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A Novel Orai1 Calcium Channel Inhibitor as Oral Therapy for Colitis
known as AAV8-TBGp-Cre. AAV8-TBGp-mediated transgenes are expressed specifically in hepatocytes. The expression of hepatic PPARγ in aHepPPARgkd was only 5% of that in control mice (PPARγfl/fl mice treated with AAV8-TBGp-Null). Expression of PPARγ in extrahepatic tissues (intestine and adipose tissue) was not reduced in aHepPPARγkd mice as compared to that of controls. Similar to HepPPARγKO mice, we observed elevated postprandial plasma TAG levels in aHepPPARγkd mice; however this was observed just seven days after loss of hepatocyte PPARγ expression. In order to isolate the mechanism(s) dysregulated by aHepPPARγkd that lead to postprandial dyslipidemia we used control and aHepPPARγkd mice one to two weeks after AAV treatments. To assess if aHepPPARγkd: 1) impairs TAG clearance, mice were ip injected with an emulsion of TAG (Intralipid® 20%, Medline); 2) increases VLDL production rate, mice were ip injected with tyloxapol 500mg/kg (to block lipoprotein-lipase mediated TAG clearance); 3) increases intestinal lipid absorption, mice were given an oral gavage of olive oil (200µl/mouse) without or with tyloxapol. Blood samples were obtained before and sequentially after every treatment to determine plasma TAG levels. In addition, we measured the expression of hepatic genes involved in TAG clearance. aHepPPARγkd did not impact TAG clearance when TAG were ip injected. Also, VLDL production was similar between control and aHepPPARγkd mice. However, intestinal lipid absorption was enhanced in aHepPPARγkd mice, which was associated with a relative impairment of TAG clearance when TAG derived from digestion. Also, hepatic expression of Cd36, a PPARγ-target gene positively associated with hepatic uptake of chylomicrons remnants, was reduced in refed aHepPPARγkd mice. Taken together, these results suggest that hepatocyte PPARγ may control signals and/or mechanisms that regulate both intestinal lipid absorption and/or hepatic TAG clearance in the postprandial state to regulate lipid homeostasis.
REVERSION OF DEFA4Cre-EXPRESSING PANETH CELLS TO A STEM CELL STATE IN RESPONSE TO NOTCH ACTIVATION OR INTESTINAL INJURY Jennifer C. Jones, Constance D. Brindley, Michael Rajala, Martin G. Myers, Peter J. Dempsey Introduction. Injury to Lgr5+ CBCs provides permissive conditions for different progenitor cell populations to mobilize and revert to a stem cell state. However, little is known about the extracellular signals that regulate stem cell reversion. In this study, we utilized a novel Paneth cell (PC)-specific Cre line to examine the role of Notch signaling in PC plasticity and transformation and for the ability of PCs to revert to a stem cell state following intestinal injury. Methods. Lineage tracing and adenoma studies were performed using defensin alpha 4 (Defa4) knock-in Cre mice bred to different Rosa26 reporter, Notch loss-of-function (LOF, ADAM10f/f), Notch gain-of-function (GOF, Rosa26NICD) and APCf/f mice, respectively. Injury models included whole-body 12Gy irradiation or doxorubicin treatment (DXR, 15mg/kg, ip). Results. In adult Defa4Cre;Rosa26Tomato mice, Tomhi+ cells faithfully co-express PC marker lysozyme and no Tom+ crypt/villus lineage tracing was detected. Consistent with these results, Defa4Cre;Rosa26Tomato enteroids only showed patches of Tom+ cells and none became completely Tom+. In Notch LOF studies using Defa4Cre;Adam10f/f;Rosa26Tomato mice, no effect on PC differentiation was observed. By contrast, in Notch GOF studies using Defa4Cre;Rosa26NICD mice, constitutive Notch activation readily produced NICD+ crypt/ villus lineage tracing throughout the small intestine and was associated with a loss of secretory differentiation and expanded crypt cell proliferation. Moreover, Defa4Cre;Rosa26 NICD enteroids were completely NICD+ confirming that Notch activation was acting cell autonomously within multipotent stem cells. To validate the above findings, adenoma initiation studies were performed. In Defa4Cre;APCf/f mice, no adenoma formation was detected. By contrast, Defa4Cre;APCf/f;Rosa26NICD mice developed robust adenomas, indicating that Notch activation leads to PC dedifferentiation into stem/progenitor cells susceptible to APCmediated transformation. Several intestinal injury models have been used to study stem cell reversion. Following irradiation in Defa4Cre;Rosa26YFP mice, a rare subset of YFP+/ EdU+ cells were detected indicating re-entry into the cell cycle. Robust, albeit variable, Tom+ crypt/ villus lineage tracing was detected following DXR treatment in Defa4Cre;Rosa26Tomato mice. However, in Notch LOF Defa4Cre;Adam10f/f;Rosa26Tomato mice, no ADAM10-deficient lineage tracing was observed after DXR. Conclusions. In the adult small intestine, Defa4Cre-expressing cells are mature PCs and do not lineage trace. However, constitutive Notch activation in Defa4Cre cells provides permissive conditions for PCs to dedifferentiate and regain stemness and these cells are susceptible to transformation. Following intestinal injury, Defa4Creexpressing cells can also undergo stem cell reversion and can contribute to intestinal regeneration.
