Liquid Biopsy for Colorectal Adenoma: Is the Exosomal miRNA Derived From Organoid a Potential Diagnostic Biomarker?

INTRODUCTION: MicroRNAs (miRNAs) can serve as tumor biomarkers; however, their role in evaluating colorectal adenoma (CRA) is unclear. Recently, the organoid culture system enabled long-term expansion of human colon epithelium. This study aimed to examine the potential of exosomal miRNAs extracted from CRA organoids as biomarkers in the clinical liquid biopsy CRA test. METHODS: We established organoid cultures from normal colon and CRA using resected specimens. Exosomes were isolated from the conditioned medium organoids. MiRNAs were isolated from the exosomes, and their expression profiles were compared using microarray analysis. To identify miRNA candidates for liquid biopsy, we prospectively compared changes in their expression in serum and exosomes before and after endoscopic resection in 26 patients with CRA. RESULTS: Seven exosomal miRNAs were overexpressed in CRA organoids: miR-4323, miR-4284, miR-1268a, miR-1290, miR-6766-3p, miR-21-5p, and miR-1246. The expression levels of 4 exosomal miRNAs (miR-4323, miR-4284, miR-1290, and miR-1246) and 2 serum miRNAs (miR-1290 and miR-1246) were significantly lower in posttreatment sera. The combined expression of 4 exosomal miRNAs could identify both CRA and large-size (>12.6 cm2) CRA with respective areas under the curve of 0.698 (95% confidence interval [CI] = 0.536-0.823) and 0.834 (95% CI = 0.660-0.929). Combinations of 2-serum miRNA expression values could identify both CRA and large-size CRA with respective area under the curves of 0.691 (95% CI = 0.528-0.817) and 0.834 (95% CI = 0.628-0.938). DISCUSSION: We found that exosomal miRNAs derived from the CRA organoid culture could be potential diagnostic biomarkers for CRA.

Localized intestinal AL amyloidosis detected as bright green using autofluorescence endoscopy.

Amyloidosis is classifiable as systemic, with amyloid deposition in organs throughout the body, or localized, involving only one organ. Amyloidosis localized in the intestinal tract is rare. This report describes three cases of localized AL amyloidosis in the intestinal tract and presents their clinical characteristics, endoscopic findings, and prognoses. All three cases were asymptomatic, and were found accidentally during endoscopy for closer examination after a positive fecal occult blood test. Endoscopic findings included patchy redness and meandering dilated vessels of the lesion. Using autofluorescence (AFI) endoscopy, the lesion of amyloid deposition was enhanced as bright green. We used fluorescence microscopy to observe unstained specimens obtained from an amyloid deposition site with excitation light. Autofluorescence was detected with the broad excitation wavelength at amyloid deposition lesion sites of the specimen. Results revealed that AL amyloid has autofluorescence that engenders its detection by AFI endoscopy as bright green. In none of the three cases was systemic amyloidosis or organ failure observed. The long-term course of all the cases was favorable.

Comprehensive Analysis of microRNA Profiles in Organoids Derived from Human Colorectal Adenoma and Cancer.

INTRODUCTION: Exosomes are membrane-enclosed nanovesicles, which are increasingly being recognized as important cell communication components for their role in transmitting microRNAs (miRNAs). No previous study has addressed the exosomal miRNA profile in colorectal adenomas (CRAs) because the long-term culture of CRA is challenging. This study aimed to identify the miRNA signature in organoid exosomes derived from human CRA and colorectal cancer (CRC) samples. METHODS: Organoid cultures were developed from resected colorectal tissues of patients with CRA or CRC undergoing surgery or endoscopic mucosal resection. Exosomes were prepared from the conditioned medium of the organoids. miRNAs were prepared from the exosomes and their source organoids. The miRNA expression profiles were compared using microarray analysis. The impact of alteration of miRNA expression on cell proliferation was examined using miRNA mimics or inhibitors in HT-29 human CRC cells. RESULTS: We established 6 organoid lines from CRC and 8 organoid lines from CRA. Exosomal miRNA signatures were different between the organoids derived from CRA and CRC. Both exosomal and cellular miR-1246 expressions were upregulated in CRC-derived organoids compared to their expression in CRA-derived organoids. Alteration of miR-1246 expression by the miR-1246 mimic or inhibitor increased or decreased cell proliferation in HT-29 cells, respectively. CONCLUSIONS: We report for the first time the miRNA profiles of exosomes in CRA- and CRC-derived organoids. The upregulation of miR-1246 might play a role in increased cell proliferation in the process of CRA-carcinoma transition.

