염증성 장질환 모델에서 soluble Siglec-9의 NF-κB 신호전달 차단을 통한 장염 호전 효과

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의학과, 2021. 2. 김주성.Background: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a superfamily of immunoreceptors recognizing sialic acid. Siglec-9 has been shown to mediate inhibitory immune responses. The aim of this study was to evaluate the effect of a soluble form of Siglec-9 (sSiglec-9) on inflamed intestinal epithelial cells (IECs), murine macrophages, and experimental murine colitis models. Methods: COLO 205 human IECs and RAW 264.7 murine macrophages were pretreated with sSiglec-9 and then stimulated with TNF-α or lipopolysaccharides, respectively. The expression of proinflammatory cytokines such as IL-8 and TNF-α was measured using real-time RT-PCR and ELISA. To demonstrate the inhibitory effects of sSiglec-9 on the NF-κB pathway, IκBα phosphorylation/degradation was determined using western blotting and the DNA binding activity of NF-κB was evaluated using an electrophoretic mobility shift assay. Further, mouse models with dextran sulfate sodium-induced acute colitis and piroxicam-induced IL-10-/- chronic colitis were generated. Intraperitoneal injections of sSiglec-9 were performed, and body weight, colon length, and histopathologic findings were examined. Results: sSiglec-9 suppressed IL-8 and TNF-α gene expression in stimulated COLO 205 and RAW 264.7 cells. sSiglec-9 inhibited IκBα phosphorylation/degradation and the DNA binding activity of NF-κB. sSiglec-9 injections significantly ameliorated weight loss, colon shortening, and the severity of intestinal inflammation in acute and chronic colitis mouse models. Conclusion: sSiglec-9 may inhibit NF-κB activation in IECs and macrophages and alleviate experimental colitis in mice, suggesting that sSiglec-9 is a potential therapeutic agent for the treatment of inflammatory bowel disease.배경: 시알산 (Sialic acid)에 결합하는 면역글로불린 유사 렉틴 (Siglec)은 면역세포의 표면에 존재하는 수용체이다. Siglec의 일종인 Siglec-9은 억제 면역 반응을 매개하며 항염증 효과를 가지고 있다. 하지만 Siglec-9의 수용성 형태인 soluble Siglec-9 (sSiglec-9)이 장내 염증반응에 대해 항염증 효과를 나타내는지는 잘 알려져 있지 않다. 본 연구에서는 sSiglec-9이 염증을 유발한 장상피세포, 대식세포에 미치는 영향과 마우스 대장염 모델에 미치는 영향을 평가하고 nuclear factor-kappa B (NF-κB)신호전달체계에 미치는 영향을 알아보고자 하였다. 방법: 인체 장상피세포주인 COLO 205, 쥐의 대식세포주인 RAW 264.7 에 sSiglec-9을 전처치 후 염증을 유발하여 real time RT-PCR, 효소면역측정법을 통해 IL-8, TNF-α 와 같은 염증성 사이토카인의 분비를 측정하였다. sSiglec-9이 NF-κB 신호전달체계에 미치는 영향을 알아보기 위하여 wetern blot과 electrophorectic mobility shift assay 방법을 통해 IκBα 인산화와 분해 정도를 측정하였다. 동물실험으로 Dextran sulfate sodium (DSS)를 경구투여하여 급성 장염을 유도한 마우스 장염 모델과 IL-10-/- 마우스 모델에 piroxicam을 경구투여하여 만성 장염을 유도한 모델에 sSiglec-9 의 효과를 시험하였다. 복강내 주사로 sSiglec-9을 투여하여 대조군과 비교하여 체중변화, 장길이, 병리학적 소견을 평가하였다. 결과: sSiglec-9은 염증을 유발한 COLO 205세포주와 RAW 264.7세포주에서 IL-8, TNF-α의 유전자 발현을 감소시켰다. sSiglec-9은 IκBα 인산화와 분해를 억제하여 NF-κB 활성을 저해하였다. 동물실험에서 sSiglec-9을 복강 내 투여하였을 때 체중감소, 장 길이 단축, 병리학적 염증 소견이 모두 호전되는 양상을 보였다. 이러한 결과는 급성 장염의 예방모델 (DSS-induced acute colitis model)과 만성 장염의 치료모델 (IL-10-/- chronic colitis model)에서 모두 확인되었다. 결론: sSiglec-9은 장상피세포와 대식세포에서 NF-κB 신호전달체계를 억제하며 대장염을 유발한 마우스에서 장염 억제 효과를 보였다. 향후 sSiglec-9이 염증성 장질환의 치료에 활용될 수 있는 새로운 후보 물질로 개발될 수 있을 것으로 기대된다.Abstract ...................................................................... ⅰ - ⅱ Contents ..............................................................................ⅲ List of figures .........................................................ⅳ - ⅵ Introduction ......................................................................... 1 Material and Methods ........................................................ 3 Results................................................................................... 8 Discussion .......................................................................... 22 References .......................................................................... 26 Abstract in Korean ........................................................... 33Docto