Su2090 SUCCESSFUL TREATMENT OF MURINE COLITIS AND ACUTE GvHD BY GUT-LOCALIZED PD-L1 EXPRESSION Ghania Chikh, Shauna Dauphinee, Connor McCarthy, Jeremy Dupal-Chicoine, Natalie Tam, Eric Hsu, Anthony Cheung Immunoregulatory T cell costimulation by programmed death 1 (PD- 1) receptor and PD ligand 1 (PD-L1) can provide a negative signal to inhibit T cell proliferation, mediate tolerance and prevent inflammation. Therefore, we hypothesize that modulation of PD-L1 levels in the gastrointestinal tract (GIT) provides a novel mechanism to control intestinal inflammation in colitis and acute GvHD which are caused by overactive cytotoxic T cells. We have developed a proprietary modified oligomeric chitosan, dually-derivitized chitosan (DDchitosan), capable of packaging plasmid DNA into nanoparticles for in vivo delivery to mucosal tissues of the gastrointestinal tract. Codon optimized gene sequences of human membrane bound full-length PD-L1 or soluble PD-L1 protein (extracellular region fused to non-lytic human IgG1-Fc) were sub-cloned into the pVax expression plasmid. PD-L1 and PD-L1-Fc plasmids were formulated in DD-chitosan resulting in nanoparticles. Protein expression and potency was confirmed following in vitro transfection. PD-L1-Fc was purified with Protein G affinity chromatography, quantified by ELISA and the purity confirmed by Coomassie stain. Full length membrane bound PD-L1 was quantified in cell lysate. In vitro potency of the PD-L1-Fc construct was assessed based on inhibition of T cell activation using T cells isolated from mouse splenocytes. In vitro potency of the membrane bound PD-L1 was assessed by PD-1 receptor binding assay using NIH/3T3 cells transfected to express PD-L1. NIH/3T3 cells expressing PD-L1 were incubated with or without PD-1 at various concentrations and binding of anti-PD-L1 or anti-PD-1 fluorescent antibodies was assed using flow cytometry. In vivo expression of PD-L1-Fc and PD-L1 in the colon was confirmed using RT-qPCR following intracolonic instillation of the polyplex to C57Bl/6 mice. Using T cell transfer model of colitis, we have shown statistically significant improvement in weight loss, survival and clinical signs with mice treated with PD-L1-Fc-PP and PD-L1-PP compared to mice treated with controls. Target engagement assessment showed a significant increase in FOXP3 expression by CD4+CD25+FOXP3+ regulatory T cells in the mesenteric lymph node and splenocytes of mice treated with PD-L1-PP compared to plasmid control. Striking therapeutic response was also observed in an acute GvHD mouse model. Treatment with PD-L1-Fc-PP and PD-L1PP demonstrated significant survival, weight and clinical signs benefit over controls. The data presented herein suggests that gut-localized delivery of PDL1 using our novel DD-chitosan carrier improves clinical manifestations of disease in a mouse model of GvHD and colitis. These findings support further development of this novel gene therapy strategy of gut-localized delivery of immune checkpoint proteins to treat Tcell mediated immune disorders of the GIT.
Su2088 A NOVEL ORAI1 CALCIUM CHANNEL INHIBITOR AS ORAL THERAPY FOR COLITIS Jonathan Skupsky, Shivashankar Othy, Andriy Yeromin, Tobias Dong, Andrew Newman, Milton Greenberg, Michael Cahalan The Orai1 calcium channel is an exciting new pharmacological target for treatment of Inflammatory Bowel Disease (IBD), because it is upstream in the inflammatory cascade and absolutely necessary for appropriate immune responses. Calcium-selective Orai1 channels (formerly called CRAC channels) become activated when T cells recognize antigen and allow calcium influx to the cytoplasm leading to increased NF-κB and NFAT-mediated gene expression driving inflammation. Orai1 blockers have shown promising results for multiple sclerosis and psoriasis with limited side-effects. To target this pathway for the treatment of IBD, we have developed an Orai1 inhibitor called VV2003. The drug was designed for oral administration as a next-generation alternative to mesalamine. In-vitro studies demonstrate that the drug has minimal toxicity and that it potently suppresses T cell proliferation and calcium signaling. Permeability studies show that VV2003 has limited ability to cross the GI mucosa restricting its anti-inflammatory effects to sites of ulceration and active colitis. Since Orai1 expression is limited, we expect to see little if any systemic side effects. We tested the drug in the preclinical adoptive transfer model for colitis. In this model, immunodeficient Rag knock-out mice are hosts for adoptive transfer of naïve CD4+ T cells. In the absence of regulatory T cells, the transferred cells expand and differentiate in the colon causing disease. Without treatment, these mice develop signs of colitis including weight loss, altered crypt architecture and infiltration of the lamina propria by week four. When given a daily oral gavage of 20 mg/kg VV2003 starting week two, treated mice showed a significant increase in weight over controls by week six and improved histological scores. These preliminary studies demonstrate that VV2003 has potential to become a novel nextgeneration oral therapy for colitis because it effectively limits leukocyte function in-vitro and in-vivo with minimal systemic absorption and minimal expected side-effects.