Factors Associated with Fibrosis during Colorectal Endoscopic Submucosal Dissection: Does Pretreatment Biopsy Potentially Elicit Submucosal Fibrosis and Affect Endoscopic Submucosal Dissection Outcomes?

BACKGROUND: Submucosal fibrosis observed during colorectal endoscopic submucosal dissection (ESD) is an important factor related to incomplete resection. Biopsy is generally accepted as having the potential to elicit submucosal fibrosis, but few reports have presented definitive proof. This study investigated the relation between submucosal fibrosis and colorectal ESD outcomes and assessed factors related to fibrosis, including pretreatment biopsy. METHODS: After reviewing 369 records of colorectal ESD performed between January 2011 and December 2016, we assessed the relation between fibrosis and ESD outcomes. Multiple logistic regression analysis revealed fibrosis risk factors. RESULTS: Severe fibrosis was related significantly to ESD outcomes such as the mean procedure time (p < 0.001), en bloc resection rate (p < 0.001), and R0 resection rate (p = 0.011). Multivariate analyses indicated residual lesions (ORs 175.4, p < 0.001), pretreatment biopsy (ORs 8.30, p = 0.002), nongranular-type laterally spreading tumors (LST-NG; ORs 5.86, p = 0.025), and invasive carcinoma (ORs 5.83, p = 0.03) as independent risk factors of severe fibrosis. In each macroscopic type, LST-NG was more strongly related to fibrosis induced by pretreatment than granular-type laterally spreading tumors with adjust ORs of 50.8 and 4.69. CONCLUSIONS: Pretreatment biopsy causes submucosal fibrosis resulting in prolonged procedure times and incomplete resection. These findings suggest important benefits of avoiding biopsy before ESD.

An Integrated Genomic and Transcriptomic Analysis Reveals Candidates of Susceptibility Genes for Crohn's Disease in Japanese Populations.

Expression quantitative trait locus (eQTL) analyses have enabled us to predict the function of disease susceptibility SNPs. However, eQTL for the effector memory T cells (TEM) located in the lamina propria mononuclear cells (LPMCs), which play an important role in Crohn's disease (CD), are not yet available. Thus, we conducted RNA sequencing and eQTL analyses of TEM cells located in the LPMCs from IBD patients (n = 20). Genome-wide association study (GWAS) was performed using genotyping data of 713 Japanese CD patients and 2,063 controls. We compared the results of GWAS and eQTL of TEM, and also performed a transcriptome-wide association study using eQTL from Genotype Tissue Expression project. By eQTL analyses of TEM, correlations of possible candidates were confirmed in 22,632 pairs and 2,463 genes. Among these candidates, 19 SNPs which showed significant correlation with tenascin-XA (TNXA) expression were significantly associated with CD in GWAS. By TWAS, TNFSF15 (FDR = 1.35e-13) in whole blood, ERV3-1 (FDR = 2.18e-2) in lymphocytes, and ZNF713 (FDR = 3.04e-2) in the sigmoid colon was significantly associated with CD. By conducting integration analyses using GWAS and eQTL data, we confirmed multiple gene transcripts are involved in the development of CD.

Long-Term Prognosis of Japanese Patients with Crohn's Disease Treated by Switching Anti-Tumor Necrosis Factor-α Antibodies.

