Regulation Of Intestinal Homeostasis Research Articles

Intestinal Barrier Protection by Probiotic Lactobacillus-Associated Indoles in Caco-2 and Enteroids

Gut bacteria generate from dietary tryptophan (trp) indole compounds, which act as potent signaling molecules intricately involved in the regulation of intestinal homeostasis and barrier integrity. Lactobacillus rhamnosus GG (LGG), a widely used probiotic, has demonstrated ability to restore the gut barrier in animal models of colitis and sepsis, as well as in children with irritable bowel syndrome. To investigate the hypothesis that LGG-derived indoles are responsible for its gut barrier-enhancing properties, we fed gnotobiotic mice trp-suffcient and trp-deficient diets. We then monocolonized them with LGG, using PBS gavaged mice as a control, and analyzed the ileal transcriptome by bulk RNA sequencing and both fecal and serum metabolomes by LC-MS. Using our novel bioinformatic tool METRCA, which correlates metabolites and gene networks, we uncovered an unexpected and substantial influence of indole spatial distribution on genes associated with gut integrity: some were significantly positively linked to tight junction (TJ) genes exclusively from the luminal side, some exclusively from the serum, and others from both. Specifically, we identified nine indoles—indole-acetamide (IAM), indole-carboxaldehyde (IAL), indole-3-lactic acid (ILA), indole-3-propionic acid (IPA), indole-3-acrylic acid (IAR), indole-3-carbinol (ICB), indole-acetic acid (IAA), indole-3-acetonitrile (IAN), and indole-3-carboxylic acid (ICA)—that demonstrated the potential to maintain gut barrier function. To assess their impact, we used a high-throughput system that measured permeability through transepithelial electrical resistance (TEER) and FITC-dextran (4 kDa) in polarized Caco-2 cells. By luminally challenging cells with Clostridium diffcile toxins (TOX), we induced barrier disruption to model leaky gut. Intriguingly, when applied luminally, IAA, IAN, ICA, and ILA rescued the TEER of Caco-2 cells exposed to TOX, while IAM, IAL, and ILA were effective when applied basolaterally, suggesting a strong polar component to their barrier repair capacity. FITC transport revealed a significant basolateral rescue effect of IAM and luminal rescue effect of IAA and ICA, indicating that different sets of indoles are required to repair the pore and leak pathways. Furthermore, we confirmed that basolateral IAM rescued FITC permeability and induced increased expression of TJ genes Tjp1 and Ocln in enteroids challenged with lipopolysaccharides. In summary, our research highlights the potential beneficial therapeutic application of certain indoles in repairing disrupted gut barriers. NIHR01-AT010243 (NG, RF), R01DK119198 (NG), NSF 1754783 (RF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Decreased expression of the NLRP6 inflammasome is associated with increased intestinal permeability and inflammation in obesity with type 2 diabetes

BackgroundObesity-associated dysfunctional intestinal permeability contributes to systemic chronic inflammation leading to the development of metabolic diseases. The inflammasomes constitute essential components in the regulation of intestinal homeostasis. We aimed to determine the impact of the inflammasomes in the regulation of gut barrier dysfunction and metabolic inflammation in the context of obesity and type 2 diabetes (T2D).MethodsBlood samples obtained from 80 volunteers (n = 20 normal weight, n = 21 OB without T2D, n = 39 OB with T2D) and a subgroup of jejunum samples were used in a case–control study. Circulating levels of intestinal damage markers and expression levels of inflammasomes as well as their main effectors (IL-1β and IL-18) and key inflammation-related genes were analyzed. The impact of inflammation-related factors, different metabolites and Akkermansia muciniphila in the regulation of inflammasomes and intestinal integrity genes was evaluated. The effect of blocking NLRP6 by using siRNA in inflammation was also studied.ResultsIncreased circulating levels (P < 0.01) of the intestinal damage markers endotoxin, LBP, and zonulin in patients with obesity decreased (P < 0.05) after weight loss. Patients with obesity and T2D exhibited decreased (P < 0.05) jejunum gene expression levels of NLRP6 and its main effector IL18 together with increased (P < 0.05) mRNA levels of inflammatory markers. We further showed that while NLRP6 was primarily localized in goblet cells, NLRP3 was localized in the intestinal epithelial cells. Additionally, decreased (P < 0.05) mRNA levels of Nlrp1, Nlrp3 and Nlrp6 in the small intestinal tract obtained from rats with diet-induced obesity were found. NLRP6 expression was regulated by taurine, parthenolide and A. muciniphila in the human enterocyte cell line CCL-241. Finally, a significant decrease (P < 0.01) in the expression and release of MUC2 after the knockdown of NLRP6 was observed.ConclusionsThe increased levels of intestinal damage markers together with the downregulation of NLRP6 and IL18 in the jejunum in obesity-associated T2D suggest a defective inflammasome sensing, driving to an impaired epithelial intestinal barrier that may regulate the progression of multiple obesity-associated comorbidities.

