Blocking common γ chain cytokine signaling ameliorates T cell-mediated pathogenesis in disease models.

The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.

High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

We recently demonstrated that capturing human monoclonal antibodies (hmAbs) using high affinity anti-human Fc (AHC) antibodies allows reliable characterization of antibody-antigen interactions. Here, we characterized six human Fc specific mouse monoclonal antibodies (mAbs) and compared their binding profiles with three previously characterized goat AHC polyclonal antibodies (pAbs), exhibiting properties of a good capture reagent. All six mouse AHC mAbs specifically bound with high affinity to the Fc region of hIgG1, hIgG2, hIgG4 and to 43 different hIgG variants, containing substitutions and/or mutations in the hinge and/or Fc region, that have been reported to exhibit modified antibody effector function and/or pharmacokinetics. Biacore sensor surfaces individually derivatized with mouse AHC mAbs exhibited >2.5-fold higher hIgG binding capacity compared to the three goat AHC pAb surfaces and reproducibly captured hIgG over 300 capture-regeneration cycles. The results of the capture kinetic analyses performed on 31 antibody-antigen interactions using surfaces derivatized with either of the two highest affinity AHC mAbs (REGN7942 or REGN7943) were in concordance with those performed using goat AHC pAb surfaces. Our data demonstrate that AHC mAbs such as REGN7942 and REGN7943 that have properties superior than the three goat AHC pAbs are highly valuable research reagents, especially to perform capture kinetic analyses of antibody-antigen interactions on optical biosensors.

Overexpression of Vitamin D Receptor in Intestinal Epithelia Protects Against Colitis via Upregulating Tight Junction Protein Claudin 15.

BACKGROUND AND AIMS: Dysfunction of the vitamin D receptor [VDR] contributes to the aetiology of IBD by regulating autophagy, immune response, and mucosal permeability. VDR directly controls the paracellular tight junction protein Claudin-2. Claudin-2 and Claudin-15 are unique in maintaining paracellular permeability. Interestingly, claudin-15 mRNA was downregulated in patients with ulcerative colitis. However, the exact mechanism of Claudin-15 regulation in colitis is still unknown. Here, we investigated the protective role of VDR against intestinal inflammation via upregulating Claudin-15. METHODS: We analysed the correlation of Claudin-15 with the reduction of VDR in human colitis. We generated intestinal epithelial overexpression of VDR [O-VDR] mice to study the gain of function of VDR in colitis. Intestinal epithelial VDR knockout [VDR∆IEC] mice were used for the loss of function study. Colonoids and SKCO15 cells were used as in vitro models. RESULTS: Reduced Claudin-15 was significantly correlated with decreased VDR along the colonic epithelium of human IBD. O-VDR mice showed decreased susceptibility to chemically and bacterially induced colitis and marked increased Claudin-15 expression [both mRNA and protein] in the colon. Correspondingly, colonic Claudin-15 was reduced in VDR∆IEC mice, which were susceptible to colitis. Overexpression of intestinal epithelial VDR and vitamin D treatment resulted in a significantly increased Claudin-15. ChIP assays identified the direct binding of VDR to the claudin-15 promoter, suggesting that claudin-15 is a target gene of VDR. CONCLUSION: We demonstrated the mechanism of VDR upregulation of Claudin-15 to protect against colitis. This might enlighten the mechanism of barrier dysfunction in IBD and potential therapeutic strategies to inhibit inflammation.

Preclinical PET imaging with the novel human antibody 89Zr-DFO-REGN3504 sensitively detects PD-L1 expression in tumors and normal tissues.

BACKGROUND: Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) blocking antibodies including cemiplimab have generated profound clinical activity across diverse cancer types. Tumorous PD-L1 expression, as assessed by immunohistochemistry (IHC), is an accepted predictive marker of response to therapy in some cancers. However, expression is often dynamic and heterogeneous, and therefore not reliably captured by IHC from tumor biopsies or archival samples. Thus, there is significant need for accurate whole-body quantification of PD-L1 levels. METHODS: We radiolabeled the novel human anti-PD-L1 antibody REGN3504 with zirconium-89 (89Zr) using the chelator p-SCN-Bn-Deferoxamine to enable non-invasive immuno-positron emission tomography (immuno-PET) of PD-L1 expression. PET imaging assessed the localization of 89Zr-REGN3504 to multiple human tumor xenografts. Mice genetically humanized for PD-1 and PD-L1 were used to assess the biodistribution of 89Zr-REGN3504 to normal tissues and the estimated human radiation dosimetry of 89Zr-REGN3504 was also determined. Pharmacokinetics of REGN3504 was assessed in monkeys. RESULTS: Clear localization of 89Zr-REGN3504 to human tumor xenografts was observed via PET imaging and ex vivo biodistribution studies demonstrated high (fourfold to sixfold) tumor:blood ratios. 89Zr-REGN3504 specifically localized to spleen and lymph nodes in the PD-1/PD-L1 humanized mice. 89Zr-REGN3504 immuno-PET accurately detected a significant reduction in splenic PD-L1 positive cells following systemic treatment with clodronate liposomes. Radiation dosimetry suggested absorbed doses would be within guidelines for other 89Zr radiolabeled, clinically used antibodies. Pharmacokinetics of REGN3504 was linear. CONCLUSION: This work supports the clinical translation of 89Zr-REGN3504 immuno-PET for the assessment of PD-L1 expression. Future clinical studies will aim to investigate the utility of 89Zr-REGN3504 immuno-PET for predicting and monitoring response to anti-PD-1 therapy.

