Gastroenterology
Abstract
Genetic variants in lipid metabolism influence the risk of developing metabolic dysfunction-associated steatotic liver disease (MASLD), cirrhosis, and end-stage liver disease (ESLD). The mechanisms by which these variants drive disease are poorly understood. Because of the PNPLA3-I148M variant's strong correlation with all stages of the MASLD spectrum and the lack of tractable therapeutic targets, we sought to understand its impact on cellular function and liver metabolism. Primary human hepatocytes (HAH) and iPSC-derived hepatocytes (iHeps) from healthy individuals possessing the PNPLA3-I148M mutation were characterized for changes in lipid metabolism, cellular stress, and survival. Using lipidomics, metabolomics, stable isotope tracing, and flux propensity analysis, we created a comprehensive metabolic profile of the changes associated with the PNPLA3-I148M variant. Functional analysis showed that the presence of the PNPLA3-I148M variant increased endoplasmic reticulum stress, mitochondrial dysfunction, and peroxisomal β-oxidation, ultimately leading to cell death via ferroptosis. Nutritional interventions, ferroptosis-specific inhibitors, and genetic approaches modulating GPX4 activity in PNPLA3-I148M HAH and iHeps decreased programmed cell death. Our findings indicate that therapies targeting ferroptosis in patients carrying the PNPLA3-I148M variant could affect the development of MASLD and ESLD and highlight the utility of iPSC-based models for the study of genetic contributions to hepatic disorders.
Authors
Rodrigo M. Florentino, Olamide Animasahun, Nils Haep, Minal Nenwani, Kehinde Omoloja, Leyla Nurcihan Altay, Abhinav Achreja, Kazutoyo Morita, Takashi Motomura, Ricardo Diaz-Aragon, Lanuza AP Faccioli, Yiyue Sun, Zhenghao Liu, Zhiping Hu, Bo Yang, Fulei Wuchu, Ajay Shankaran, Miya Paserba, Annalisa M. Baratta, Shohrat Arazov, Zehra N. Kocas-Kilicarslan, Noah Meurs, Jaideep Behari, Edgar N. Tafaleng, Jonathan Franks, Alina Ostrowska, Takahiro Tomiyama, Kyohei Yugawa, Akinari Morinaga, Zi Wang, Kazuki Takeishi, Dillon C. Gavlock, Mark Miedel, D. Lansing Taylor, Ira J. Fox, Tomoharu Yoshizumi, Deepak Nagrath, Alejandro Soto-Gutierrez
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a rapidly metastasizing cancer characterized by a dense desmoplastic stroma comprised of extracellular matrix (ECM) proteins, which complicates treatment. Upon stimulation, pancreatic stellate cells (PSCs) differentiated into cancer-associated fibroblasts (CAFs) that are the source of ECM and cytokines in PDAC. We previously reported that mechanical stress activates PSCs and induces fibrosis through mechanical ion channel PIEZO1-mediated TRPV4 channel activation, but its role in PDAC remains unclear. Here we report that pathological activation of PIEZO1 differentiated human PSCs into an inflammatory CAF phenotype that expresses chemoresistance and cancer stemness markers CD10 and GPR77. In an orthotopic PDAC model, TRPV4 knockout mice exhibited a significant reduction in tumor size, circulating inflammatory cytokines, tissue inhibitor of metalloproteinases-1 (TIMP1), and pre-metastatic niche markers, serum amyloid A (SAA) proteins. A similar trend was observed in mice lacking functional PIEZO1 in PSCs. The livers of TRPV4 knockout mice exhibited fewer cancer cell microlesions, lacked macro tumors, produced lower levels of inflammatory protein S100A8, and developed fewer inflammatory cell clusters. In orthotopic and genetically engineered models of PDAC, these mice also had improved survival, suggesting that blocking TRPV4 channels may be a promising therapeutic target for PDAC.