Unraveling the genomic background of resilience based on variability in milk yield and milk production levels in North American Holstein cattle through genome-wide association study and Mendelian randomization analyses

Breeding more resilient animals will benefit the dairy cattle industry in the long term, especially as global climate changes become more severe. Previous studies have reported genetic parameters for various milk yield-based resilience indicators, but the underlying genomic background of these traits remain unknown. In this study, we conducted genome-wide association studies of 62,029 single nucleotide polymorphisms (SNPs) with 4 milk yield-based resilience indicators, including the weighted occurrence frequency (wfPert) and accumulated milk losses (dPert) of milk yield perturbations, and log-transformed variance (LnVar) and lag-1 autocorrelation (rauto) of daily yield residuals. These variables were previously derived from 5.6 million daily milk yield records from 21,350 lactations (parities 1 to 3) of 11,787 North American Holstein cows. The average daily milk yield (ADMY) throughout lactation was also included to compare the shared genetic background of resilience indicators with milk yield. The differential genetic background of these indicators was first revealed by the significant genomic regions identified and significantly-enriched biological pathways of positional candidate genes, which confirmed the genetic difference among resilience indicators. Interestingly, the functional analyses of candidate genes suggested that the regulation of intestinal homeostasis is most likely affecting resilience derived based on variability in milk yield. Based on Mendelian randomization analyses of multiple instrumental SNPs, we further found an unfavorable causal association of ADMY with LnVar. In conclusion, the resilience indicators evaluated are genetically different traits and there are causal associations of milk yield with some of the resilience indicators evaluated. In addition to providing biological insights into the molecular regulation mechanisms of resilience derived based on variability in milk yield, this study also indicates the need for developing selection indexes combining multiple indicator traits and taking into account their genetic relationship for breeding more resilient dairy cattle.

Genetic variants of MUC4 are associated with susceptibility to and mortality of colorectal cancer and exhibit synergistic effects with LDL-C levels.

As a disease with high mortality and prevalence rates worldwide, colorectal cancer (CRC) has been thoroughly investigated. Mucins are involved in the induction of CRC and the regulation of intestinal homeostasis but a member of the mucin gene family MUC4 has a controversial role in CRC. MUC4 has been associated with either decreased susceptibility to or a worse prognosis of CRC. In our study, the multifunctional aspects of MUC4 were elucidated by genetic polymorphism analysis in a case-control study of 420 controls and 464 CRC patients. MUC4 rs1104760 A>G polymorphism had a protective effect on CRC risk (AG, AOR = 0.537; GG, AOR = 0.297; dominant model, AOR = 0.493; recessive model, AOR = 0.382) and MUC4 rs2688513 A>G was associated with an increased mortality rate of CRC (5 years, GG, adjusted HR = 6.496; recessive model, adjusted HR = 5.848). In addition, MUC4 rs1104760 A>G showed a high probability of being a potential biomarker for CRC patients with low-density lipoprotein cholesterol (LDL-C) in the risk range while showing a significant synergistic effect with the LDL-C level. This is the first study to indicate a significant association between MUC4 genetic polymorphisms and CRC prevalence, suggesting a functional genetic variant with the LDL-C level, for CRC prevention.

Gut microbiota and oleoylethanolamide in the regulation of intestinal homeostasis.

