LRRK2 but not ATG16L1 is associated with Paneth cell defect in Japanese Crohn’s disease patients

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Abstract

BACKGROUND. Morphological patterns of Paneth cells are a prognostic biomarker in Western Crohn’s disease (CD) patients, and are associated with autophagy-associated ATG16L1 and NOD2 variants. We hypothesized that genetic determinants of Paneth cell phenotype in other ethnic CD cohorts are distinct but also involved in autophagy.

METHODS. We performed a hypothesis-driven analysis of 56 single nucleotide polymorphisms (SNPs) associated with CD susceptibility or known to affect Paneth cell function in 110 Japanese CD patients who underwent ileal resection. We subsequently performed a genome-wide association analysis. Paneth cell phenotype was determined by defensin-5 immunofluorescence. Selected genotype–Paneth cell defect correlations were compared to a Western CD cohort (n = 164).

RESULTS. The average percentage of abnormal Paneth cells in Japanese CD was similar to Western CD (P = 0.87), and abnormal Paneth cell phenotype was also associated with early recurrence (P = 0.013). In contrast to Western CD, ATG16L1 T300A was not associated with Paneth cell defect in Japanese CD (P = 0.20). Among the 56 selected SNPs, only LRRK2 M2397T showed significant association with Paneth cell defect (P = 3.62 × 10^–4), whereas in the Western CD cohort it was not (P = 0.76). Pathway analysis of LRRK2 and other candidate genes with P less than 5 × 10^–4 showed connections with known CD susceptibility genes and links to autophagy and TNF-α networks.

CONCLUSIONS. We found dichotomous effects of ATG16L1 and LRRK2 on Paneth cell defect between Japanese and Western CD. Genes affecting Paneth cell phenotype in Japanese CD were also associated with autophagy. Paneth cell phenotype also predicted prognosis in Japanese CD.

FUNDING. Helmsley Charitable Trust, Doris Duke Foundation (grant 2014103), Japan Society for the Promotion of Science (KAKENHI grants JP15H04805 and JP15K15284), Crohn’s and Colitis Foundation grant 274415, NIH (grants 1R56DK095820, K01DK109081, and UL1 TR000448).

Authors

Ta-Chiang Liu, Takeo Naito, Zhenqiu Liu, Kelli L. VanDussen, Talin Haritunians, Dalin Li, Katsuya Endo, Yosuke Kawai, Masao Nagasaki, Yoshitaka Kinouchi, Dermot P.B. McGovern, Tooru Shimosegawa, Yoichi Kakuta, Thaddeus S. Stappenbeck

Lymphatic deletion of calcitonin receptor–like receptor exacerbates intestinal inflammation

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Abstract

Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor–like receptor (Calcrl), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrl^fl/fl/Prox1-CreER^T2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrl^fl/fl/Prox1-CreER^T2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl/adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.

Authors

Reema B. Davis, Daniel O. Kechele, Elizabeth S. Blakeney, John B. Pawlak, Kathleen M. Caron

Preferential TNFα signaling via TNFR2 regulates epithelial injury and duct obstruction in experimental biliary atresia

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Abstract

Biliary atresia is an obstructive cholangiopathy of infancy that progresses to end-stage cirrhosis. Although the pathogenesis of the disease is not completely understood, previous reports link TNFα to apoptosis of the bile duct epithelium in the presence of IFNγ. Here, we investigate if TNFα signaling regulates pathogenic mechanisms of biliary atresia. First, we quantified the expression of TNFA and its receptors TNFR1 and TNFR2 in human livers and found an increased expression of the receptors at the time of diagnosis. In mechanistic experiments using a neonatal mouse model of rhesus rotavirus–induced (RRV-induced) biliary atresia, the expression of the ligand and both receptors increased 6- to 8-fold in hepatic DCs and NK lymphocytes above controls. The activation of tissue NK cells by RRV-primed DCs was independent of TNFα-TNFR signaling. Once activated, the expression of TNFα by NK cells induced lysis of 55% ± 2% of bile duct epithelial cells, which was completely prevented by blocking TNFα or TNFR2, but not TNFR1. More notably, antibody-mediated or genetic disruption of TNFα-TNFR2 signaling in vivo decreased apoptosis and epithelial injury; suppressed the infiltration of livers by T cells, DCs, and NK cells; prevented extrahepatic bile duct obstruction; and promoted long-term survival. These findings point to a key role for the TNFα/TNFR2 axis on pathogenesis of experimental biliary atresia and identify new therapeutic targets to suppress the disease phenotype.

