Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.
Jessica B. Sarthi, Annie M. Trumbull, Shayda M. Abazari, Vincent van Unen, Joshua E. Chan, Yanfen Jiang, Jesse Gammons, Marc O. Anderson, Onur Cil, Calvin J. Kuo, Zachary M. Sellers
Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T-cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett’s (BE) to dysplasia to EAC, investigated gene (RNAseq), protein (tissue microarray) expression and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We show that the loss of both CD3-ε expression and CD3+ T-cell number are correlated with worse OS in EAC. The GRAIL (gene related to anergy in lymphocytes) isoform 1 (GRAIL1), which is the prominent isoform in EACs, degrades (ε, γ, δ) CD3s and inactivates T-cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilizes CD3s. Further, GRAIL1 mediated CD3 degradation is facilitated by interferon stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, either the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-LRAA mutant can increase CD3 levels. Together, we identified that an ISG15→GRAIL1→mutant p53 amplification loop negatively influencing CD3 levels and T-cell activity, thus promoting immunosuppression in EAC.
Dyke P. McEwen, Paramita Ray, Derek J. Nancarrow, Zhuwen Wang, Srimathi Kasturirangan, Saeed Abdullah, Ayushi Balan, Rishi Hoskeri, Dafydd Thomas, Theodore S. Lawrence, David G. Beer, Kiran H. Lagisetty, Dipankar Ray
Fibroblast growth factor 15/19 (FGF15/19, mouse/human ortholog) is expressed in the ileal enterocytes of the small intestine and released postprandially in response to bile acid absorption. Previous reports of FGF15–/– mice have limited our understanding of gut-specific FGF15’s role in metabolism. Therefore, we studied the role of endogenous gut-derived FGF15 in bile acid, cholesterol, glucose, and energy balance. We found that circulating levels of FGF19 were reduced in individuals with obesity and comorbidities, such as type 2 diabetes and metabolic dysfunction–associated fatty liver disease. Gene expression analysis of ileal FGF15-positive cells revealed differential expression during the obesogenic state. We fed standard chow or a high-fat metabolic dysfunction-associated steatohepatitis–inducing diet to control and intestine-derived FGF15-knockout (FGF15INT-KO) mice. Control and FGF15INT-KO mice gained similar body weight and adiposity and did not show genotype-specific differences in glucose, mixed meal, pyruvate, and glycerol tolerance. FGF15INT-KO mice had increased systemic bile acid levels but decreased cholesterol levels, pointing to a primary role for gut-derived FGF15 in regulating bile acid and cholesterol metabolism when exposed to obesogenic diet. These studies show that intestinal FGF15 plays a specific role in bile acid and cholesterol metabolism regulation but is not essential for energy and glucose balance.
Nadejda Bozadjieva-Kramer, Jae Hoon Shin, Ziru Li, Alan C. Rupp, Nicole Miller, Stace Kernodle, Nicolas Lanthier, Paulina Henry, Nikhil Seshadri, Andriy Myronovych, Ormond A. MacDougald, Robert W. O’Rourke, Rohit Kohli, Charles F. Burant, Amy E. Rothberg, Randy J. Seeley
Crohn’s disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology, and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-Seq of patient-matched colon and ileum biopsies from treatment-naive pediatric patients with CD (n = 169) and a control cohort (n = 108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we found that pediatric patients with CD with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 upregulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.
Alexandria J. Shumway, Michael T. Shanahan, Emilie Hollville, Kevin Chen, Caroline Beasley, Jonathan W. Villanueva, Sara Albert, Grace Lian, Moises R. Cure, Matthew Schaner, Lee-Ching Zhu, Surekha Bantumilli, Mohanish Deshmukh, Terrence S. Furey, Shehzad Z. Sheikh, Praveen Sethupathy
The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis–specific SNPs were enriched in distal colon–related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.