A Novel Role of SLC26A3 in the Maintenance of Intestinal Epithelial Barrier Integrity.

BACKGROUND & AIMS: The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. METHODS: Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. RESULTS: DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction. CONCLUSIONS: To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis.

Vitamin D Receptor Protects Against Dysbiosis and Tumorigenesis via the JAK/STAT Pathway in Intestine.

BACKGROUND & AIMS: Vitamin D exerts regulatory roles via vitamin D receptor (VDR) in mucosal immunity, host defense, and inflammation involving host factors and microbiome. Human Vdr gene variation shapes the microbiome and VDR deletion leads to dysbiosis. Low VDR expression and diminished vitamin D/VDR signaling are observed in colon cancer. Nevertheless, how intestinal epithelial VDR is involved in tumorigenesis through gut microbiota remains unknown. We hypothesized that intestinal VDR protects mice against dysbiosis via modulating the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in tumorigenesis. METHODS: To test our hypothesis, we used an azoxymethane/dextran sulfate sodium-induced cancer model in intestinal VDR conditional knockout (VDRΔIEC) mice, cell cultures, stem cell-derived colonoids, and human colon cancer samples. RESULTS: VDRΔIEC mice have higher numbers of tumors, with the location shifted from the distal to proximal colon. Fecal microbiota analysis showed that VDR deletion leads to a bacterial profile shift from normal to susceptible carcinogenesis. We found enhanced bacterial staining in mouse and human tumors. Microbial metabolites from VDRΔIEC mice showed increased secondary bile acids, consistent with observations in human CRC. We further identified that VDR protein bound to the Jak2 promoter, suggesting that VDR transcriptionally regulated Jak2. The JAK/STAT pathway is critical in intestinal and microbial homeostasis. Fecal samples from VDRΔIEC mice activate the STAT3 signaling in human and mouse organoids. Lack of VDR led to hyperfunction of Jak2 in response to intestinal dysbiosis. A JAK/STAT inhibitor abolished the microbiome-induced activation of STAT3. CONCLUSIONS: We provide insights into the mechanism of VDR dysfunction leading to dysbiosis and tumorigenesis. It indicates a new target: microbiome and VDR for the prevention of cancer.

Vitamin D receptor promotes healthy microbial metabolites and microbiome.

Microbiota derived metabolites act as chemical messengers that elicit a profound impact on host physiology. Vitamin D receptor (VDR) is a key genetic factor for shaping the host microbiome. However, it remains unclear how microbial metabolites are altered in the absence of VDR. We investigated metabolites from mice with tissue-specific deletion of VDR in intestinal epithelial cells or myeloid cells. Conditional VDR deletion severely changed metabolites specifically produced from carbohydrate, protein, lipid, and bile acid metabolism. Eighty-four out of 765 biochemicals were significantly altered due to the Vdr status, and 530 significant changes were due to the high-fat diet intervention. The impact of diet was more prominent due to loss of VDR as indicated by the differences in metabolites generated from energy expenditure, tri-carboxylic acid cycle, tocopherol, polyamine metabolism, and bile acids. The effect of HFD was more pronounced in female mice after VDR deletion. Interestingly, the expression levels of farnesoid X receptor in liver and intestine were significantly increased after intestinal epithelial VDR deletion and were further increased by the high-fat diet. Our study highlights the gender differences, tissue specificity, and potential gut-liver-microbiome axis mediated by VDR that might trigger downstream metabolic disorders.

All-trans Retinoic Acid Counteracts Diarrhea and Inhibition of Downregulated in Adenoma Expression in Gut Inflammation.

BACKGROUND: Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. AIMS: Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. METHODS: Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. RESULTS: All-trans retinoic acid (10 µM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1ß and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. CONCLUSION: Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.

Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins.

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 106  oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and ß-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.

Regulation of Microbiota by Vitamin D Receptor: A Nuclear Weapon in Metabolic Diseases.

