Colonoids From Patients With Pediatric Inflammatory Bowel Disease Exhibit Decreased Growth Associated With Inflammation Severity and Durable Upregulation of Antigen Presentation Genes.
- 作者列表："Kelsen JR","Dawany N","Conrad MA","Karakasheva TA","Maurer K","Wei JM","Uman S","Dent MH","Behera R","Bryant LM","Ma X","Moreira L","Chatterji P","Shraim R","Merz A","Mizuno R","Simon LA","Muir AB","Giraudo C","Behrens EM","Whelan KA","Devoto M","Russo PA","Andres SF","Sullivan KE","Hamilton KE
BACKGROUND:Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids. METHODS:We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture. RESULTS:Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD. CONCLUSIONS:We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.
背景: 确定上皮细胞对炎症性肠病 (IBD) 的贡献对于开发急需的屏障修复疗法至关重要。极早发 (VEO)-IBD 患儿比大龄儿童和成人 IBD 患儿有更广泛、严重和难治的疾病，在某些情况下，屏障功能有缺陷。因此，我们使用三维、活检来源的类器官评价了儿童 IBD (VEO 和老年发病) 和非 IBD 上皮之间的功能和转录组差异。 方法: 我们测量了患者肠样 (回肠) 和结肠 (结肠) 系的生长效率，与组织病理学和临床参数相关。我们对患者结肠进行了 RNA 测序，并在多次传代后进行了流式细胞术，以评价培养中持续存在的变化。 结果: 与非 IBD 对照组相比，IBD 患儿的肠道和结肠显示与组织学炎症相关的生长减少。我们观察到儿童 IBD 患者结肠中 LYZ 表达增加，这在成人 IBD 患者中已有报道。我们还观察到抗原呈递基因 HLA-DRB1 和 HLA-DRA 的上调，这些基因在儿童 IBD 患者长期传代后持续存在。 结论: 我们首次对 VEO-IBD 患者和发病年龄较大的儿童 IBD 患者的肠道和结肠进行了功能评价，其中一部分表现出生长不良。患者结肠中增强的、持久的上皮抗原呈递基因表达支持上皮细胞内在差异可能导致 IBD 发病的观点。
METHODS:Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal death worldwide. After the pathogen is orally ingested, it invades and replicates within the colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are lacking. Here, we found that orogastric inoculation of infant rabbits with Shigella flexneri resulted in diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely arise through S. flexneri spreading from cell to cell. Robust S. flexneri intestinal colonization, invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, a factor required for bacterial spreading and adhesion in vitro Expression of the proinflammatory chemokine interleukin 8 (IL-8), detected with in situ mRNA labeling, was higher in animals infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting that epithelial invasion promotes expression of this chemokine. Collectively, our findings suggest that oral infection of infant rabbits offers a useful experimental model for studies of the pathogenesis of shigellosis and for testing of new therapeutics.IMPORTANCEShigella species are the leading bacterial cause of diarrheal death globally. The pathogen causes bacillary dysentery, a bloody diarrheal disease characterized by damage to the colonic mucosa and is usually spread through the fecal-oral route. Small animal models of shigellosis that rely on the oral route of infection are lacking. Here, we found that orogastric inoculation of infant rabbits with S. flexneri led to a diarrheal disease and colonic pathology reminiscent of human shigellosis. Diarrhea, intestinal colonization, and pathology in this model were dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis of shigellosis and to develop and test new therapeutics.
METHODS:PURPOSE:To quantify the effects of absorbed radiation dose on healthy liver parenchyma following radioembolisation (RE) using [99mTc]TcMebrofenin to analyse both global and regional liver function. METHODS:Patients having RE to treat hepatic disease underwent a [99mTc]TcMebrofenin hepatobilliary scintigraphy (HBS) study at both baseline and 8 weeks following treatment. Changes in global liver uptake rate were compared with healthy liver absorbed dose measures derived from the post-treatment 90Y PET/CT, including average dose, minimum dose to 70% of the volume (D70) and volume receiving at least 50 Gy (V50). Changes in functional burden associated with treatment and spared liver volumes in patients receiving lobar RE were also assessed, as were changes experienced by regional volumes corresponding to various dose ranges. Standard liver function pathology tests (LFTs) (bilirubin, albumin, ALP, AST, ALT and GGT) were examined for changes between baseline and post-treatment. RESULTS:Thirty-five patients were included in the study, of which, 9 had lobar treatment. A significant linear correlation was found between both baseline global liver uptake rate (negative) and D70 with change in global liver uptake rate. Patients undergoing lobar treatments demonstrated a shift in functional burden, and a significant difference was seen between the mean dose corresponding to liver volumes that increased their functional burden (9 Gy) and those that decreased their functional burden (35 Gy). No baseline LFTs predicted a decrease in global liver function; however, D70 demonstrated a linear correlation with changes in bilirubin and GGT. CONCLUSIONS:Given the significant negative relationship between baseline and change in global liver uptake rate, baseline HBS studies should not be used alone to disqualify patients considered for RE. In terms of treatment planning and evaluation, D70 may be the most appropriate metric of dose, with values greater than 15 Gy indicative of a likely drop in global liver function. The evidence of increasing functional burden in spared liver volumes suggests that patients at risk of complications could benefit from a lobar approach to treatment.
METHODS:NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for inflammatory bowel diseases. In this study, we found that Libertellenone M (Lib M), a secondary metabolite from the endophytic fungus Phomopsis sp. S12, has anti-inflammatory potential both in vitro and in vivo. Lib M selectively inhibited the expression of proinflammatory cytokine IL-1β and IL-18 in LPS-activated macrophages. The cleavage of pro-caspase 1 was remarkably reduced by Lib M in macrophages stimulated with three NLRP3 inflammasome activators. Administering Lib M attenuated dextran sulfate sodium-induced experimental acute colitis in mice and significantly reduced the production of these cytokines and cleaved caspase 1 in colon tissues. Apart from inhibition of NLRP3 inflammasome assembly, Lib M also suppressed NF-κB nuclear translocation in macrophages. Taken together, these findings suggest that Lib M-mediated inhibition of NLRP3 inflammasome activation could protect against colitis-like inflammatory diseases, and that this compound derived from a plant-associated fungus might inspire the exploration of novel immunosuppressive agents.