Enteroendocrine cell regulation of the gut-brain axis.

Enteroendocrine cells (EECs) are an essential interface between the gut and brain that communicate signals about nutrients, pain, and even information from our microbiome. EECs are hormone-producing cells expressed throughout the gastrointestinal epithelium and have been leveraged by pharmaceuticals like semaglutide (Ozempic, Wegovy), terzepatide (Mounjaro), and retatrutide (Phase 2) for diabetes and weight control, and linaclotide (Linzess) to treat irritable bowel syndrome (IBS) and visceral pain. This review focuses on role of intestinal EECs to communicate signals from the gut lumen to the brain. Canonically, EECs communicate information about the intestinal environment through a variety of hormones, dividing EECs into separate classes based on the hormone each cell type secretes. Recent studies have revealed more diverse hormone profiles and communication modalities for EECs including direct synaptic communication with peripheral neurons. EECs known as neuropod cells rapidly relay signals from gut to brain via a direct communication with vagal and primary sensory neurons. Further, this review discusses the complex information processing machinery within EECs, including receptors that transduce intraluminal signals and the ion channel complement that govern initiation and propagation of these signals. Deeper understanding of EEC physiology is necessary to safely treat devastating and pervasive conditions like irritable bowel syndrome and obesity.

Intestinal neuropod cell GUCY2C regulates visceral pain.

Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP(cGMP), which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we revealed that intestinal GUCY2C was selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2Chi neuropod cells associated with cocultured dorsal root ganglia neurons and induced hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient - but not GUCY2C-deficient - neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling in VP that was comparable with chemically induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.

Chimeric adenoviral (Ad5.F35) and listeria vector prime-boost immunization is safe and effective for cancer immunotherapy.

Strategies to augment immunity to self/neoantigens expressed by cancers are urgently needed to expand the proportion of patients benefiting from immunotherapy, particularly for GI cancers where only a fraction of patients respond to immunotherapies. However, current vaccine strategies are limited by poor immunogenicity, pre-existing vector-specific immunity, and vaccine-induced vector-specific immunity. Here, we examined a prime-boost strategy using a chimeric adenoviral vector (Ad5.F35) that resists pre-existing immunity followed by recombinant Listeria monocytogenes (Lm) to amplify immunity to the GI cancer antigen GUCY2C. This previously unexplored combination enhanced the quantity, avidity, polyfunctionality, and antitumor efficacy of GUCY2C-specific effector CD8+ T cells, without toxicity in any tissue, including GUCY2C-expressing intestines and brain. Importantly, this combination was partially resistant to pre-existing immunity to Ad5 which is endemic in human populations and vector-specific immunity did not limit the ability of multiple Lm administrations to repeatedly enhance GUCY2C-specific responses. Broadly, these findings suggest that cancer patient immunizations targeting self/neoantigens, as well as immunizations for difficult infectious diseases (HIV, malaria, etc), may be most successful using a combination of Ad5.F35-based priming, followed by Lm-based boosting. More specifically, Lm-GUCY2C may be utilized to amplify GUCY2C-specific immunity in patients receiving adenovirus-based GUCY2C vaccines currently in clinical trials to prevent or treat recurrent GI cancer.

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C.

The Gram-positive bacterium Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8+ T-cell responses towards the dominant H-2Kd-restricted epitope, GUCY2C254-262. However, Lm-GUCY2C produced robust CD8+ T-cell responses towards Lm-derived peptides suggesting that GUCY2C254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C254-262 immunity completely suppressed GUCY2C254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2Kd compared to GUCY2C254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2Kd binding, producing GUCY2CF255Y, significantly improved stability with H-2Kd and rescued GUCY2C254-262 immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines.

Guanylyl cyclase C as a biomarker for immunotherapies for the treatment of gastrointestinal malignancies.

Gastrointestinal cancers encompass a diverse class of tumors arising in the GI tract, including esophagus, stomach, pancreas and colorectum. Collectively, gastrointestinal cancers compose a high fraction of all cancer deaths, highlighting an unmet need for novel and effective therapies. In this context, the transmembrane receptor guanylyl cyclase C (GUCY2C) has emerged as an attractive target for the prevention, detection and treatment of many gastrointestinal tumors. GUCY2C is an intestinally-restricted protein implicated in tumorigenesis that is universally expressed by primary and metastatic colorectal tumors as well as ectopically expressed by esophageal, gastric and pancreatic cancers. This review summarizes the current state of GUCY2C-targeted modalities in the management of gastrointestinal malignancies, with special focus on colorectal cancer, the most incident gastrointestinal malignancy.

From leptin to lasers: the past and present of mouse models of obesity.

Introduction: Obesity is a prevalent condition that accounts for significant morbidity and mortality across the globe. Despite substantial effort, most obesity pharmacotherapies have proven unsafe or ineffective. The use of obese mouse models provides unique insight into the hormones and mechanisms that regulate appetite and metabolism. Paramount among these models are the 'obese' and 'diabetic' mice that revealed the powerful satiety hormone leptin, revolutionizing obesity research.Areas Covered: In this article, the authors discuss work on leptin therapy, and the clinical response to leptin in humans. The authors describe the use of modern mouse genetics to study targetable mechanisms for genetic forms of human obesity. Additionally, they describe mouse models of neuromodulation and their utility in unraveling neural circuits that govern appetite and metabolism.Expert opinion: Combining past and present models of obesity is required for the development of safe, effective, and impactful obesity therapy. Current research in obesity can benefit from repositories of genetically engineered mouse models to discover interactions between appetitive systems and circuits. Combining leptin therapy with other satiety signals comprising the gut-brain axis is a promising approach to induce significant enduring weight loss.

