Diarrhea is a major side effect of ErbB receptor tyrosine kinase inhibitors (TKIs) in cancer chemotherapy. Here, we show that the primary mechanism of ErbB TKI diarrhea is activation of basolateral membrane potassium (K+) channels and apical membrane chloride (Cl-) channels in intestinal epithelia, and demonstrate the efficacy of channel blockers in a rat model of TKI diarrhea. Short-circuit current in colonic epithelial cells showed that the TKIs gefitinib, lapatinib and afatinib do not affect basal secretion, but amplify carbachol-stimulated secretion by 2 to 3 fold. Mechanistic studies with the second-generation TKI afatinib showed that the amplifying effect on Cl- secretion was Ca2+ and cAMP independent, blocked by CFTR and K+ channel inhibitors, and involved the EGF receptor binding and ERK signaling. Afatinib-amplified activation of basolateral K+ and apical Cl- channels was demonstrated by selective membrane permeabilization, ion substitution and channel inhibitors. Rats administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from ~60% to >80%, which was reduced by up to 75% the K+ channel inhibitors clotrimazole or senicapoc, or the CFTR inhibitor (R)-BPO-27. These results indicate a mechanism for TKI diarrhea involving K+ and Cl- channel activation, and support the therapeutic efficacy of channel inhibitors.
Tianying Duan, Onur Cil, Jay R. Thiagarajah, Alan S Verkman
Molecular mechanisms that control leukocyte migration across the vascular endothelium (transendothelial migration; TEndoM) have been extensively characterized in vivo, but details of leukocyte transepithelial migration (TEpM) and its dysregulation (a pathologic feature of many mucosal diseases) are missing due to the lack of suitable animal models. Here, we describe a murine model that utilizes a vascularized proximal colonic segment (pcLoop) and enables quantitative studies of leukocyte trafficking across colonic epithelium. Consistent with previous in vitro studies, intraluminal injection of antibodies against integrin CD11b/CD18 reduced recruitment of polymorphonuclear neutrophils (PMN) into the lumen of pcLoops, and it increased subepithelial accumulation of PMN. We extended studies using the pcLoop to determine contributions of Junctional Adhesion Molecule-A (JAM-A, or F11R) in PMN TEpM and confirmed that mice with total loss of JAM-A or mice with intestinal epithelial selective loss of JAM-A had increased colonic permeability. Furthermore, there was reduced PMN migration into the colonic lumen that paralleled subepithelial accumulation of PMN in global-KO mice, as well as in intestinal epithelial-targeted JAM-A–deficient mice. These findings highlight a potentially novel role for JAM-A in regulating PMN TEpM in vivo and demonstrate utility of this model for identifying receptors that may be targeted in vivo to reduce pathologic intestinal inflammation.
Sven Flemming, Anny-Claude Luissint, Asma Nusrat, Charles A. Parkos
Intestinal epithelial barrier repair is vital for remission in inflammatory bowel disease (IBD). Extracellular adenosine signaling has been implicated in promoting restoration of epithelial barrier function. Currently, no clinically approved agents target this pathway. Adenosine signaling is terminated by uptake from the extracellular space via equilibrative nucleoside transporters (ENTs). We hypothesized that ENT inhibition could dampen intestinal inflammation. Initial studies demonstrated transcriptional repression of ENT1 and ENT2 in IBD biopsies or in murine IBD models. Subsequent studies in mice with global Ent1 or Ent2 deletion revealed selective protection of Ent2–/– mice. Elevated intestinal adenosine levels in conjunction with abolished protection following pharmacologic blockade of A2B adenosine receptors implicate adenosine signaling as the mechanism of gut protection in Ent2–/– mice. Additional studies in mice with tissue-specific deletion of Ent2 uncovered epithelial Ent2 as the target. Moreover, intestinal protection provided by a selective Ent2 inhibitor was abolished in mice with epithelium-specific deletion of Ent2 or the A2B adenosine receptor. Taken together, these findings indicate that increased mucosal A2B signaling following repression or deletion of epithelial Ent2 coordinates the resolution of intestinal inflammation. This study suggests the presence of a targetable purinergic network within the intestinal epithelium designed to limit tissue inflammation.