Authors
Joelle M.-J. Romac, Sandip M. Swain, Nidula Mullappilly, Bandana Bindhani, Rodger A. Liddle
Abstract
Clinical trials have identified 2 distinct eosinophilic esophagitis (EoE) treatment phenotypes: those that show proton pump inhibitor (PPI) responsiveness (PPI-R) and those that show PPI unresponsiveness (PPI-UR). Comprehensive clinical, endoscopic, and RNA-Seq analyses of patients with EoE prior to and following PPI therapy have not previously been performed to our knowledge. We showed that clinical, endoscopic, and histologic evaluation of esophageal biopsies from pediatric PPI-R and PPI-UR individuals with EoE prior to PPI therapy (diagnosis) were indistinguishable. RNA-Seq analyses revealed common immune and inflammatory transcriptional signatures in both PPI-R EoE and PPI-UR EoE esophageal biopsy samples at diagnosis and distinct signatures enriched for processes related to neuropeptide signaling and cell cycle and division. PPI therapy induced histologic, endoscopic, and transcriptional remission in PPI-R EoE, but not in PPI-UR EoE. Persistent disease in PPI-UR EoE was associated with the presence of Th2 inflammatory and dedifferentiated esophageal epithelial transcriptomic signatures, while PPI-R EoE revealed genes enriched in cellular responses to LPS, host defense against viruses, and type I IFN signaling. In silico analyses identified common and unique EoE disease gene drivers in PPI-R and PPI-UR EoE. These studies indicate that the 2 EoE phenotypes have unique transcriptomic elements that underlie the molecular nature of PPI-R and PPI-UR EoE disease.
Authors
Somdutta Chakraborty, Ankit Sharma, Sahiti Marella, Christian F. Rizza, Patrick A. O’Brien, Varsha Ganesan, Gila Idelman, Susie Min, Mayee Chen, Talaya McCright-Gill, Nancy Gonzalez, Alexandros D. Polydorides, Paul S. Foster, Simon P. Hogan, Mirna Chehade
Abstract
Intestinal epithelial barrier-integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the epithelial cytoskeleton is critical for maintaining gut barrier-integrity, the role of septins- a family of GTP-binding, cytoskeletal proteins- is largely unknown. This highlights an important knowledge gap as dysfunction of septins, and specifically septin 9 (SEPT9), is associated with intestinal pathologies. We determined that SEPT9 localizes to the apical junctions of intestinal epithelial cells (IECs), overlapping with both tight and adherens junctions. IEC-specific ablation of SEPT9 in mice resulted in leaky gut, due to mislocalization of junctional proteins, and increased susceptibility to experimental colitis. Consistently, SEPT9 expression was significantly reduced in intestinal mucosa of inflammatory bowel disease (IBD) patients. Using affinity-purification mass spectrometry, super-resolution imaging, and genetic knockout, we determined that SEPT9 interacts with and is necessary to recruit non-muscle myosin IIC (NMIIC) to the IEC peri-junctional actomyosin belt. Loss of NMIIC also caused IEC barrier disruption. In summary, SEPT9 regulates intestinal barrier-integrity by supporting the assembly of tight and adherens junctions through NMIIC recruitment to the actomyosin belt. The septin cytoskeleton safeguards the intestinal mucosa during acute inflammation, and its disruption in IBD suggests a loss of this protective function.
Authors
Nayden G. Naydenov, Gaizun Hu, Dominik Robak, Atif Zafar, Khosiyat Makhmudova, Susana Lechuga, Yuta Ohno, Naseer Sangwan, Saikat Bandyopadhyay, Ryan Musich, Erin Jeffery, Lei Sun, Armando Marino-Melendez, Florian Rieder, Gloria Sheynkman, Andrei I. Ivanov, Seham Ebrahim
Abstract
Bowel smooth muscle experiences mechanical stress constantly during normal function, and pathologic mechanical stressors in disease states. We tested the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. To test this hypothesis, primary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-ε-caprolactone nano-fibrous scaffolds, were subjected to pathologic, high frequency (1 Hz) uniaxial 3% cyclic stretch (loaded) or kept unloaded in culture for 6 hours. RNA sequencing, qRT-PCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. These studies showed loading induced differential expression of 4537 HISMC genes. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors, and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle and on other cells in the bowel wall. These data show HISMCs undergo remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMCs into proliferative, fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.