Silencing the intestinal GUCY2C tumor suppressor axis requires APC loss of heterozygosity.

Most sporadic colorectal cancer reflects acquired mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, while germline heterozygosity for mutant APC produces the autosomal dominant disorder Familial Adenomatous Polyposis (FAP) with a predisposition to colorectal cancer. In these syndromes, loss of heterozygosity (LOH) silences the remaining normal allele of APC, through an unknown mechanism, as the initiating step in transformation. Guanylyl cyclase C receptor (GUCY2C) and its hormones, uroguanylin and guanylin, have emerged as a key signaling axis opposing mutations driving intestinal tumorigenesis. Indeed, uroguanylin and guanylin are among the most commonly repressed genes in colorectal cancer. Here, we explored the role of APC heterozygosity in mechanisms repressing hormone expression which could contribute to LOH. In genetic mouse models of APC loss, uroguanylin and guanylin expression were quantified following monoallelic or biallelic deletion of the Apc gene. Induced biallelic loss of APC repressed uroguanylin and guanylin expression. However, monoallelic APC loss in Apcmin/+ mice did not alter hormone expression. Similarly, in FAP patients, normal colonic mucosa (monoallelic APC loss) expressed guanylin while adenomas and an invasive carcinoma (biallelic APC loss) were devoid of hormone expression. Thus, uroguanylin and guanylin expression by normal intestinal epithelial cells persists in the context of APC heterozygosity and is lost only after tumor initiation by APC LOH. These observations reveal a role for loss of the hormones silencing the GUCY2C axis in tumor progression following biallelic APC loss, but not in mechanisms creating the genetic vulnerability in epithelial cells underlying APC LOH initiating tumorigenesis.

Molecular characterization of the human lens epithelium-derived cell line SRA01/04.

Cataract-associated gene discovery in human and animal models have informed on key aspects of human lens development, homeostasis and pathology. Additionally, in vitro models such as the culture of permanent human lens epithelium-derived cell lines (LECs) have also been utilized to understand the molecular biology of lens cells. However, these resources remain uncharacterized, specifically regarding their global gene expression and suitability to model lens cell biology. Therefore, we sought to molecularly characterize gene expression in the human LEC, SRA01/04, which is commonly used in lens studies. We first performed short tandem repeat (STR) analysis and validated SRA01/04 LEC for its human origin, as recommended by the eye research community. Next, we used Illumina HumanHT-12 v3.0 Expression BeadChip arrays to gain insights into the global gene expression profile of SRA01/04. Comparative analysis of SRA01/04 microarray data was performed using other resources such as the lens expression database iSyTE (integrated Systems Tool for Eye gene discovery), the cataract gene database Cat-Map and the published lens literature. This analysis showed that SRA01/04 significantly expresses >40% of the top iSyTE lens-enriched genes (313 out of 749) across different developmental stages. Further, SRA01/04 also significantly expresses ~53% (168 out of 318) of cataract-associated genes in Cat-Map. We also performed comparative gene expression analysis between SRA01/04 cells and the previously validated mouse LEC 21EM15. To gain insight into whether SRA01/04 reflects epithelial or fiber cell characteristics, we compared its gene expression profile to previously reported differentially expressed genes in isolated mouse lens epithelial and fiber cells. This analysis suggests that SRA01/04 has reduced expression of several fiber cell-enriched genes. In agreement with these findings, cell culture analysis demonstrates that SRA01/04 has reduced potential to initiate spontaneous lentoid body formation compared to 21EM15 cells. Next, to independently validate SRA01/04 microarray gene expression, we subjected several candidate genes to RT-PCR and RT-qPCR assays. This analysis demonstrates that SRA01/04 supports expression of many key genes associated with lens development and cataract, including CRYAB, CRYBB2, CRYGS, DKK3, EPHA2, ETV5, GJA1, HSPB1, INPPL1, ITGB1, PAX6, PVRL3, SFRP1, SPARC, TDRD7, and VIM, among others, and therefore can be relevant for understanding the mechanistic basis of these factors. At the same time, SRA01/04 cells do not exhibit robust expression of several genes known to be important to lens biology and cataract such as ALDH1A1, COL4A6, CP, CRYBA4, FOXE3, HMX1, HSF4, MAF, MEIS1, PITX3, PRX, SIX3, and TRPM3, among many others. Therefore, the present study offers a rich transcript-level resource for case-by-case evaluation of the potential advantages and limitations of SRA01/04 cells prior to their use in downstream investigations. In sum, these data show that the human LEC, SRA01/04, exhibits lens epithelial cell-like character reflected in the expression of several lens-enriched and cataract-associated genes, and therefore can be considered as a useful in vitro resource when combined with in vivo studies to gain insight into specific aspects of human lens epithelial cells.