Authors

Pranavkumar Shivakumar, Tatsuki Mizuochi, Reena Mourya, Sridevi Gutta, Li Yang, Zhenhua Luo, Jorge A. Bezerra

BET inhibitors block pancreatic stellate cell collagen I production and attenuate fibrosis in vivo

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Abstract

The fibrotic reaction, which can account for over 70%–80% of the tumor mass, is a characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with activation and proliferation of pancreatic stellate cells (PSCs), which are key regulators of collagen I production and fibrosis in vivo. In this report, we show that members of the bromodomain and extraterminal (BET) family of proteins are expressed in primary PSCs isolated from human PDAC tumors, with BRD4 positively regulating, and BRD2 and BRD3 negatively regulating, collagen I expression in primary cancer-associated PSCs. We show that the inhibitory effect of pan-BET inhibitors on collagen I expression in primary cancer-associated PSCs is through blocking of BRD4 function. Importantly, we show that FOSL1 is repressed by BRD4 in primary cancer-associated PSCs and negatively regulates collagen I expression. While BET inhibitors do not affect viability or induce PSC apoptosis or senescence, BET inhibitors induce primary cancer-associated PSCs to become quiescent. Finally, we show that BET inhibitors attenuate stellate cell activation, fibrosis, and collagen I production in the EL-Kras^G12D transgenic mouse model of pancreatic tumorigenesis. Our results demonstrate that BET inhibitors regulate fibrosis by modulating the activation and function of cancer-associated PSCs.

Authors

Krishan Kumar, Brian T. DeCant, Paul J. Grippo, Rosa F. Hwang, David J. Bentrem, Kazumi Ebine, Hidayatullah G. Munshi

Characterization of candidate genes in inflammatory bowel disease–associated risk loci

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Abstract

GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn’s disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis.

Authors

Joanna M. Peloquin, Gautam Goel, Lingjia Kong, Hailiang Huang, Talin Haritunians, R. Balfour Sartor, Mark J. Daly, Rodney D. Newberry, Dermot P. McGovern, Vijay Yajnik, Sergio A. Lira, Ramnik J. Xavier

FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities

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Abstract

Recent gene-profiling analyses showed significant upregulation of the folate hydrolase (FOLH1) gene in the affected intestinal mucosa of patients with inflammatory bowel disease (IBD). The FOLH1 gene encodes a type II transmembrane glycoprotein termed glutamate carboxypeptidase II (GCPII). To establish that the previously reported increased gene expression was functional, we quantified the glutamate carboxypeptidase enzymatic activity in 31 surgical specimens and report a robust 2.8- to 41-fold increase in enzymatic activity in the affected intestinal mucosa of IBD patients compared with an uninvolved area in the same patients or intestinal mucosa from healthy controls. Using a human-to-mouse approach, we next showed a similar enzymatic increase in two well-validated IBD murine models and evaluated the therapeutic effect of the potent FOLH1/GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) (IC₅₀ = 300 pM). In the dextran sodium sulfate (DSS) colitis model, 2-PMPA inhibited the GCPII activity in the colonic mucosa by over 90% and substantially reduced the disease activity. The significance of the target was confirmed in FOLH1^–/– mice who exhibited resistance to DSS treatment. In the murine IL-10^–/– model of spontaneous colitis, daily 2-PMPA treatment also significantly reduced both macroscopic and microscopic disease severity. These results provide the first evidence of FOLH1/GCPII enzymatic inhibition as a therapeutic option for IBD.

Authors

Rana Rais, Weiwei Jiang, Huihong Zhai, Krystyna M. Wozniak, Marigo Stathis, Kristen R. Hollinger, Ajit G. Thomas, Camilo Rojas, James J. Vornov, Michael Marohn, Xuhang Li, Barbara S. Slusher

Dual epithelial and immune cell function of Dvl1 regulates gut microbiota composition and intestinal homeostasis

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Abstract

Homeostasis of the gastrointestinal (GI) tract is controlled by complex interactions between epithelial and immune cells and the resident microbiota. Here, we studied the role of Wnt signaling in GI homeostasis using Disheveled 1 knockout (Dvl1^–/–) mice, which display an increase in whole gut transit time. This phenotype is associated with a reduction and mislocalization of Paneth cells and an increase in CD8⁺ T cells in the lamina propria. Bone marrow chimera experiments demonstrated that GI dysfunction requires abnormalities in both epithelial and immune cells. Dvl1^–/– mice exhibit a significantly distinct GI microbiota, and manipulation of the gut microbiota in mutant mice rescued the GI transit abnormality without correcting the Paneth and CD8⁺ T cell abnormalities. Moreover, manipulation of the gut microbiota in wild-type mice induced a GI transit abnormality akin to that seen in Dvl1^–/– mice. Together, these data indicate that microbiota manipulation can overcome host dysfunction to correct GI transit abnormalities. Our findings illustrate a mechanism by which the epithelium and immune system coregulate gut microbiota composition to promote normal GI function.