Authors
Sharon M. Wolfson, Katherine Beigel, Sierra E. Anderson, Brooke Deal, Molly Weiner, Se-Hwan Lee, Deanne M. Taylor, Su Chin Heo, Robert O. Heuckeroth, Sohaib K. Hashmi
Abstract
The intestinal mucosal epithelium forms a barrier between luminal contents and the body. microRNAs (miRNAs) regulate mucosal homeostasis by controlling inflammatory responses and structural integrity. Here, we discovered a protective role for miR147 in intestinal inflammation using a miR147tdTomato reporter mouse. miR147 was enriched in the intestines, with the highest expression in the colonic epithelial cells at the luminal surface, with prominent expression in differentiated enterocytes. Mice with general or intestinal epithelial deletion of miR147 showed increased intestinal inflammation and diminished mucosal healing during colitis. RNA sequencing of miR147-deficient cells showed dysregulated immune signaling, with upregulated pro-inflammatory cytokine pathways and reduced type I interferon responses and revealed Ndufa4 as a likely miR147 target. Ndufa4, a mitochondrial protein regulating energy metabolism and inflammation, is elevated at the crypt base, inversely correlating with miR147. Mice lacking the miR147 binding site in Ndufa4’s 3′ untranslated region phenocopied miR147-deficient mice during colitis. Spatial and single-cell transcriptomic analyses in murine and human colons showed mutually exclusive miR147 and Ndufa4 expression, consistent with a regulatory relationship in epithelial differentiation and metabolism. These findings underscore miR147’s role in intestinal homeostasis and mucosal healing, suggesting it as a therapeutic target for inflammatory bowel disease.
Authors
Agnieszka K. Czopik, Arash Dabiri, Chia-Hao Tung, Victoria Vaughn, Xiangsheng Huang, Jinlian Wang, Hui Li, Nicolas F. Moreno, Natalia V. Piwko, Katherine Figarella, Hongfang Liu, Zhongming Zhao, Xiaoyi Yuan, Holger K. Eltzschig
Abstract
In celiac disease (CeD), a gluten-dependent autoimmune disorder, transglutaminase 2 (TG2) deamidates selected glutamine residues in gluten peptides, while HLA-DQ2 presents deamidated antigens to inflammatory T cells. The cellular sources of pathogenic TG2 and DQ2 are unclear. Using chemical biology tools, we show that intestinal CD103+ dendritic cells (DCs) couple cell-surface TG2 to the endocytic LRP1 receptor to simultaneously deamidate gluten antigens and concentrate them in lysosomes. In DQ2-transgenic mice, CD103+ DCs loaded with deamidated antigens migrate from intestinal lamina propria and Peyer’s patches into mesenteric lymph nodes, where they engage T cells. In turn, gluten antigen presentation upregulates intestinal TG2 activity. The tool (HB-230) used to establish a role of CD103+ DCs in gluten antigen presentation and TG2 activation in mice also revealed that the TG2/LRP1 pathway is active in human CD14+ monocytes. Within this population of circulating monocytes, a DC subset with the gut-homing β7-integrin marker is elevated in CeD patients with active disease compared to non-celiac controls or patients on a gluten-free diet. Our findings not only inform the cellular basis for gluten toxicity in CeD but they also highlight the immunologic role of an enigmatic protein of growing therapeutic relevance in CeD and other immune disorders.