INTRODUCTION: The long-term prognosis of Japanese patients with Crohn's disease (CD) treated by switching anti-tumor necrosis factor-α (anti-TNFα) antibodies remains unclear. OBJECTIVE: This study aimed to clarify the long-term prognosis and clinical factors that affect the long-term prognosis and outcomes of such patients. METHODS: This retrospective, observational, single-center cohort study analyzed Japanese patients with CD treated by switching between infliximab and adalimumab in the Tohoku University Hospital between March 2003 and December 2017. Cumulative relapse-free survival and cumulative surgery-free survival rates were analyzed using the Kaplan-Meier method. Clinical factors that affected the long-term outcomes were identified using both a log-rank test and the Cox proportional hazards model. RESULTS: The cumulative relapse-free survival rates were 68.6, 33.7, and 22.9% at 1, 3, and 5 years, respectively. The surgery-free survival rates were 91.7, 75.7, and 57.4% at 1, 3, and 5 years, respectively. The cumulative relapse-free survival rate was significantly higher in the group with ileal lesions (HR = 0.12; 95% CI 0.0066-0.64, p = 0.0086), stricture (HR = 0.24; 95% CI 0.0094-0.59, p = 0.0021), and a penetrating type (HR = 0.34; 95% CI 0.14-0.84, p = 0.020). Intolerance (HR = 0.29; 95% CI 0.12-0.63, p = 0.0013) and switching after surgery (HR = 0.41; 95% CI 0.17-0.87, p = 0.019) were clinical factors that reduced the risk of recurrence. The cumulative surgery-free survival rate was significantly higher in the group that switched after surgery (HR = 0.28; 95% CI 0.074-0.91, p = 0.034) and used concomitant thiopurine (HR = 0.32; 95% CI 0.10-0.90, p = 0.030). CONCLUSION: We should clarify the reason for switching anti-TNFα antibodies and investigate bowel complications before switching. Surgical reset of bowel complications including stricture and fistula could reduce the risk of recurrence after switching anti-TNFα antibodies. Concomitant thiopurine administration might reduce the risk of bowel surgery after switching anti-TNFα antibodies.

Identification of two major autoantigens negatively regulating endothelial activation in Takayasu arteritis.

The presence of antiendothelial cell antibodies (AECAs) has been documented in Takayasu arteritis (TAK), a chronic granulomatous vasculitis. Here, we identify cell-surface autoantigens using an expression cloning system. A cDNA library of endothelial cells is retrovirally transfected into a rat myeloma cell line from which AECA-positive clones are sorted with flow cytometry. Four distinct AECA-positive clones are isolated, and endothelial protein C receptor (EPCR) and scavenger receptor class B type 1 (SR-BI) are identified as endothelial autoantigens. Autoantibodies against EPCR and SR-BI are detected in 34.6% and 36.5% of cases, respectively, with minimal overlap (3.8%). Autoantibodies against EPCR are also detected in ulcerative colitis, the frequent comorbidity of TAK. In mechanistic studies, EPCR and SR-BI function as negative regulators of endothelial activation. EPCR has also an effect on human T cells and impair Th17 differentiation. Autoantibodies against EPCR and SR-BI block the functions of their targets, thereby promoting pro-inflammatory phenotype.

Endoscopic radial incision and cutting for Crohn's Disease-associated intestinal stricture: a pilot study.

Background and study aims Intestinal stricture associated with Crohn's disease (CD) is usually treated by endoscopic balloon dilation (EBD) or stricture plasty. Although EBD is effective and safe for such strictures, refractory stricture after EBD poses a problem. Hence, other novel approaches for these refractory strictures are required. On the other hand, the efficacy of radial incision and cutting (RIC) method for esophageal stricture after esophagogastric surgery is reported. In this pilot study, we adopted the RIC technique for five CD patients with refractory intestinal stricture to dilate their strictures. We conducted the RIC procedure using an electric needle knife with a ceramic tip on the top of the needle. Four cases were of anastomotic stricture after ileocecal resection and the remaining one case was of stricture due to mucosal healing. The RIC procedure was successful for all five patients. Average procedure time was 18.6 minutes. One patient developed delayed bleeding after RIC. There were no cases of perforation. RIC could be an alternative therapy for intestinal stricture associated with CD. Further studies should be conducted to clarify its efficacy and safety.

Serum C-reactive protein and albumin are useful biomarkers for tight control management of Crohn's disease in Japan.