Metabolic syndrome is a multi-faceted disease. The microbiota, as a newly discovered organ, contributes to the pathogenesis and progression of metabolic syndrome. Recent studies have demonstrated that nuclear receptors play critical roles in metabolic diseases. In the current review, we discuss the general role of the microbiome in health and metabolic syndrome. We summarize the functions of the nuclear receptor vitamin D receptor (VDR) in metabolism. The focus of this review is the novel roles of vitamin D/VDR signaling in regulating inflammation and the microbiome, especially in obesity. Furthermore, we extend our discussion of potential gut-liver axis mediated by VDR signaling and microbiota in obesity. Finally, we discuss the potential clinical application of probiotics and fecal microbiota transplantation in prevention and treatment of metabolic syndrome. Insights into nuclear receptors in metabolism and metabolic diseases will allow us to develop new strategies for fighting metabolic diseases.

Activation of Nuclear Factor-κB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3.

BACKGROUND & AIMS: Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects. METHODS: We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6-24 hours). We purified nuclear extracts and quantified nuclear factor-κB (NF-κB) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl- for HCO3- was measured by uptake of 125I. Expression of the NF-κB inhibitor α (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 µg/mouse for 3-6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. RESULTS: Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-κB inhibitor α in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA, at the regions of -935 to -629 and -375 to -84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein. CONCLUSIONS: In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl- / HCO3- exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease-associated diarrhea.

CDX2 upregulates SLC26A3 gene expression in intestinal epithelial cells.

SLC26A3 [downregulated in adenoma (DRA)] plays a key role in mammalian intestinal NaCl absorption, in that it mediates apical membrane Cl-/[Formula: see text] exchange. DRA function and expression are significantly decreased in diarrhea associated with inflammatory bowel disease. DRA is also considered to be a marker of cellular differentiation and is predominantly expressed in differentiated epithelial cells. Caudal-type homeobox protein-2 (CDX2) is known to regulate genes involved in intestinal epithelial differentiation and proliferation. Reduced expression of both DRA and CDX2 in intestinal inflammation prompted us to study whether the DRA gene is directly regulated by CDX2. Our initial studies utilizing CDX2 knockout (CDX2fV/fV;Cre+) mice showed a marked reduction in DRA mRNA and protein levels in proximal and distal colon. In silico analysis of the DRA promoter showed two consensus sites for CDX2 binding. Therefore, we utilized Caco-2 cells as an in vitro model to examine if DRA is a direct target of CDX2 regulation. siRNA-mediated silencing of CDX2 in Caco-2 cells resulted in a marked (~50%) decrease in DRA mRNA and protein levels, whereas ectopic overexpression of CDX2 upregulated DRA expression and also stimulated DRA promoter activity, suggesting transcriptional regulation. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated direct binding of CDX2 to one of the two putative CDX2 binding sites in the DRA promoter (+645/+663). In summary, our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2, implying that reduced expression of DRA in inflammatory bowel disease-associated diarrhea may, in part, be due to downregulation of CDX2 in the inflamed mucosa.NEW & NOTEWORTHY SLC26A3 [downregulated in adenoma (DRA)] mediates intestinal luminal NaCl absorption and is downregulated in inflammatory bowel disease-associated diarrhea. Since both DRA and caudal-type homeobox protein-2 (CDX2) are reduced in intestinal inflammation and the DRA promoter harbors CDX2 binding sites, we examined whether the DRA gene is regulated by CDX2. Our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2 via direct binding to the DRA promoter, suggesting that reduced expression of DRA in inflammatory bowel disease-associated diarrhea could, in part, be attributed to downregulation of CDX2.

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

Preclinical Activity of the Novel Anti-Prolactin Receptor (PRLR) Antibody-Drug Conjugate REGN2878-DM1 in PRLR-Positive Breast Cancers.

The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.

GLP-1 nanomedicine alleviates gut inflammation.

The gut hormone, glucagon like peptide-1 (GLP-1) exerts anti-inflammatory effects. However, its clinical use is limited by its short half-life. Previously, we have shown that GLP-1 as a nanomedicine (GLP-1 in sterically stabilized phospholipid micelles, GLP-1-SSM) has increased in vivo stability. The current study was aimed at testing the efficacy of this GLP-1 nanomedicine in alleviating colonic inflammation and associated diarrhea in dextran sodium sulfate (DSS) induced mouse colitis model. Our results show that GLP-1-SSM treatment markedly alleviated the colitis phenotype by reducing the expression of pro-inflammatory cytokine IL-1ß, increasing goblet cells and preserving intestinal epithelial architecture in colitis model. Further, GLP-1-SSM alleviated diarrhea (as assessed by luminal fluid) by increasing protein expression of intestinal chloride transporter DRA (down regulated in adenoma). Our results indicate that GLP-1 nanomedicine may act as a novel therapeutic tool in alleviating gut inflammation and associated diarrhea in inflammatory bowel disease (IBD).