A vast literature strongly suggests that the endocannabinoid (eCB) system and related bioactive lipids (the paracannabinoid system) contribute to numerous physiological processes and are involved in pathological conditions such as obesity, type 2 diabetes, and intestinal inflammation. The gut paracannabinoid system exerts a prominent role in gut physiology as it affects motility, permeability, and inflammatory responses. Another important player in the regulation of host metabolism is the intestinal microbiota, as microorganisms are indispensable to protect the intestine against exogenous pathogens and potentially harmful resident microorganisms. In turn, the composition of the microbiota is regulated by intestinal immune responses. The intestinal microbial community plays a fundamental role in the development of the innate immune system and is essential in shaping adaptive immunity. The active interplay between microbiota and paracannabinoids is beginning to appear as potent regulatory system of the gastrointestinal homeostasis. In this context, oleoylethanolamide (OEA), a key component of the physiological systems involved in the regulation of dietary fat consumption, energy homeostasis, intestinal motility, and a key factor in modulating eating behavior, is a less studied lipid mediator. In the small intestine namely duodenum and jejunum, levels of OEA change according to the nutrient status as they decrease during food deprivation and increase upon refeeding. Recently, we and others showed that OEA treatment in rodents protects against inflammatory events and changes the intestinal microbiota composition. In this review, we briefly define the role of OEA and of the gut microbiota in intestinal homeostasis and recapitulate recent findings suggesting an interplay between OEA and the intestinal microorganisms.

Maintenance of Intestinal Homeostasis in Diarrhea-Predominant Irritable Bowel Syndrome by Electroacupuncture Through Submucosal Enteric Glial Cell-Derived S-Nitrosoglutathione.

Objective: To determine whether electroacupuncture (EA) maintains intestinal homeostasis in diarrhea-predominant irritable bowel syndrome (IBS-D) rats by repairing intestinal barrier function through enteric glial cell (EGC)-derived S-nitrosoglutathione (GSNO). Methods: Sprague–Dawley rats were randomly divided into a control group (n = 10) and an IBS-D group (n = 20). These rats received senna solution by gavage and chronic unpredictable mild stress for 14 days and were further divided into a model group (n = 10) and an EA group (n = 10). Rats in the EA group were electroacupunctured at ST25 (Tianshu), ST36 (Zusanli), and LR3 (Taichong) for 20 min every day for 14 days. The abdominal withdrawal reflex (AWR), the percentage of time spent in open arms (OT%) in the elevated plus maze test, and the diarrhea index (DI) were measured. Histopathological examination was performed to evaluate the pathological features of the colon after sacrificing the rats. Transmission electron microscopy was used to observe the EGC in the muscle and submucosal layers. Enzyme-linked immunosorbent assay was performed to detect GSNO expression in the colon. Double immunofluorescence labeling was used to detect the colocalized GFAP and GSNO expressions in the muscle and submucosal layers. Plasma FITC-dextran was used to measure intestinal permeability, whereas western blot was used to detect ZO-1 and occludin expressions in the colon. Results: OT% and ZO-1 and occludin expressions were significantly lower than those of the control group, whereas AWR scores, DI, GSNO expression in the colon, colocalized GFAP and GSNO expressions in the submucosal layer, and intestinal permeability were significantly higher than those of the control group. Structural EGC abnormalities were observed in the model group. After EA treatment, OT% and ZO-1 and occludin expressions increased significantly, whereas AWR scores, DI, GSNO expression, colocalized GFAP and GSNO expressions in the submucosal layer, and intestinal permeability decreased significantly. The EGC structure was then restored to its normal state. Conclusion: EA treatment downregulates the submucosal EGC–derived GSNO expressions, repairs the intestinal barrier by upregulating the ZO-1 and occludin expression, and improves IBS-D symptoms, including visceral hypersensitivity, anxiety, and diarrhea, suggesting a potential role for EGC-derived GSNO in the regulation of intestinal homeostasis in IBS-D rats.