The Prognostic Value of Residual Nonrectal Inflammation in Ulcerative Colitis

ope

Extracellular Vesicles Derived from Kefir Grain Lactobacillus Ameliorate Intestinal Inflammation via Regulation of Proinflammatory Pathway and Tight Junction Integrity

The aim of this study was to demonstrate the anti-inflammatory effect of Lactobacillus kefirgranum PRCC-1301-derived extracellular vesicles (PRCC-1301 EVs) on intestinal inflammation and intestinal barrier function. Human intestinal epithelial cells (IECs) Caco-2 were treated with PRCC-1301 EVs and then stimulated with dextran sulfate sodium (DSS). Real-time RT-PCR revealed that PRCC-1301 EVs inhibited the expression of pro-inflammatory cytokines in Caco-2 cells. PRCC-1301 EVs enhanced intestinal barrier function by maintaining intestinal cell integrity and the tight junction. Loss of Zo-1, claudin-1, and occludin in Caco-2 cells and the colitis tissues was recovered after PRCC-1301 EVs treatment, as evidenced by immunofluorescence analysis. Acute murine colitis was induced using 4% DSS and chronic colitis was generated in piroxicam-treated IL-10-/- mice. PRCC-1301 EVs attenuated body weight loss, colon shortening, and histological damage in acute and chronic colitis models in mice. Immunohistochemistry revealed that phosphorylated NF-κB p65 and IκBα were reduced in the colon tissue sections treated with PRCC-1301 EVs. Our results suggest that PRCC-1301 EVs may have an anti-inflammatory effect on colitis by inhibiting the NF-κB pathway and improving intestinal barrier function.ope

