures are representative of 2 independent experiments (original magnification ×400). Veh, treated with vehicle; GGS, treated with guggulsterone (20 μM); LPS, treated with LPS (100 ng/mL); α-TREM-1, treated with TREM-1 agonist antibody (0.4 μg/mL); IgG, immunoglobulin G isotype control (0.4 μg/mL). #p < 0.05 vs. WT, ##p < 0.01 vs. WT, ###p < 0.005 vs. WT,*p < 0.05 vs. LPS,***p < 0.005 vs. LPS
표 Guggulsterone promotes wound healing by modulating differentiation of human monocytes and macrophages. (a) GGS increases CD206+ cells in human monocytes. Monocytes from the peripheral blood mononuclear cells (PBMCs) of healthy subjects were treated with or without LPS for 4 h after GGS pretreatment for 1 h and stained with immunofluorescent antibodies against CD206 (Alexa 555 stained), and calculated as the CD206+percent of total cells (DAPI stained). Datar epresent means ±S.D. (n=2). (b) GGS induces M2 polarisation of human monocyte-derived macrophages. Differentiated macrophages in M1 conditions were generated by treating monocytes isolated from PBMCs of healthy subjects with granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL) for 7 days followed by treatment with LPS for 4 h after GGS pretreatment for 1 h. Total RNA was isolated, and quantitative real-time PCR was performed using M1 (TNFA) and M2 markers (CD206 and IL10). Data represent means ± S.D. (n = 3–4). (c–d) GGS enhances wound healing by inflammatory bowel disease (IBD) patient-derived macrophages. Macrophages differentiated in M1 conditions by treating monocytes isolated from the PBMCs of IBD patients using granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL) for 7 days were co-cultured with scratched HT-29 cells and used in wound healing assays. (c) Representative images for wound closure (original magnification ×100). (d)Comparison of wound closure. Data represent means ± S.D (n = 4). Veh, treated with vehicle; GGS, treated with guggulsterone (20 μM); LPS, treated with lipopolysaccharide (100 ng/mL); α-TREM-1, treated with TREM-1 agonist antibody (0.4 μg/mL); IgG,immunoglobulin G isotype control (0.4 μg/mL). ##p < 0.01 vs. WT, ###p < 0.005 vs. WT,*p < 0.05 vs. LPS, **p < 0.01 vs. LPS, ***p < 0.005 vs. LPS
표 Guggulsterone modulates mRNA expression of macrophage M1 and M2 markers in inflamed colon tissues. Mouse colons were homogenized, total RNA was isolated, and quantitative real-time PCR was performed using (a) Tlr4, Irak1, and Il17 as M1 markers and (b) Pparg as a M2 marker. (c) Flow cytometric analysis of laminar propria macrophages in the colon. Arrows indicate single cell and F4/80+CD11b+cell gating flow. Veh, administered with vehicle; GGS, administered with guggulsterone; TNBS, injected with TNBS. Data are expressed as the mean ± S.D. (n = 4)
표 Guggulsterone suppresses M1 markers but induces M2 markers in RAW264.7 cells. RAW264.7 cells were pre-treated with GGS and treated with LPS in the presence or absence of anti-TREM-1 antibody. Total RNA was isolated, and quantitative real-time PCR was performed using M1 markers (a: Tnfa, iNos, Il1b) and M2 markers (b: Cd206, Arg1, Tgfb1, Pparg; c: Il10, and Il10rb). GGS, treated with guggulsterone (20μM); LPS, treated with lipopolysaccharide (10 μg/mL); α-TREM-1, treated with TREM-1 agonist antibody (0.4 μg/mL); IgG, immunoglobulin G isotype control (0.4 μg/mL); ns, not significant. Data represent the means ± S.D. (n = 3–4). *p < 0.05 vs. LPS, **p < 0.01 vs. LPS, ***p < 0.005 vs. LPS