Effects of oxidation-based tea processing on the characteristics of the derived polysaccharide conjugates and their regulation of intestinal homeostasis in DSS-induced colitis mice

Different cultivars and processing technologies involved in producing tea result in the high heterogeneity of derived polysaccharide conjugates, which limits the understanding of their composition and structure, and biological activity. Here, raw tea leaves from the same cultivar were used to produce dried fresh tea leaves, green tea, and black tea, and three polysaccharide conjugates derived from dried fresh tea leaves (FTPS), green tea (GTPS), and black tea (BTPS) were prepared accordingly. Their physiochemical characteristics and bioactivities were investigated. The results showed that the oxidation during tea processing increased the phenolics and proteins while decreasing the GalA in the derived TPS conjugates; meanwhile, it reduced the molecular weight and particle size of BTPS but enhanced their antioxidant activity in vitro. Furthermore, all three TPS conjugates improved intestinal homeostasis by reducing TJ protein loss and inflammation and alleviated DSS-induced colitis symptoms in mice. In addition, the three TPS conjugates showed differential regulation of the intestinal microbiome and altered the produced SCFAs, which contributed to the prevention of colitis. Our findings suggest that TPS conjugates could be applied in colitis prevention in association with the regulation of gut microbiota, and their efficacy could be optimized by employing suitable tea processing technologies.

Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis.

The maintenance of intestinal homeostasis is fundamentally important to health. Intestinal barrier function and immune regulation are key determinants of intestinal homeostasis and are therefore tightly regulated by a variety of signaling mechanisms. The endocannabinoid system is a lipid mediator signaling system widely expressed in the gastrointestinal tract. Accumulating evidence suggests the endocannabinoid system is a critical nexus involved in the physiological processes that underlie the control of intestinal homeostasis. In this review we will illustrate how the endocannabinoid system is involved in regulation of intestinal permeability, fluid secretion, and immune regulation. We will also demonstrate a reciprocal regulation between the endocannabinoid system and the gut microbiome. The role of the endocannabinoid system is complex and multifaceted, responding to both internal and external factors while also serving as an effector system for the maintenance of intestinal homeostasis.

Xenobiotic receptors and the regulation of intestinal homeostasis: harnessing the chemical output of the intestinal microbiota.

The commensal bacteria that reside in the gastrointestinal tract exist in a symbiotic relationship with the host, driving the development of the immune system and maintaining metabolic and tissue homeostasis in the local environment. The intestinal microbiota has the capacity to generate a wide array of chemical metabolites to which the cells of the intestinal mucosa are exposed. Host cells express xenobiotic receptors, such as the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR), that can sense and respond to chemicals that are generated by nonhost pathways. In this review, we outline the physiological and immunological processes within the intestinal environment that are regulated by microbial metabolites through the activation of the AhR and the PXR, with a focus on ligands generated by the stepwise catabolism of tryptophan.

3,5,7,3',4'-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells.

The intestinal tight junction (TJ) barrier plays a pivotal role in the regulation of intestinal homeostasis. This study investigated the effects of 3,5,7,3',4'-pentamethoxyflavone (PMF), a major polymethoxyflavone found in black ginger, on TJ barrier regulation using intestinal Caco-2 cells. PMF treatment enhanced the TJ barrier integrity in Caco-2 cells, indicated by increased transepithelial electrical resistance (control, 1261 ± 36 Ω·cm2; 100 μM PMF, 1383 ± 55 Ω·cm2 at 48 h, p < 0.05) and decreased permeability to fluorescein-conjugated dextran (control, 24.2 ± 1.8 pmol/(cm2 × h); 100 μM PMF, 18.6 ± 1.0 pmol/(cm2 × h), p < 0.05). Immunoblot analysis revealed that PMF increased the cytoskeletal association and cellular expression of the TJ proteins, zonula occludens-1, claudin-3, and claudin-4 (e.g., occludin; control, 1.00 ± 0.2; 100 μM PMF, 3.69 ± 0.86 at 48 h, p < 0.05). Quantitative reverse transcriptase-polymerase chain reaction analysis and a luciferase promoter assay showed that PMF enhanced the transcription of occludin, claudin-3, and claudin-4. The promoter assay with site-directed mutagenesis indicated that PMF-induced occludin and claudin-3 transcription was mediated by transcription factors, KLF5 and EGR1, respectively, while PMF activated claudin-4 transcription through GATA1 and AP1. Taken together, the transcriptional regulation of TJ proteins is involved in PMF-mediated promotion of the intestinal barrier in vitro.