간이식 환자에서 대장 용종 및 대장암의 유병률 및 특성

학위논문 (석사)-- 서울대학교 대학원 : 임상의과학과, 2017. 2. 김병관.배경: 장기 이식 환자는 이식 후 장기간의 면역 억제제 사용으로 인해 이식 후 악성 종양 발생의 위험도가 높다. 본 연구는 간 이식 환자에서 이식 후 진행성 대장 종양 및 대장암의 유병률이 증가하는 지의 여부와 면역 억제제가 미치는 영향을 평가하기 위하여 시행되었다. 방법: 1991년 1월부터 2012년 12월까지 간 이식을 시행한 환자 중 이식 후 대장 내시경을 시행한 총 348명을 대상으로 하였다. 연령, 성별이 일치하는 대조군을 선정하여 양 군의 진행성 대장 종양 및 대장암의 발생률을 비교하였다. 결과: 간 이식 후 대장 내시경을 시행한 총 348명 중 17명 (4.9%) 에서 진행성 대장종양이 확인되었고, 그 중 9명 (2.6%) 은 대장암이었다. 대조군에 비해 진행성 대장 종양의 유병률은 3.6배 높았으며 (승산 비, 3.57895% 신뢰구간, 1.578-8.115P = 0.001) 대장암의 유병률은 간 이식 환자군에서 대조군에 비해 8.4배 더 높았다 (승산 비, 8.41695% 신뢰구간, 1.808-39.172P = 0.001). 결론: 간 이식 환자에서 진행성 대장 종양의 유병률은 대조군에 비해 높았다. 그러므로 본 연구진은 간 이식 후 대장 내시경을 이용한 감시를 권장하며 면역 억제제 치료가 대장암으로의 악성화를 촉진할 가능성이 있음을 제시한다.Background and Aim: Liver transplant patients are in high risk of malignancy because of the prolonged immunosuppression after transplantation. The aim of this study was to determine whether the prevalence of advanced colorectal neoplasia increased in liver transplant recipients and to define the effect of immunosuppression on the advanced colorectal neoplasia. Method: Our study consisted of 348 liver transplant patients who underwent a colonoscopy at Seoul National University Hospital from 1991 to 2012. Age- and sex-matched controls were identified from a population of asymptomatic individuals. Results: Of the 348 patients (Median age, 58male gender, 79.9%), seventeen (4.9%) patients had advanced colorectal neoplasms including colorectal cancers (9 patients, 2.6%) after liver transplantation. The odds of advanced colorectal neoplasia occurring in transplant patients were 3.6 times greater than in controls (OR 3.57895% CI 1.578-8.115P = 0.001). The risk of developing colon cancer in transplant patients was 8.4 times greater than in controls (OR 8.41695% CI 1.808-39.172P = 0.001). However, there was no significant difference in the prevalence of non-advanced adenoma between the two groups. Conclusions: Liver transplant patients were in high risk of colorectal cancer. Therefore, colonoscopy surveillance after liver transplantation is recommended. Immunosuppressive therapy could facilitate colorectal cancer carcinogenesis.Introduction 1 Material and Methods 3 Results 7 Discussion 23 References 28 Abstract in Korean 32Maste

Anemia is associated with the risk of Crohn's disease, not ulcerative colitis: A nationwide population-based cohort study

Anemia is a common manifestation of inflammatory bowel disease (IBD), but it remains unclear whether anemia is associated with the development of IBD. We assessed the risk of developing IBD in anemic patients, and stratified the results with respect to their hemoglobin concentrations. A population-based study was conducted using the National Healthcare Insurance Service database in South Korea. We included individuals over 20 years' old who participated in the national health screening program in 2009 (n = 9,962,064). Anemia was defined as a hemoglobin level less than 13 g/dL in men and less than 12 g/dL in women. We compared the rate of newly diagnosed IBD in anemic patients and non-anemic individuals. Newly diagnosed IBD was identified using both the ICD-10 medical code and specialized V codes for rare intractable diseases in South Korea. During the mean follow-up period of 7.3 years, the incidences of CD and UC in anemic patients were 2.89 and 6.88 per 100,000 person-years, respectively. The risk of CD was significantly higher in anemic patients than in non-anemic individuals [adjusted hazard ratio (aHR), 2.084; 95% confidence interval (CI), 1.769-2.455]. The risk of CD development was inversely proportional to the hemoglobin concentration. A J-curve relationship was observed between age and the risk of CD in anemic patients. The risk of CD in male anemic patients was significantly higher than that in female anemic patients (aHR, 1.432 vs. 1.240, respectively). By contrast, there was no statistically significant difference in the risk of developing UC in anemic and non-anemic individuals (aHR, 0.972; 95% CI, 0.880-1.073). This work indicates that anemia is related to the development of CD, and this risk was inversely proportional to the hemoglobin concentration.ope

Periodontitis combined with smoking increases risk of the ulcerative colitis: A national cohort study