Carol M. Aherne, Colm B. Collins, Caroline R. Rapp, Kristine E. Olli, Loni Perrenoud, Paul Jedlicka, Jessica L. Bowser, Tingting W. Mills, Harry Karmouty-Quintana, Michael R. Blackburn, Holger K. Eltzschig
BACKGROUND. Crohn’s disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity. METHODS. We performed small RNA sequencing of adult colon tissue from CD and NIBD controls. Colonic epithelial cells and immune cells were isolated from colonic tissues, and microRNA-31 (miR-31) expression was measured. miR-31 expression was measured in colonoid cultures generated from controls and patients with CD. We performed small RNA-sequencing of formalin-fixed paraffin-embedded colon and ileum biopsies from treatment-naive pediatric patients with CD and controls and collected data on disease features and outcomes. RESULTS. Small RNA-sequencing and microRNA profiling in the colon revealed 2 distinct molecular subtypes, each with different clinical associations. Notably, we found that miR-31 expression was a driver of these 2 subtypes and, further, that miR-31 expression was particularly pronounced in epithelial cells. Colonoids revealed that miR-31 expression differences are preserved in this ex vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery were associated with worse disease outcome as measured by need for an end ileostomy and recurrence of disease in the neoterminal ileum. In pediatric patients, lower miR-31 expression at the time of diagnosis was associated with future development of fibrostenotic ileal CD requiring surgery CONCLUSIONS. These findings represent an important step forward in designing more effective clinical trials and developing personalized CD therapies. FUNDING. This work was supported by CCF Career Development Award (SZS), R01-ES024983 from NIEHS (SZS and TSF), 1R01DK104828-01A1 from NIDDK (SZS and TSF), P01-DK094779-01A1 from NIDDK (SZS), P30-DK034987 from NIDDK (SZS), 1-16-ACE-47 ADA Pathway Award (PS), UNC Nutrition Obesity Research Center Pilot & Feasibility Grant P30DK056350 (PS), CCF PRO-KIIDS NETWORK (SZS and PS), UNC CGIBD T32 Training Grant from NIDDK (JBB), T32 Training Grant (5T32GM007092-42) from NIGMS (MH), and SHARE from the Helmsley Trust (SZS). The UNC Translational Pathology Laboratory is supported, in part, by grants from the National Cancer Institute (3P30CA016086) and the UNC University Cancer Research Fund (UCRF) (PS).
Benjamin P. Keith, Jasmine B. Barrow, Takahiko Toyonaga, Nevzat Kazgan, Michelle Hoffner O’Connor, Neil D. Shah, Matthew S. Schaner, Elisabeth A. Wolber, Omar K. Trad, Greg R. Gipson, Wendy A. Pitman, Matthew Kanke, Shruti J. Saxena, Nicole Chaumont, Timothy S. Sadiq, Mark J. Koruda, Paul A. Cotney, Nancy Allbritton, Dimitri G. Trembath, Francisco Sylvester, Terrence S. Furey, Praveen Sethupathy, Shehzad Z. Sheikh
Irritable bowel syndrome (IBS) patients suffer from chronic abdominal pain and extraintestinal comorbidities, including overactive bladder (OAB) and interstitial cystitis/painful bladder syndrome (IC-PBS). Mechanistic understanding of the cause and time course of these comorbid symptoms is lacking, as are clinical treatments. Here, we report that colitis triggers hypersensitivity of colonic afferents, neuroplasticity of spinal cord circuits, and chronic abdominal pain, which persists after inflammation. Subsequently, and in the absence of bladder pathology, colonic hypersensitivity induces persistent hypersensitivity of bladder afferent pathways, resulting in bladder-voiding dysfunction, indicative of OAB/IC-PBS. Daily administration of linaclotide, a guanylate cyclase-C (GC-C) agonist that is restricted to and acts within the gastrointestinal tract, reverses colonic afferent hypersensitivity, reverses neuroplasticity-induced alterations in spinal circuitry, and alleviates chronic abdominal pain in mice. Intriguingly, daily linaclotide administration also reverses persistent bladder afferent hypersensitivity to mechanical and chemical stimuli and restores normal bladder voiding. Linaclotide itself does not inhibit bladder afferents, rather normalization of bladder function by daily linaclotide treatment occurs via indirect inhibition of bladder afferents via reduced nociceptive signaling from the colon. These data support the concepts that cross-organ sensitization underlies the development and maintenance of visceral comorbidities, while pharmaceutical treatments that inhibit colonic afferents may also improve urological symptoms through common sensory pathways.