Tight control management of Crohn's disease (CD) based on biomarkers is more effective than conventional clinical management; however, fecal calprotectin is not allowed in Asian and some Western countries. To investigate whether tight control management based on readily available serum biomarkers results in better outcomes, we retrospectively reviewed treatment courses of consecutive Japanese CD patients treated with anti-tumor necrosis factor agents between 2003 and 2018. The association between failure of tight control (C-reactive protein (CRP) ≥ 0.5 mg/dL or albumin (Alb) < 3.8 g/dL at week 8 or 24) and subsequent major adverse outcomes (MAOs; hospitalization related to CD worsening, surgery, and discontinuation due to treatment failure) were analyzed. Among 223 patients followed for >8 weeks, 88 patients experienced MAOs. Multivariate analysis identified penetrating type, CRP ≥ 0.5 mg/dL and Alb < 3.8 g/dL at week 8 as independent risk factors (hazard ratios: 2.16, 2.06, and 2.08, respectively). Among 204 patients followed for >24 weeks, 80 patients experienced MAOs. Penetrating type, CRP ≥ 0.5 mg/dL, and Alb < 3.8 g/dL at week 24 were identified as independent risk factors (2.39, 1.90, and 2.20, respectively). Even in settings without fecal calprotectin, tight control management based on serum CRP and Alb may help avoid MAOs.

Ustekinumab treatment for hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a follicular occlusive inflammatory skin disease that occurs in the axilla, groin, buttocks and vulval region. Control of the intractable inflammation is a primary goal of HS treatments. Benefit of anti-tumor necrosis factor (TNF) antibodies against HS have been reported, and adalimumab has been approved for HS in Europe, the USA and Japan. However, the alternative therapies for anti-TNF antibodies have not been established yet. We experienced a case of HS which developed during the infliximab treatment for Crohn's disease (CD) and was well managed by ustekinumab (UST). We reviewed the articles relating to ustekinumab treatments for HS. Twenty-four HS patients, 16 women and eight men, have been treated with ustekinumab. The average age was 35.7 ± 10.8 years (mean ± SD). All were of Hurley stage II or III. Ten (10/24, 41.6%) had received anti-TNF drugs including infliximab, adalimumab and etanercept prior to UST treatment for HS. Although the initial doses varied from 45 mg s.c. to 390 mg i.v., all cases were treated with 45 or 90 mg s.c. every 8 or 12 weeks at the regular dose, by following the regimen for psoriasis or CD. HS in most of the cases started to improve after 3-5 months of UST initiation, and some achieved complete remission. To our knowledge, our case is the first Asian HS patient improved by UST. Overall, UST is useful for HS and could be an alternative treatment if HS patients do not respond to other medications including anti-TNF drugs.

The indole compound MA-35 attenuates tumorigenesis in an inflammation-induced colon cancer model.

In inflammatory bowel disease, chronic inflammation results in the development of colon cancer known as colitis-associated cancer. This disease is associated with tumor necrosis factor-α (TNF-α) signaling. In addition, intestinal fibrosis is a common clinical complication that is promoted by transforming growth factor ß1 (TGF-ß1). In our previous study, MA-35 attenuated renal fibrosis by inhibiting both TNF-α and TGF-ß1 signaling. This study aimed to identify the possible antitumor effects and antifibrotic effects of MA-35 using an AOM/DSS mouse model. MA-35 was orally administered every day for 70 days in the AOM/DSS mouse model. There was no difference in weight loss between the AOM/DSS group and the AOMDSS + MA-35 group, but the disease activity index score and the survival rate were improved by MA-35. MA-35 blocked the anemia and shortening of the colon induced by AOM/DSS. MA-35 reduced the macroscopic formation of tumors in the colon. In the microscopic evaluation, MA-35 reduced inflammation and fibrosis in areas with dysplasia. Furthermore, the TNF-α mRNA level in the colon tended to be reduced, and the interleukin 6, TGF-ß1 and fibronectin 1 mRNA levels in the colon were significantly reduced by MA-35. These results suggested that MA-35 inhibited AOM/DSS-induced carcinogenesis by reducing inflammation and fibrosis.

A Genome-wide Association Study Identifying RAP1A as a Novel Susceptibility Gene for Crohn's Disease in Japanese Individuals.