Background: Periodontitis is a chronic inflammation of periodontal tissues. The effect of periodontitis on the development of inflammatory bowel disease (IBD) remains unclear. Aim: To assessed the risk of IBD among patients with periodontitis, and the risk factors for IBD related to periodontitis. Methods: A nationwide population-based cohort study was performed using claims data from the Korean National Healthcare Insurance Service. In total, 9950548 individuals aged ≥ 20 years who underwent national health screening in 2009 were included. Newly diagnosed IBD [Crohn's disease (CD), ulcerative colitis (UC)] using the International Classification of Disease 10th revision and rare intractable disease codes, was compared between the periodontitis and non-periodontitis groups until 2017. Results: A total of 1092825 individuals (11.0%) had periodontitis. Periodontitis was significantly associated with older age, male gender, higher body mass index, quitting smoking, not drinking alcohol, and regular exercise. The mean age was 51.4 ± 12.9 years in the periodontitis group and 46.6 ± 14.2 years in the non-periodontitis group (P < 0.01), respectively. The mean body mass index was 23.9 ± 3.1 and 23.7 ± 3.2 in the periodontitis and non-periodontitis groups, respectively (P < 0.01). Men were 604307 (55.3%) and 4844383 (54.7%) in the periodontitis and non-periodontitis groups, respectively. The mean follow-up duration was 7.26 years. Individuals with periodontitis had a significantly higher risk of UC than those without periodontitis [adjusted hazard ratio: 1.091; 95% confidence interval (CI): 1.008-1.182], but not CD (adjusted hazard ratio: 0.879; 95% confidence interval: 0.731-1.057). The risks for UC were significant in the subgroups of age ≥ 65 years, male gender, alcohol drinker, current smoker, and reduced physical activity. Current smokers aged ≥ 65 years with periodontitis were at a 1.9-fold increased risk of UC than non-smokers aged ≥ 65 years without periodontitis. Conclusion: Periodontitis was significantly associated with the risk of developing UC, but not CD, particularly in current smokers aged ≥ 65 years.ope

Development of a Clinical and Genetic Prediction Model for Early Intestinal Resection in Patients with Crohn's Disease: Results from the IMPACT Study

Early intestinal resection in patients with Crohn's disease (CD) is necessary due to a severe and complicating disease course. Herein, we aim to predict which patients with CD need early intestinal resection within 3 years of diagnosis, according to a tree-based machine learning technique. The single-nucleotide polymorphism (SNP) genotype data for 337 CD patients recruited from 15 hospitals were typed using the Korea Biobank Array. For external validation, an additional 126 CD patients were genotyped. The predictive model was trained using the 102 candidate SNPs and seven sets of clinical information (age, sex, cigarette smoking, disease location, disease behavior, upper gastrointestinal involvement, and perianal disease) by employing a tree-based machine learning method (CatBoost). The importance of each feature was measured using the Shapley Additive Explanations (SHAP) model. The final model comprised two clinical parameters (age and disease behavior) and four SNPs (rs28785174, rs60532570, rs13056955, and rs7660164). The combined clinical-genetic model predicted early surgery more accurately than a clinical-only model in both internal (area under the receiver operating characteristic (AUROC), 0.878 vs. 0.782; n = 51; p < 0.001) and external validation (AUROC, 0.836 vs. 0.805; n = 126; p < 0.001). Identification of genetic polymorphisms and clinical features enhanced the prediction of early intestinal resection in patients with CD.ope

Comparative effectiveness of second-line biological therapies for ulcerative colitis and Crohn's disease in patients with prior failure of anti-tumour necrosis factor treatment

Background: Therapeutic options for inflammatory bowel disease (IBD) have increased since the introduction of tumour necrosis factor (TNF) inhibitors a few decades ago. However, direct comparisons of the effectiveness of second-line biological agents in patients with ulcerative colitis (UC) and Crohn's disease (CD) are lacking. Methods: Patients with UC or CD who experienced anti-TNF treatment failure and subsequently used vedolizumab, ustekinumab, or tofacitinib as a second-line drug were retrospectively recruited. The primary outcomes were the clinical remission rate at week 16 and the cumulative relapse rate 48 weeks after receiving induction therapy. Results: A total of 94 patients with UC or CD experienced anti-TNF treatment failure and received vedolizumab (UC: 37; CD: 28), ustekinumab (CD: 16), or tofacitinib (UC: 13). The clinical remission rates were not significantly different between the vedolizumab and tofacitinib groups in UC patients (56.8% vs. 46.2%, p = 0.509). In CD patients, the clinical remission rates were not significantly different between the vedolizumab and ustekinumab groups (53.6% vs. 50.0%, p = 0.820). Moreover, the cumulative rates of clinical relapse were not significantly different between the vedolizumab and tofacitinib groups in UC patients and between the vedolizumab and ustekinumab groups in CD patients (p = 0.396 and p = 0.692, respectively). Safety profiles were also similar among the treatment groups in both UC and CD patients. Conclusions: After prior anti-TNF therapy failure, vedolizumab and tofacitinib in UC patients and vedolizumab and ustekinumab in CD patients were not significantly different in terms of the efficacy in inducing and maintaining a clinical response.ope