표 Guggulsterone suppresses M1 markers but induces M2 markers in mouse bone marrow-derived macrophages. Bone marrow-derived macrophages (BMDMs) were isolated from wild type (WT), Il0-/-, Tlr4-/-, and MyD88-/-mice. BMDMs were pre-treated with GGS and treated with LPS in the presence or absence of anti-TREM-1 antibody for 4h. Total RNA was isolated, and quantitative real-time PCR was performed using M1 markers, iNos (a), Tlr4 and Il1b (b), and Irak1 (c), and M2 markers, Arg1and Pparg (d). GGS, treated with guggulsterone (20 μM); LPS, treated with LPS (100ng/mL); α-TREM-1, treated with TREM-1 agonist antibody (0.4 μg/mL); IgG, immunoglobulin G isotype control (0.4 μg/mL). Data represent the means ± S.D. (n = 3–4). #p < 0.05 vs. WT, ##p < 0.01 vs. WT, ###p < 0.005 vs. WT,***p < 0.005 vs. LPS
표 GGuggulsterone suppresses M1 markers but induces M2 markers in human monocytes. Representative images of CD206+cells (original magnification×200). Differentiated macrophages in M1 conditions were generated by treating monocytes isolated from PBMC of healthy subjects with granulocyte macrophage-colony stimulating factor (GM-CSF, 100ng/mL) for 7days. Macrophages were treated with LPS for 4 h after GGS pretreatment for 1 h and stained with immunofluorescent antibodies against CD206 (red). Figures are representative of 2 independent experiments. DAPI (blue) staining shows the nuclei. Original magnification: ×40. Veh, treated with vehicle; GGS, treated with guggulsterone (20 μM); LPS, treated with lipopolysaccharide (100 ng/mL)
표 Guggulsterone suppresses M1 markers, but induces M2 markers in monocyte-derived macrophages. Differentiated macrophages into M1 conditions were generated by treating monocytes isolated from PBMC of inflammatory bowel disease (IBD) patients (a) or healthy subjects (b) using granulocyte macrophage-colony stimulating factor (GM-CSF, 100 ng/mL) for 7 days followed by treatment with LPS for 4 h with or without TREM-1 agonist antibody after GGS pretreatment for 1 h. (a) Morphology changes of macrophages derived from PBMCs of IBD patients. Arrow indicates M1-like morphology of macrophages. Figures are representative of 2 independent experiments. Original magnification: ×400. (b) mRNA expression changes of macrophages derived from PBMC of healthy subjects. Total RNA was isolated, and quantitative real-time PCR was performed using M1 markers (TREM1, iNOS). Data represent the means ± S.D. (n = 3–4). Detailed methods are described in the Methods. Veh, treated with vehicle; GGS, treated with guggulsterone (20 μM); LPS, treated with lipopolysaccharide (100 ng/mL); α-TREM-1, treated with TREM-1 agonist antibody (0.4μg/mL)
표 Guggulsterone promotes wound healing by M2 polarization of macrophage in IBD patients. Differentiated macrophages in M1 conditions were generated by treating monocytes isolated from PBMC of IBD patients with granulocyte macrophage-colony stimulating factor (GM-CSF, 100 ng/mL) for 7 days followed by treatment with LPS for 4 h after GGS pretreatment for 1h. (a) CD206+cellstainin HT-29 monolayer.(b)CD206+cells tainin the wound. After wound healing assay, cells were stained with immunofluorescent antibodies against CD206 (Alexa555 stained) and DAPI (blue). Arrow indicates M1-like morphology of macrophages. Figures are representative of 2 independent experiments. Original magnification: ×200. Veh, treated with vehicle; GGS,treated with guggulsterone (20μM); LPS, treated with lipopolysaccharide (100 ng/mL); α -TREM-1, treated with TREM-1 agonist antibody (0.4 μg/mL); IgG, immunoglobulin G isotype control (0.4 μg/mL)
표 UPGMA Dendrogram of IBD. BD: Behçet disease. UPGMA: Unweighted Pair Group Method with Arithmetic
표 Taxonomic composition of IBD from Phylum. BD: Behçet disease
표 Taxonomic composition of IBD from Class. BD: Behçet disease
표 Diversity indices of IBD BD: Behçet disease

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