BACKGROUND AND AIMS: Genome-wide association studies [GWASs] of European populations have identified numerous susceptibility loci for Crohn's disease [CD]. Susceptibility genes differ by ethnicity, however, so GWASs specific for Asian populations are required. This study aimed to clarify the Japanese-specific genetic background for CD by a GWAS using the Japonica array [JPA] and subsequent imputation with the 1KJPN reference panel. METHODS: Two independent Japanese case/control sets (Tohoku region [379 CD patients, 1621 controls] and Kyushu region [334 CD patients, 462 controls]) were included. GWASs were performed separately for each population, followed by a meta-analysis. Two additional replication sets [254 + 516 CD patients and 287 + 565 controls] were analysed for top hit single nucleotide polymorphisms [SNPs] from novel genomic regions. RESULTS: Genotype data of 4 335 144 SNPs from 713 Japanese CD patients and 2083 controls were analysed. SNPs located in TNFSF15 (rs78898421, Pmeta = 2.59 × 10-26, odds ratio [OR] = 2.10), HLA-DQB1 [rs184950714, pmeta = 3.56 × 10-19, OR = 2.05], ZNF365, and 4p14 loci were significantly associated with CD in Japanese individuals. Replication analyses were performed for four novel candidate loci [p <1 × 10-6], and rs488200 located upstream of RAP1A was significantly associated with CD [pcombined = 4.36 × 10-8, OR = 1.31]. Transcriptome analysis of CD4+ effector memory T cells from lamina propria mononuclear cells of CD patients revealed a significant association of rs488200 with RAP1A expression. CONCLUSIONS: RAP1A is a novel susceptibility locus for CD in the Japanese population.

Long-term prognosis of Japanese patients with biologic-naïve Crohn's disease treated with anti-tumor necrosis factor-α antibodies.

BACKGROUND/AIMS: Few reports have described the long-term treatment outcomes of the anti-tumor necrosis factor-α antibody for Japanese Crohn's disease (CD) patients. The aim of this study was to evaluate them and clarify the clinical factors that affect the long-term prognosis of the anti-tumor necrosis factor-α treatments. METHODS: This was a retrospective, observational, single-center cohort study. Japanese CD patients treated with either infliximab or adalimumab as a first-line therapy were analyzed. The cumulative retention rates of the biologics, relapse-free survival, and surgery-free survival were analyzed using Kaplan-Meier methods. The clinical factors associated with the long-term outcomes were estimated by both the log-rank test and Cox proportional hazard model. RESULTS: The cumulative retention rate was significantly higher in the group with a concomitant elemental diet of ≥900 kcal/day, baseline C-reactive protein (CRP) levels <2.6 mg/dL, and baseline serum albumin levels ≥3.5 g/dL, respectively. The baseline serum albumin levels were also associated with both relapse-free and surgery-free survival. The lack of concomitant use of an elemental diet ≥900 kcal/day was identified as the only independent risk factor for the withdrawal of the biologics. CONCLUSIONS: Baseline CRP levels and serum albumin levels could affect the long-term outcomes in CD patients. Concomitant elemental diet of ≥900 kcal/day could have a positive influence on clinical treatment course.

Inflammation-independent TL1A-mediated intestinal fibrosis is dependent on the gut microbiome.

Tumor necrosis factor-like cytokine 1A (TL1A, TNFSF15) is implicated in inflammatory bowel disease (IBD), modulating the location and severity of intestinal inflammation and fibrosis. TL1A expression is increased in inflamed gut mucosa and associated with fibrostenosing Crohn's disease. Tl1a-overexpression in mice lead to spontaneous ileitis, and exacerbated induced proximal colitis and fibrosis. IBD is associated with shifts in the gut microbiome, but the effect of differing microbial populations and their interaction with TL1A on fibrosis has not been investigated. We demonstrate that the pro-fibrotic and inflammatory phenotype resulting from Tl1a-overexpression is abrogated in the absence of resident microbiota. To evaluate if this is due to the absence of a unique bacterial population, as opposed to any bacteria per se, we gavaged germ-free (GF) wild-type and Tl1a-transgenic (Tl1a-Tg) mice with stool from specific pathogen free (SPF) mice and a healthy human donor (Hu). Reconstitution with SPF, but not Hu microbiota, resulted in increased intestinal collagen deposition and fibroblast activation in Tl1a-Tg mice. Notably, there was reduced fibroblast migration and activation under GF conditions compared to native conditions. We then identified several candidate organisms that correlated directly with increased fibrosis in reconstituted mice and showed that these organisms directly impact fibroblast function in vitro. Thus, Tl1a-mediated intestinal fibrosis and fibroblast activation are dependent on specific microbial populations.