Tumor-Suppressive Effect of Metformin via the Regulation of M2 Macrophages and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment of Colorectal Cancer

Myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the tumor microenvironment contribute to tumor progression by inducing immune tolerance to tumor antigens and cancer cells. Metformin, one of the most common diabetes drugs, has shown anti-inflammatory and anti-tumor effects. However, the effects of metformin on inflammatory cells of the tumor microenvironment and its underlying mechanisms remain unclarified. In this study, we investigated the effect of metformin on M2 macrophages and MDSCs using monocyte THP-1 cells and a dextran sodium sulfate (DSS)-treated ApcMin/+ mouse model of colon cancer. Metformin decreased the fractions of MDSCs expressing CD33 and arginase, as well as M2 macrophages expressing CD206 and CD163. The inhibitory effect of metformin and rapamycin on MDSCs and M2 macrophages was reversed by the co-treatment of Compound C (an AMP-activated protein kinase (AMPK) inhibitor) or mevalonate. To examine the effect of protein prenylation and cholesterol synthesis (the final steps of the mevalonate pathway) on the MDSC and M2 macrophage populations, we used respective inhibitors (YM53601; SQLE inhibitor, FTI-277; farnesyl transferase inhibitor, GGTI-298; geranylgeranyl transferase inhibitor) and found that the MDSC and M2 populations were suppressed by the protein prenylation inhibitors. In the DSS-treated ApcMin/+ mouse colon cancer model, metformin reduced the number and volume of colorectal tumors with decreased populations of MDSCs and M2 macrophages in the tumor microenvironment. In conclusion, the inhibitory effect of metformin on MDSCs and M2 macrophages in the tumor microenvironment of colon cancers is mediated by AMPK activation and subsequent mTOR inhibition, leading to the downregulation of the mevalonate pathway.ope

Rebleeding Rate and Risk Factors for Rebleeding after Device-Assisted Enteroscopy in Patients with Obscure Gastrointestinal Bleeding: A KASID Multicenter Study

Introduction: The impact of device-assisted enteroscopy (DAE) on long-term rebleeding in patients with obscure gastrointestinal bleeding (OGIB) exhibiting detectable small-bowel lesions remains unclear. We investigated the long-term rebleeding rate and predictive factors for DAE in patients with OGIB. Method: Patients with OGIB with small bowel lesions detected through DAE were enrolled at three Korean tertiary hospitals. Predictive risk factors associated with rebleeding were analyzed using the Cox regression analysis. Results: From April 2008 to April 2021, 141 patients were enrolled, including 38 patients (27.0%) with rebleeding. The rebleeding rates at 1, 2, and 3 years were 25.0%, 29.6%, and 31.1%, respectively. The Cox regression analysis revealed that multiple small-bowel lesions (hazard ratio [HR]: 2.551, 95% confidence interval [CI]: 1.157-5.627, p = 0.020), the need for more than five packed red blood cells (RBC) transfusions (HR: 2.704, 95% CI: 1.412-5.181, p = 0.003), and ulcerative lesions (HR: 1.992, 95% CI: 1.037-3.826, p = 0.039) were positively associated with rebleeding. Therapeutic interventions for patients with detectable lesions, overt bleeding (vs. occult bleeding), comorbidities, and medications were not associated with rebleeding. Conclusion: More than 25% of patients with OGIB having detectable small-bowel lesions had rebleeding. Patients with multiple lesions, a requirement of more than five packed RBC transfusions, and ulcerative lesions were associated with a higher risk of rebleeding.ope