Lactobacillus acidophilus counteracts inhibition of NHE3 and DRA expression and alleviates diarrheal phenotype in mice infected with Citrobacter rodentium.

Impaired absorption of electrolytes is a hallmark of diarrhea associated with inflammation or enteric infections. Intestinal epithelial luminal membrane NHE3 (Na+/H+ exchanger 3) and DRA (Down-Regulated in Adenoma; Cl-/HCO3- exchanger) play key roles in mediating electroneutral NaCl absorption. We have previously shown decreased NHE3 and DRA function in response to short-term infection with enteropathogenic E coli (EPEC), a diarrheal pathogen. Recent studies have also shown substantial downregulation of DRA expression in a diarrheal model of infection with Citrobacter rodentium, the mouse counterpart of EPEC. Since our previous studies showed that the probiotic Lactobacillus acidophilus (LA) increased DRA and NHE3 function and expression and conferred protective effects in experimental colitis, we sought to evaluate the efficacy of LA in counteracting NHE3 and DRA inhibition and ameliorating diarrhea in a model of C rodentium infection. FVB/N mice challenged with C rodentium [1 × 109 colony-forming units (CFU)] with or without administration of live LA (3 × 109 CFU) were assessed for NHE3 and DRA mRNA and protein expression, mRNA levels of carbonic anhydrase, diarrheal phenotype (assessed by colonic weight-to-length ratio), myeloperoxidase activity, and proinflammatory cytokines. LA counteracted C rodentium-induced inhibition of colonic DRA, NHE3, and carbonic anhydrase I and IV expression and attenuated diarrheal phenotype and MPO activity. Furthermore, LA completely blocked C rodentium induction of IL-1ß, IFN-γ, and CXCL1 mRNA and C rodentium-induced STAT3 phosphorylation. In conclusion, our data provide mechanistic insights into antidiarrheal effects of LA in a model of infectious diarrhea and colitis.

Endothelial lipid phosphate phosphatase-3 deficiency that disrupts the endothelial barrier function is a modifier of cardiovascular development.

AIMS: Lipid phosphate phosphatase-3 (LPP3) is expressed at high levels in endothelial cells (ECs). Although LPP3 is known to hydrolyse the phosphate group from lysolipids such as spingosine-1-phosphate and its structural homologues, the function of Lpp3 in ECs is not completely understood. In this study, we investigated how tyrosine-protein kinase receptor (TEK or Tie2) promoter-dependent deletion of Lpp3 alters EC activities. METHODS AND RESULTS: Lpp3(fl/fl) mice were crossed with the tg.Tie2(Cre) transgenic line. Vasculogenesis occurred normally in embryos with Tie2(Cre)-mediated deletion of Lpp3 (called Lpp3(ECKO)), but embryonic lethality occurred in two waves, the first wave between E8.5 and E10.5, while the second between E11.5 and E13.5. Lethality in Lpp3(ECKO) embryos after E11.5 was accompanied by vascular leakage and haemorrhage, which likely resulted in insufficient cardiovascular development. Analyses of haematoxylin- and eosin-stained heart sections from E11.5 Lpp3(ECKO) embryos showed insufficient heart growth associated with decreased trabeculation, reduced growth of the compact wall, and absence of cardiac cushions. Staining followed by microscopic analyses of Lpp3(ECKO) embryos revealed the presence of apoptotic ECs. Furthermore, Lpp3-deficient ECs showed decreased gene expression and protein levels of Cyclin-D1, VE-cadherin, Fibronectin, Klf2, and Klf4. To determine the underlying mechanisms of vascular leakage and barrier disruption, we performed knockdown and rescue experiments in cultured ECs. LPP3 knockdown decreased transendothelial electrical resistance and increased permeability. Re-expression of ß-catenin cDNA in LPP3-knockdown ECs partially restored the effect of the LPP3 loss, whereas re-expression of p120ctn cDNA did not. CONCLUSION: These findings demonstrate the essential roles of LPP3 in the maturation of EC barrier integrity and normal cardiovascular development.

Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

Mechanisms of Intestinal Serotonin Transporter (SERT) Upregulation by TGF-ß1 Induced Non-Smad Pathways.

TGF-ß1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-ß1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-ß1 on SERT. Model human intestinal Caco-2 cells grown as monolayer's or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-ß1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-ß1 receptor (TGFRI) as SB-431542, a specific TGF-ßRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-ß1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 µM) attenuated the TGF-ß1-mediated increase in SERT function. TGF-ß1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-ß1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-ß1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-ß1 offers a novel therapeutic strategy to treat GI disorders.