John L. Johnson, Davit Sargsyan, Eric M. Neiman, Amy Hart, Aleksandar Stojmirovic, Roman Kosoy, Haritz Irizar, Mayte Suárez-Fariñas, Won-Min Song, Carmen Argmann, Stefan Avey, Liraz Shmuel-Galia, Tim Vierbuchen, Gerold Bongers, Yu Sun, Leonard Edelstein, Jacqueline Perrigoue, Jennifer E. Towne, Aisling O’Hara Hall, Katherine A. Fitzgerald, Kasper Hoebe
The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett’s esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/TP53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Streptococcus parasanguinis, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1β, Lbp, Lcn2, Myd88, Nfkb1, Tlr2, and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.
Katherine M. Weh, Connor L. Howard, Yun Zhang, Bridget A. Tripp, Jennifer L. Clarke, Amy B. Howell, Joel H. Rubenstein, Julian A. Abrams, Maria Westerhoff, Laura A. Kresty
Bile acids (BAs) affect the intestinal environment by ensuring barrier integrity, maintaining microbiota balance, regulating epithelium turnover, and modulating the immune system. As a master regulator of BA homeostasis, farnesoid X receptor (FXR) is severely compromised in patients with inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). At the front line, gut macrophages react to the microbiota and metabolites that breach the epithelium. We aim to study the role of the BA/FXR axis in macrophages. This study demonstrates that inflammation-induced epithelial abnormalities compromised FXR signaling and altered BAs’ profile in a mouse CAC model. Further, gut macrophage–intrinsic FXR sensed aberrant BAs, leading to pro-inflammatory cytokines’ secretion, which promoted intestinal stem cell proliferation. Mechanistically, activation of FXR ameliorated intestinal inflammation and inhibited colitis-associated tumor growth, by regulating gut macrophages’ recruitment, polarization, and crosstalk with Th17 cells. However, deletion of FXR in bone marrow or gut macrophages escalated the intestinal inflammation. In summary, our study reveals a distinctive regulatory role of FXR in gut macrophages, suggesting its potential as a therapeutic target for addressing IBD and CAC.
Xingchen Dong, Ming Qi, Chunmiao Cai, Yu Zhu, Yuwenbin Li, Sally Coulter, Fei Sun, Christopher Liddle, Nataliya V. Uboha, Richard Halberg, Wei Xu, Paul Marker, Ting Fu
Circular RNAs (circRNAs) are highly expressed in the mammalian intestinal epithelium, but their functions remain largely unknown. Here we identified the circRNA Cdr1as as a repressor of intestinal epithelial regeneration and defense. Cdr1as levels increase in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with inflammatory bowel diseases and sepsis. Ablation of the Cdr1as locus from the mouse genome enhances renewal of the intestinal mucosa, promotes injury-induced epithelial regeneration, and protects the mucosa against colitis. We found approximately 40 microRNAs, including microRNA miR-195, differentially express between intestinal mucosa of Cdr1as knockout (–/–) versus littermate mice. Increasing the levels of Cdr1as inhibits intestinal epithelial repair after wounding in cultured cells and represses growth of intestinal organoids cultured ex vivo, but this inhibition is abolished by miR-195 silencing. The reduction in miR-195 levels in the Cdr1as–/– intestinal epithelium is the result of reduced stability and processing of the precursor miR-195. These findings indicate that Cdr1as reduces proliferation and repair of the intestinal epithelium at least in part via interaction with miR-195 and highlight a role for induced Cdr1as in the pathogenesis of unhealed wounds and disrupted renewal of the intestinal mucosa.
Hee Kyoung Chung, Lan Xiao, Naomi Han, Jason Chen, Vivian Yao, Cassandra M. Cairns, Benjamin Raufman, Jaladanki N. Rao, Douglas J. Turner, Rosemary Kozar, Myriam Gorospe, Jian-Ying Wang
Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson’s disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein–seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.
Rashmi Chandra, Arpine Sokratian, Katherine R. Chavez, Stephanie King, Sandip M. Swain, Joshua C. Snyder, Andrew B. West, Rodger A. Liddle
Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.
Yan Zhu, Dengyong Zhang, Pooja Shukla, Young-Ho Jung, Prit Benny Malgulwar, Sharmeen Chagani, Medina Colic, Sarah Benjamin, John A. Copland III, Lin Tan, Philip L. Lorenzi, Milind Javle, Jason T. Huse, Jason Roszik, Traver Hart, Lawrence N. Kwong
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