Authors
Fu-Chen Yang, Harrison A. Besser, Hye Rin Chun, Megan Albertelli, Nielsen Q. Fernandez-Becker, Bana Jabri, Chaitan Khosla
Abstract
BACKGROUND. Glucagon-like peptide-2 (GLP-2) analogs are used clinically to enhance nutrient absorption in patients with short bowel syndrome (SBS); however, the precise mechanism remains unclear. To address this, the study aimed to clarify the dynamics of intestinal epithelial cells and immune cells in patients with SBS treated with GLP-2 analogs. METHODS. Five male patients diagnosed with SBS, all of whom received treatment with the GLP-2 analog teduglutide, were included in the study. We conducted longitudinal single-cell RNA sequencing (scRNA-seq) analysis of intestinal tissue from SBS patients over a year, integrating microbiome composition analysis. RESULTS. After treatment, the alpha diversity of the gut microbiome increased, indicating a more varied microbial environment. ScRNA-seq analysis revealed a reduction of T helper 2 cells and an increase in regulatory T (Treg) cells, suggesting a shift towards an immunoregulatory intestinal environment. Additionally, nutrient-absorbing enterocyte-Top2 and middle clusters expanded, enhancing the absorption capacity, whereas major histocompatibility complex class I/II-expressing enterocyte-Top1 cells declined, potentially modulating immune responses. CONCLUSION. The study findings indicate that GLP-2 analogs reshape intestinal immunity and microbiota, fostering a less inflammatory environment while promoting nutrient uptake efficiency. These insights offer a deeper understanding of the role of GLP-2 analogs in gut adaptation and provide a foundation for refining clinical strategies for SBS treatment. FUNDING. This work was supported by Sakaguchi Memorial Foundation, Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) (21K18272, 23H03665, 23H02899, 23K27590, 25K22627, 23K08037), JST FOREST(21457195), and the Takeda Japan Medical Office Funded Research Grant 2022.
Authors
Yumi Kudo, Kentaro Miyamoto, Shohei Suzuki, Akihiko Chida, Anna Tojo, Mai Hasegawa, Arina Shigehara, Ikuko Koya, Yoshinari Ando, Masayasu Sato, Aya Kondo, Tomoko Kumagai, Harunori Deguchi, Yoshiki Sugiyama, Yoko Ito, Koji Shirosaki, Satoko Yamagishi, Yutaro Maeda, Hiroki Kanamori, Motohiro Kano, Mototoshi Kato, Hanako Tsujikawa, Yusuke Yoshimatsu, Kaoru Takabayashi, Koji Okabayashi, Takanori Kanai, Naoki Hosoe, Motohiko Kato, Jonathan Moody, Chung-Chau Hon, Tatsuo Kuroda, Yohei Yamada, Akihiro Fujino, Tomohisa Sujino
Abstract
Authors
Veethika Pandey, Heike R. Doeppler, Ligia I. Bastea, Alicia K. Fleming Martinez, Barath Shreeder, Brandy H. Edenfield, Keith L. Knutson, DeLisa Fairweather, Peter Storz
Abstract
Glycans are one of the 4 major macromolecules essential for life and are the most abundant family of organic molecules. However, in contrast with DNA and RNA, glycan structures have no template; this results in limited tools to study this challenging macromolecule with a diversity of glycan structures. A central bottleneck in studying glycosylation in vivo is that inhibitors and complete KOs are lethal. In a forward genetic screen, we identified a viable, hypomorphic mutation at a conserved site in mannose phosphate isomerase (Mpi) that causes a multisystemic phenotype affecting RBCs, liver, stomach, intestines, skin, size, fat, and fluid balance in mice. The phenotype could be rescued with mannose. Analyses of glycopeptides in mice with this mutation showed a 500% increase in unoccupied N-glycan sites. This is equivalent to a “glycan knockdown,” which would be useful for examining the role of glycans in biology and disease. Therefore, we report an in vivo tool to study global N-glycosylation deficiency with tissue-specific targeting and a rescue mechanism with mannose.
Authors
Elisa B. Lin, Steve Meregini, Zhao Zhang, Avishek Roy, Tandav Argula, James M. Mitchell, William J. Israelsen, Sara Ludwig, Jamie Russell, Jiexia Quan, Sara Hildebrand, Evan Nair-Gill, Bruce Beutler, Jeffrey A. SoRelle
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