Authors

Haim Belinson, Adam K. Savage, Douglas Fadrosh, Yien-Ming Kuo, Din Lin, Ricardo Valladares, Ysbrand Nusse, Anthony Wynshaw-Boris, Susan V. Lynch, Richard M. Locksley, Ophir D. Klein

PRL3-zumab, a first-in-class humanized antibody for cancer therapy

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Abstract

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3⁺, but not PRL-3^–, orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3⁺ tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become “extracellular oncotargets” that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery.

Authors

Min Thura, Abdul Qader Omer Al-Aidaroos, Wei Peng Yong, Koji Kono, Abhishek Gupta, You Bin Lin, Kousaku Mimura, Jean Paul Thiery, Boon Cher Goh, Patrick Tan, Ross Soo, Cheng William Hong, Lingzhi Wang, Suling Joyce Lin, Elya Chen, Sun Young Rha, Hyun Cheol Chung, Jie Li, Sayantani Nandi, Hiu Fung Yuen, Shu-Dong Zhang, Yeoh Khay Guan, Jimmy So, Qi Zeng

Quantitation of circulating satellite RNAs in pancreatic cancer patients

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Abstract

Pancreatic ductal adenocarcinoma (Pdac) is a malignancy with a poor prognosis due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues but are difficult to measure due to their repetitive sequences and irregularities. HSATII RNA core sequence levels in sera were significantly higher in Pdac patients compared with noncancer patients (median copy number: 14.75 and 3.17 per μl in the training set and 17.35 and 2.9 in the validation set, respectively). In addition, patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion of Pdac, could also be efficiently detected. This method can be routinely applied to screen patients with Pdac and high-risk patients, facilitating the development of preventive medicine for this disease.

Authors

Takahiro Kishikawa, Motoyuki Otsuka, Takeshi Yoshikawa, Motoko Ohno, Keisuke Yamamoto, Natsuyo Yamamoto, Ai Kotani, Kazuhiko Koike

Paneth cell defects in Crohn’s disease patients promote dysbiosis

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Abstract

BACKGROUND. Paneth cell dysfunction has been implicated in a subset of Crohn’s disease (CD) patients. We previously stratified clinical outcomes of CD patients by using Paneth cell phenotypes, which we defined by the intracellular distribution of antimicrobial proteins. Animal studies suggest that Paneth cells shape the intestinal microbiome. However, it is unclear whether Paneth cell phenotypes alter the microbiome complexity in CD subjects. Therefore, we analyzed the correlation of Paneth cell phenotypes with mucosal microbiome composition and ileal RNA expression in pediatric CD and noninflammatory bowel disease (non-IBD) patients.

METHODS. Pediatric CD (n = 44) and non-IBD (n = 62) patients aged 4 to 18 were recruited prior to routine endoscopic biopsy. Ileal mucosal samples were analyzed for Paneth cell phenotypes, mucosal microbiome composition, and RNA transcriptome.

RESULTS. The prevalence of abnormal Paneth cells was higher in pediatric versus adult CD cohorts. For pediatric CD patients, those with abnormal Paneth cells showed significant changes in their ileal mucosal microbiome, highlighted by reduced protective microbes and enriched proinflammatory microbes. Ileal transcriptome profiles showed reduced transcripts for genes that control oxidative phosphorylation in CD patients with abnormal Paneth cells. These transcriptional changes in turn were correlated with specific microbiome alterations. In non-IBD patients, a subset contained abnormal Paneth cells. However, this subset was not associated with alterations in the microbiome or host transcriptome.

CONCLUSION. Paneth cell abnormalities in human subjects are associated with mucosal dysbiosis in the context of CD, and these changes are associated with alterations in oxidative phosphorylation, potentially in a feedback loop.

FUNDING. The research was funded by Helmsley Charitable Trust (to T.S. Stappenbeck, R.J. Xavier, and D.P.B. McGovern), Crohn’s and Colitis Foundation of America (to N.H. Salzman, T.S. Stappenbeck, R.J. Xavier, and C. Huttenhower), and Doris Duke Charitable Foundation grant 2014103 (to T.C. Liu).

Authors

Ta-Chiang Liu, Bhaskar Gurram, Megan T. Baldridge, Richard Head, Vy Lam, Chengwei Luo, Yumei Cao, Pippa Simpson, Michael Hayward, Mary L. Holtz, Pavlos Bousounis, Joshua Noe, Diana Lerner, Jose Cabrera, Vincent Biank, Michael Stephens, Curtis Huttenhower, Dermot P.B. McGovern, Ramnik J. Xavier, Thaddeus S. Stappenbeck, Nita H. Salzman