Clinical and genetic risk factors for decreased bone mineral density in Japanese patients with inflammatory bowel disease.

BACKGROUND AND AIM: Patients with inflammatory bowel disease (IBD) are at a high risk of low bone mineral density (BMD). Reportedly, clinical and genetic factors cause low BMD in Caucasians; however, studies in non-Caucasian populations remain scarce. METHODS: Clinical risk factors for low BMD were investigated in 266 Japanese patients with IBD, and a genome-wide association analysis (GWAS) was performed using linear regression with associated clinical factors as covariates. Genotyping was performed using a population-optimized genotyping array (Japonica array® ). After quality control, the genotype data of 4 384 682 single-nucleotide polymorphisms (SNPs) from 254 patients with IBD were used for GWAS. RESULTS: Body mass index, age, and disease duration were independently associated with the BMD of the femoral neck (P = 1.41E - 13, 1.04E - 5, and 1.58E - 3, respectively), and body mass index and sex were associated with the BMD of the lumbar spine (P = 6.90E - 10 and 6.84E - 3, respectively). In GWAS, 118 and 42 candidate SNPs of the femoral neck and lumbar spine, respectively, were identified. Among 118, 111 candidate SNPs of the femoral neck were located within the SLC22A23 gene, which is a known IBD susceptibility gene (minimum P = 1.42E - 07). Among 42, 18 candidate SNPs of the lumbar spine were located within the MECOM gene, which is associated with osteopenia (minimum P = 5.86E - 07). Interestingly, none of the known loci showed a significant association with BMD. CONCLUSIONS: Although clinical risk factors for low BMD in IBD were similar to those in the general population, genetic risk factors were rather different.

Effective and less invasive diagnostic strategy for gastrointestinal GVHD.

BACKGROUND AND STUDY AIMS: Rectosigmoidoscopy with biopsy has been regarded to be a useful procedure to diagnose gastrointestinal graft-versus-host disease (GVHD). However, little is known about the specific colonoscopic features of gastrointestinal GVHD. In this study, we focused on the 4 unique colonoscopic findings - orange peel appearance, spotty redness, small mucosal sloughing, and diffuse mucosal defect - which are possible specific findings of gastrointestinal GVHD. We aimed to estimate the usefulness of these four unique colonoscopic findings in the rectosigmoid portion to diagnose gastrointestinal GVHD. PATIENTS AND METHODS: Seventy patients who were histologically diagnosed with gastrointestinal GVHD at our institute were retrospectively enrolled. Colonoscopic findings were reviewed, focusing on the four characteristic findings. The percentage of the positive cases for the characteristic findings was calculated. The final scoping portion and the number of cases showing any of the four characteristic findings in the rectosigmoid portion were also evaluated. The relationships between biopsy sites and the histological findings were also evaluated. RESULTS: Orange peel appearance was observed in 66 cases (94.3 %). Spotty redness was observed in 45 cases (64.3 %). Small mucosal sloughing was observed in 49 cases (70.0 %). Diffuse mucosal defect was observed in six cases (8.6 %). The number of cases that were concurrently positive for one, two, and three findings were 16 (20.8 %), 20 (26.0 %), and 34 (48.6 %), respectively. Fifty-eight cases (82.9 %) were investigated up to the rectosigmoid portion, and 12 (17.1 %) were investigated beyond the sigmoid colon. All of the cases showed at least 1 of the 4 characteristics in the rectosigmoid portion. The percentage of crypt apoptosis in the biopsy specimen from orange peel appearance, spotty redness, small mucosal sloughing, and diffuse mucosal defect were 87.5 %, 83.3 %, 87.2 %, and 88.9 %, respectively. CONCLUSION: Orange peel appearance, spotty redness, small mucosal sloughing, and diffuse mucosal defect are the characteristic colonoscopic findings useful for diagnosis of gastrointestinal GVHD. These findings are frequently observed in the rectosigmoid portion. The histological detection rates for crypt cell apoptosis from these findings are high. Identifying the four characteristic findings on rectosigmoidoscopy and taking biopsies from these areas could be essential for the diagnostic strategy for gastrointestinal GVHD.