Luke Grundy, Andrea M. Harrington, Joel Castro, Sonia Garcia-Caraballo, Annemie Deiteren, Jessica Maddern, Grigori Y. Rychkov, Pei Ge, Stefanie Peters, Robert Feil, Paul Miller, Andre Ghetti, Gerhard Hannig, Caroline B. Kurtz, Inmaculada Silos-Santiago, Stuart M. Brierley
Colon cancer is a devastating illness that is associated with gut inflammation. Here, we explored the possible role of lipin-1, a phosphatidic acid phosphatase, in the development of colitis-associated tumorigenesis. Azoxymethane and dextran sodium sulfate–treated (DSS-treated) animals deficient in lipin-1 harbored fewer tumors and carcinomas than WT animals due to decreased cellular proliferation, lower expression of antiapoptotic and protumorigenic factors, and a reduced infiltration of macrophages in colon tumors. They also displayed increased resistance to DSS-induced colitis by producing less proinflammatory cytokines and experiencing less immune infiltration. Lipin-1–deficient macrophages from the colon were less activated and displayed lower phosphatidic acid phosphatase activity than WT macrophages isolated from DSS-treated animals. Transference of WT macrophages into lipin-1–deficient animals was sufficient to increase colitis burden. Furthermore, treatment of lipin-1–deficient mice with IL-23 exacerbated colon inflammation. Analysis of human databases from colon cancer and ulcerative colitis patients showed that lipin-1 expression is increased in those disorders and correlates with the expression of the proinflammatory markers CXCL1 and CXCL2. And finally, clinically, LPIN1 expression had prognostic value in inflammatory and stem-cell subtypes of colon cancers. Collectively, these data demonstrate that lipin-1 is a critical regulator of intestinal inflammation and inflammation-driven colon cancer development.
Clara Meana, Ginesa García-Rostán, Lucía Peña, Gema Lordén, África Cubero, Antonio Orduña, Balázs Győrffy, Jesús Balsinde, María A. Balboa
Paneth cells contribute to small intestinal homeostasis by secreting antimicrobial peptides and constituting the intestinal stem cell (ISC) niche. Certain T cell–mediated enteropathies are characterized by extensive Paneth cell depletion coincident with mucosal destruction and dysbiosis. In this study, mechanisms of intestinal crypt injury have been investigated by characterizing responses of mouse intestinal organoids (enteroids) in coculture with mouse T lymphocytes. Activated T cells induced enteroid damage, reduced Paneth cell and Lgr5+ ISC mRNA levels, and induced Paneth cell death through a caspase-3/7–dependent mechanism. IFN-γ mediated these effects, because IFN-γ receptor–null enteroids were unaffected by activated T cells. In mice, administration of IFN-γ induced enteropathy with crypt hyperplasia, villus shortening, Paneth cell depletion, and modified ISC marker expression. IFN-γ exacerbated radiation enteritis, which was ameliorated by treatment with a selective JAK1/2 inhibitor. Thus, IFN-γ induced Paneth cell death and impaired regeneration of small intestinal epithelium in vivo, suggesting that IFN-γ may be a useful target for treating defective mucosal regeneration in enteric inflammation.