Cell scientist to watch – Vivian Li

ABSTRACT Vivian Li obtained her PhD from the University of Hong Kong in 2008, where she investigated the molecular mechanisms of human colonic development and tumorigenesis. Funded by a Croucher Foundation Fellowship, she joined the lab of Hans Clevers at the Hubrecht Institute in the Netherlands for her postdoctoral work. There, she identified novel Wnt signalling mechanisms at different subcellular levels and characterised intestinal stem cell genes using newly created transgenic mouse models. Vivian established her group at the MRC National Institute for Medical Research, which is now part of the Francis Crick Institute, London, in February 2013. In her lab she uses genetic mouse models and organoids to investigate the regulation of intestinal homeostasis and cancer with a primary focus on the Wnt signalling pathway. Vivian was awarded a Future Leaders in Cancer Research Prize in 2018 by Cancer Research UK and is the winner of the 2021 Women in Cell Biology Early Career Medal awarded by the British Society for Cell Biology.

Estrogen-related receptor alpha (ERR\u03b1) is a key regulator of intestinal homeostasis and protects against colitis

The estrogen-related receptor alpha (ERRα) is a primary regulator of mitochondrial energy metabolism, function and dynamics, and has been implicated in autophagy and immune regulation. ERRα is abundantly expressed in the intestine and in cells of the immune system. However, its role in inflammatory bowel disease (IBD) remains unknown. Here, we report a protective role of ERRα in the intestine. We found that mice deficient in ERRα were susceptible to experimental colitis, exhibiting increased colon inflammation and tissue damage. This phenotype was mediated by impaired compensatory proliferation of intestinal epithelial cells (IEC) following injury, enhanced IEC apoptosis and necrosis and reduced mucus-producing goblet cell counts. Longitudinal analysis of the microbiota demonstrated that loss of ERRα lead to a reduction in microbiome α-diversity and depletion of healthy gut bacterial constituents. Mechanistically, ERRα mediated its protective effects by acting within the radio-resistant compartment of the intestine. It promoted disease tolerance through transcriptional control of key genes involved in intestinal tissue homeostasis and repair. These findings provide new insights on the role of ERRα in the gut and extends our current knowledge of nuclear receptors implicated in IBD.

Impact of Transcriptome and Gut Microbiome on the Response of HIV-1 Infected Individuals to a Dendritic Cell-Based HIV Therapeutic Vaccine.

Therapeutic vaccines based on dendritic cells offer a good approach to HIV-specific T-cell responses and partial control of the viral load after antiretroviral therapy interruption. The aim of the present study was to identify mRNA expression profiles and to assess the impact of the gut microbiome composition for predicting the viral load control after antiretroviral therapy interruption. We enrolled 29 patients to receive either placebo or a monocyte-derived dendritic cell vaccine. Patients with a decrease in their viral load of >0.5 log10 copies/mL by 12 weeks after antiretroviral therapy interruption were considered responders. In total, 66 genes were considered differentially expressed between responders and non-responders. Enrichment analysis revealed several upregulated pathways involved in the host defense response to a virus via the type I interferon signaling pathway. Regarding the gut microbiota, responders showed enriched levels of Bacteroidetes (p < 0.005) and Verrucomicrobia (p = 0.017), while non-responders were enriched with Tenericutes (p = 0.049) and Actinobacteria (p < 0.005). We also found important differences at the genus level. However, we did not discover any effect of the dendritic cell vaccine on the transcriptome or the gut microbiota. An alternative analysis did characterize that the microbiota from responders were associated with the metabolic production of short-chain fatty acids, which are key metabolites in the regulation of intestinal homeostasis. The evidence now consistently shows that short-chain fatty acid depletion occurs in HIV-infected individuals receiving antiretroviral treatment.