Myeloid ATG16L1 Facilitates Host-Bacteria Interactions in Maintaining Intestinal Homeostasis.

Intact ATG16L1 plays an essential role in Paneth cell function and intestinal homeostasis. However, the functional consequences of ATG16L1 deficiency in myeloid cells, particularly macrophages, are not fully characterized. We generated mice with Atg16l1 deficiency in myeloid and dendritic cells and showed that mice with myeloid Atg16l1 deficiency had exacerbated colitis in two acute and one chronic model of colitis with increased proinflammatory to anti-inflammatory macrophage ratios, production of proinflammatory cytokines, and numbers of IgA-coated intestinal microbes. Mechanistic analyses using primary murine macrophages showed that Atg16l1 deficiency led to increased reactive oxygen species production, impaired mitophagy, reduced microbial killing, impaired processing of MHC class II Ags, and altered intracellular trafficking to the lysosomal compartments. Increased production of reactive oxygen species and reduced microbial killing may be general features of the myeloid compartment, as they were also observed in Atg16l1-deficient primary murine neutrophils. A missense polymorphism (Thr300Ala) in the essential autophagy gene ATG16L1 is associated with Crohn disease (CD). Previous studies showed that this polymorphism leads to enhanced cleavage of ATG16L1 T300A protein and thus reduced autophagy. Similar findings were shown in primary human macrophages from controls and a population of CD patients carrying the Atg16l1 T300A risk variant and who were controlled for NOD2 CD-associated variants. This study revealed that ATG16L1 deficiency led to alterations in macrophage function that contribute to the severity of CD.

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

FCGR3A-158 polymorphism influences the biological response to infliximab in Crohn's disease through affecting the ADCC activity.

An association between FCGR3A-158 V/F polymorphism and biological responses to infliximab has been reported in Crohn's disease (CD) in Western countries. However, little is known about the mechanism by which gene polymorphism affects the responses to infliximab. The aims of this study were to confirm the association in Japanese CD patients and to reveal the effect of gene polymorphism on biological responses to infliximab. Japanese CD patients were examined retrospectively at weeks 8 and 30. Clinical and biological responses were assessed by the Crohn's disease activity index and C-reactive protein levels, respectively. The infliximab-binding affinity of natural killer (NK) cells from FCGR3A-158 V/V, V/F and F/F donors was examined. Infliximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activities were also determined using transmembrane TNF-α-expressing Jurkat T cells as target cells and peripheral blood mononuclear cells (PBMCs) from V/V, V/F and F/F donors as effector cells. Biological responses at week 8 were statistically higher in V/V patients, whereas no significant differences were observed in either clinical responses at weeks 8 and 30 or biological responses at week 30 among the three genotypes. NK cells and PBMCs from V/V patients also showed higher infliximab-binding affinity and infliximab-mediated ADCC activity, respectively. Our results suggest that FCGR3A-158 polymorphism is a predicting factor of biological responses to infliximab in the early phases. FCGR3A-158 polymorphism was also found to affect the infliximab-binding affinity of NK cells and infliximab-mediated ADCC activity in vitro, suggesting that an effect on ADCC activity influences biological responses to infliximab in CD patients.

Dendritic cell-specific ablation of the protein tyrosine phosphatase Shp1 promotes Th1 cell differentiation and induces autoimmunity.

Dendritic cells (DCs) promote immune responses to foreign Ags and immune tolerance to self-Ags. Deregulation of DCs is implicated in autoimmunity, but the molecules that regulate DCs to protect against autoimmunity have remained unknown. In this study, we show that mice lacking the protein tyrosine phosphatase Shp1 specifically in DCs develop splenomegaly associated with more CD11c(+) DCs. Splenic DCs from the mutant mice showed upregulation of CD86 and CCR7 expression and of LPS-induced production of proinflammatory cytokines. The mice manifested more splenic Th1 cells, consistent with the increased ability of their DCs to induce production of IFN-γ by Ag-specific T cells in vitro. The number of splenic CD5(+)CD19(+) B-1a cells and the serum concentrations of Igs M and G2a were also increased in the mutant mice. Moreover, aged mutant mice developed glomerulonephritis and interstitial pneumonitis together with increased serum concentrations of autoantibodies. Shp1 is thus a key regulator of DC functions that protects against autoimmunity.