Yoshihiro Eriguchi, Kiminori Nakamura, Yuki Yokoi, Rina Sugimoto, Shuichiro Takahashi, Daigo Hashimoto, Takanori Teshima, Tokiyoshi Ayabe, Michael E. Selsted, André J. Ouellette
Altered response to the intestinal microbiota strongly associates with inflammatory bowel disease (IBD); however, how commensal microbial cues are integrated by the host during the pathogenesis of IBD is not understood. Epigenetics represents a potential mechanism that could enable intestinal microbes to modulate transcriptional output during the development of IBD. Here, we reveal a histone methylation signature of intestinal epithelial cells isolated from the terminal ilea of newly diagnosed pediatric IBD patients. Genes characterized by significant alterations in histone H3-lysine 4 trimethylation (H3K4me3) showed differential enrichment in pathways involving immunoregulation, cell survival and signaling, and metabolism. Interestingly, a large subset of these genes was epigenetically regulated by microbiota in mice and several microbiota-sensitive epigenetic targets demonstrated altered expression in IBD patients. Remarkably though, a substantial proportion of these genes exhibited H3K4me3 levels that correlated with the severity of intestinal inflammation in IBD, despite lacking significant differential expression. Collectively, these data uncover a previously unrecognized epigenetic profile of IBD that can be primed by commensal microbes and indicate sensitive targets in the epithelium that may underlie how microbiota predispose to subsequent intestinal inflammation and disease.
Daniel Kelly, Michael Kotliar, Vivienne Woo, Sajjeev Jagannathan, Jordan Whitt, Jessica Moncivaiz, Bruce J. Aronow, Marla C. Dubinsky, Jeffrey S. Hyams, James F. Markowitz, Robert N. Baldassano, Michael C. Stephens, Thomas D. Walters, Subra Kugathasan, Yael Haberman, Nambirajan Sundaram, Michael J. Rosen, Michael Helmrath, Rebekah Karns, Artem Barski, Lee A. Denson, Theresa Alenghat
SLC26A3 (downregulated in adenoma; DRA) is a Cl–/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl– exchange with HCO3–, I–, and thiocyanate (SCN–), with an IC50 of ~0.2 μM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.
Peter M. Haggie, Onur Cil, Sujin Lee, Joseph-Anthony Tan, Amber A. Rivera, Puay-Wah Phuan, Alan S. Verkman
Adenosine-to-inosine (A-to-I) RNA editing, a process mediated by adenosine deaminases that act on the RNA (ADAR) gene family, is a recently discovered epigenetic modification dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in colorectal cancer (CRC) remain unclear. We have systematically and comprehensively investigated the significance of the expression status of ADAR1 and of the RNA editing levels of antizyme inhibitor 1 (AZIN1), one of the most frequently edited genes in cancers, in 392 colorectal tissues from multiple independent CRC patient cohorts. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues when compared with corresponding normal mucosa. High levels of AZIN1 RNA editing emerged as a prognostic factor for overall survival and disease-free survival and were an independent risk factor for lymph node and distant metastasis. Furthermore, elevated AZIN1 editing identified high-risk stage II CRC patients. Mechanistically, edited AZIN1 enhances stemness and appears to drive the metastatic processes. We have demonstrated that edited AZIN1 functions as an oncogene and a potential therapeutic target in CRC. Moreover, AZIN1 RNA editing status could be used as a clinically relevant prognostic indicator in CRC patients.
Kunitoshi Shigeyasu, Yoshinaga Okugawa, Shusuke Toden, Jinsei Miyoshi, Yuji Toiyama, Takeshi Nagasaka, Naoki Takahashi, Masato Kusunoki, Tetsuji Takayama, Yasuhide Yamada, Toshiyoshi Fujiwara, Leilei Chen, Ajay Goel
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