Anti-cancer and anti-inflammatory effects elicited by short chain fatty acids produced by Escherichia coli isolated from healthy human gut microbiota

BackgroundExtracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study.ResultsEscherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression.ConclusionSCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.

A perspective of intestinal immune-microbiome interactions in alcohol-associated Liver Disease

Uncovering the intricacies of the gut microbiome and how it interacts with the host immune system has opened up pathways in the search for the treatment of disease conditions. Alcohol-associated liver disease is a major cause of death worldwide. Research has shed light on the breakdown of the protective gut barriers, translocation of gut microbes to the liver and inflammatory immune response to microbes all contributing to alcohol-associated liver disease. This knowledge has opened up avenues for alternative therapies to alleviate alcohol-associated liver disease based on the interaction of the commensal gut microbiome as a key player in the regulation of the immune response. This review describes the relevance of the intestinal immune system, the gut microbiota, and specialized and non-specialized intestinal cells in the regulation of intestinal homeostasis. It also reflects how these components are altered during alcohol-associated liver disease and discusses new approaches for potential future therapies in alcohol-associated liver disease.

TNFAIP8 controls murine intestinal stem cell homeostasis and regeneration by regulating microbiome-induced Akt signaling

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0−/− enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.

A new insight into the intestine of Pacific white shrimp: Regulation of intestinal homeostasis and regeneration in Litopenaeus vannamei during temperature fluctuation

Litopenaeus vannamei (L. vannamei) is an essential aquaculture shrimp throughout the world, but its aquaculture industry is threatened by temperature fluctuation. In this study, our histological results indicated that the shrimp intestine has a self-repairing ability during temperature fluctuation; however the potential mechanisms were still unknown. Therefore, transcriptome profiles of the intestine were collected from shrimp at 28 °C (C28), 13 °C (T13) and 28 °C after their temperature rose back (R28) and were analyzed. A total of 2229 differentially expressed genes (DEGs) (986 up- and 1243 downregulated) were identified in the C28 group, and 1790 DEGs (933 up- and 857 downregulated) were identified in the R28 group when compared to their expression levels in the T13 group. According to the functional annotation using KEGG, we found that the immune system was the most enriched section of organismal systems and that the shrimp can mobilize the body's immune response to regulate organism homeostasis during temperature fluctuation, although cold stress decreased the immunity. Additionally, metabolic inhibition is a strategy to cope with cold stress, and the regulation of lipid metabolism was especially important for shrimp during temperature fluctuation. Remarkably, the Hippo signaling pathway might help the repair of intestinal structure. Our research provides the first histological analysis and transcriptome profiling for the L. vannamei intestine during the temperature fluctuation stage. These results enrich our understanding of the mechanism of intestinal self-repair and homeostasis and could provide guidance for shrimp farming.

Abstract A16: A GUCY2c-CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors

Abstract Background: Guanylyl cyclase C (GUCY2C) is a regulator of intestinal homeostasis and is expressed in more than 90% of colorectal cancer (CRC), as well as in other gastrointestinal malignancies. Target expression in normal tissues is largely restricted to the apical side of intestinal epithelial tight junctions, which could allow for preferential uptake of GUCY2C-targeted biologics by tumors that have disrupted tight junction architecture. Here we demonstrate tumor-selective uptake and potent efficacy with a half-life extended, GUCY2C-CD3 bispecific molecule that recruits CD3-positive T cells to induce cytotoxicity of GUCY2C-expressing tumors in several CRC models in vivo. Additionally, to address immune evasion mechanisms, rational combinations of the bispecific are explored with immune checkpoint blockade agents, as well as by blocking angiogenesis, which has been reported to enhance T-cell infiltration into tumors. Methods: GUCY2C-CD3 mediated activity was evaluated in vivo in several cell line- and patient derived-xenograft models of CRC, using adoptive transfer of human T cells in established subcutaneous and orthotopic tumors. Immunohistochemistry was performed to demonstrate biodistribution of the drug and recruitment of activated T cells in GUCY2C-expressing tumors vs. normal tissues. We also performed CYTOF analyses on GUCY2C-CD3-treated tumors and tumor-infiltrating lymphocytes (TILs) to identify markers of immune evasion. Combination studies with anti PD-1/PD-L1 checkpoint blockade agents and with an anti VEGF-A antibody were performed using the adoptive transfer model. Results: GUCY2C-CD3 bispecific showed preferential biodistribution to GUCY2C-expressing xenograft tumors compared to normal tissue. The drug demonstrated potent dose-dependent efficacy in several CRC models with no signs of toxicity. Bispecific treated tumors not only showed recruitment of activated T cells to the tumor site, but also upregulated checkpoint mechanisms, such as PD-L1 on tumors and exhaustion markers on TILs. Consequently, anti PD-1/PD-L1 checkpoint blocking antibodies provided enhanced efficacy when combined with GUCY2C-CD3. Additionally, significant combination benefit was observed when GUCY2C-CD3 was combined with an anti VEGF-A blocking antibody. Conclusions: Our preclinical data demonstrate that a GUCY2C-CD3 bispecific can selectively target colorectal tumors, including those with KRAS or BRAF mutations, which are difficult to treat with currently approved therapies. This bispecific shows combination benefits with T-cell checkpoint blockade agents, as well as antiangiogenesis agents, indicating that single-agent activity with GUCY2C-CD3 can be further enhanced with mechanisms that address immune evasion. Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Lindsay King, Jatin Narula, Kerry Kelleher, Cynthia Rohde, Lioudmila Tchistiakova, Anhco Nguyen, Puja Sapra. A GUCY2c-CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A16.

Enteroendocrine Cells: Sensing Gut Microbiota and Regulating Inflammatory Bowel Diseases.

Host sensing in the gut microbiota has been crucial in the regulation of intestinal homeostasis. Although inflammatory bowel diseases (IBDs), multifactorial chronic inflammatory conditions of the gastrointestinal tract, have been associated with intestinal dysbiosis, the detailed interactions between host and gut microbiota are still not completely understood. Enteroendocrine cells (EECs) represent 1% of the intestinal epithelium. Accumulating evidence indicates that EECs are key sensors of gut microbiota and/or microbial metabolites. They can secrete cytokines and peptide hormones in response to microbiota, either in traditional endocrine regulation or by paracrine impact on proximal tissues and/or cells or via afferent nerve fibers. Enteroendocrine cells also play crucial roles in mucosal immunity, gut barrier function, visceral hyperalgesia, and gastrointestinal (GI) motility, thereby regulating several GI diseases, including IBD. In this review, we will focus on EECs in sensing microbiota, correlating enteroendocrine perturbations with IBD, and the underlying mechanisms.

Contribution of NOTCH signaling pathway along with TNF-α in the intestinal inflammation of ulcerative colitis.

The aim of this study was to determine gene expression levels of TNF-α, NOTCH1, and HES1 in patients with UC. Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease (IBD), causing an excessive expression of pro-inflammatory cytokines such as TNF-α. Also, target genes of NOTCH signaling are involved in the regulation of intestinal homeostasis. Previous studies have demonstrated that TNF-α increases in ulcerative colitis (UC) patients, but the relationship between TNF-α and NOTCH signaling pathway in UC etiopathology needs further study. Twelve active UC patients and twelve healthy controls were enrolled in this study. RNA was extracted and the mRNA expression levels of TNF-α, NOTCH1, and HES1 were examined using real-time PCR analyses. Further, transcriptome data deposited in Gene Expression Omnibus (GEO) database were analyzed to detect the differential expression of TNF superfamily and NOTCH1 gene in IBD patients. Finally, the interaction of TNF-α and NOTCH signaling was obtained from The SIGnaling Network Open Resource 2.0 (SIGNOR 2.0) database. The transcription levels of TNF-α, NOTCH1, and HES1 genes were significantly elevated in UC patients compared with control (p < 0.05). In addition, GEO results confirmed our expression results. SIGNOR analysis showed that TNF-α interacts with NOTCH signaling components. Based on our data, we observed that NOTCH1 and HES1 in co-operation of TNF-α, may play an important role in pathogenesis of UC. The members of NOTCH signaling pathway can be ideal candidates to target the therapy of IBD.