Su2091 HEDGEHOG SIGNALING REGULATES PDL-1 EXPRESSION IN GASTRIC CANCER CELLS TO INDUCE TUMOR PROLIFERATION Jayati Chakrabarti, Loryn L. Holokai, Nina Bertaux-Skeirik, LiJyun Syu, Taylor Broda, Julie Chang, Emma L. Teal, James Wells, Andrzej A. Dlugosz, Yana Zavros
Su2089 Background: The Hedgehog (Hh) signaling pathway is often reactivated in various types of cancers including gastric, and hence raises the interest of targeting the Hh pathway as a potential therapeutic target for the treatment of gastric cancer. Tumor cells expressing programmed cell death ligand1 (PDL-1) interact with PD-1 on CD8+ cytotoxic T lymphocytes (CTLs). This interaction inhibits CTL effector function, subsequently leading to immune evasion and cancer cell proliferation. Regulation of PDL-1 may be a promising therapeutic strategy. However, the mechanism regulating PDL-1 expression remains unclear in gastric cancer. Hypothesis: Hh-induced PDL-1 inactivates effector T cell function and allows gastric cancer cell proliferation. Methods: Gastric organoids were generated from an induced pluripotent stem cell (iPSC) line from a patient with (GC HGO) and without (HGO) hereditary diffuse gastric cancer. Mouse gastric-derived organoids were generated from cancer tissue of a triple-transgenic model engineered to express an activated GLI2 allele, GLI2A, in Lgr5expressing stem cells in adult mice when treated with doxycycline (iLgr5-GLI2A mTGOs). Organoids were also derived from normal mouse gastric tissue (mGOs). Conditioned media
HEPATOCYTE PPARγ MAY REGULATE INTESTINAL ABSORPTION AND/OR HEPATIC CLEARANCE OF TRIACYLGLYCEROLS, TO PREVENT POSTPRANDIAL DYSLIPIDEMIA. Jose Cordoba Chacon Aged, 40 week-old, congenital hepatocyte PPARγ knockout (HepPPARγKO) mice show elevated plasma triacylglycerols (TAG) levels. It is thought that elevated TAG levels are consequence of impairment in the clearance of VLDL and chylomicrons. In order to assess if loss of hepatocyte PPARγ directly promotes dyslipidemia in adult mice, we have generated an adult-onset hepatocyte-specific PPARγ knockdown (aHepPPARγkd) mouse model. aHepPPARγkd mice are generated by injecting 10 week-old PPARγ floxed (PPARγfl/fl) mice with a single dose of adeno-associated virus serotype 8 (AAV8) that express a Cre recombinase driven by a thyroxine binding globulin promoter (TBGp, hepatocyte-specific promoter)
S-1311
DDW Abstracts
DDW Abstracts
Su2087
REVERSION OF DEFA4Cre-EXPRESSING PANETH CELLS TO A STEM CELL STATE IN RESPONSE TO NOTCH ACTIVATION OR INTESTINAL INJURY Jennifer C. Jones, Constance D. Brindley, Michael Rajala, Martin G. Myers, Peter J. Dempsey Introduction. Injury to Lgr5+ CBCs provides permissive conditions for different progenitor cell populations to mobilize and revert to a stem cell state. However, little is known about the extracellular signals that regulate stem cell reversion. In this study, we utilized a novel Paneth cell (PC)-specific Cre line to examine the role of Notch signaling in PC plasticity and transformation and for the ability of PCs to revert to a stem cell state following intestinal injury. Methods. Lineage tracing and adenoma studies were performed using defensin alpha 4 (Defa4) knock-in Cre mice bred to different Rosa26 reporter, Notch loss-of-function (LOF, ADAM10f/f), Notch gain-of-function (GOF, Rosa26NICD) and APCf/f mice, respectively. Injury models included whole-body 12Gy irradiation or doxorubicin treatment (DXR, 15mg/kg, ip). Results. In adult Defa4Cre;Rosa26Tomato mice, Tomhi+ cells faithfully co-express PC marker lysozyme and no Tom+ crypt/villus lineage tracing was detected. Consistent with these results, Defa4Cre;Rosa26Tomato enteroids only showed patches of Tom+ cells and none became completely Tom+. In Notch LOF studies using Defa4Cre;Adam10f/f;Rosa26Tomato mice, no effect on PC differentiation was observed. By contrast, in Notch GOF studies using Defa4Cre;Rosa26NICD mice, constitutive Notch activation readily produced NICD+ crypt/ villus lineage tracing throughout the small intestine and was associated with a loss of secretory differentiation and expanded crypt cell proliferation. Moreover, Defa4Cre;Rosa26 NICD enteroids were completely NICD+ confirming that Notch activation was acting cell autonomously within multipotent stem cells. To validate the above findings, adenoma initiation studies were performed. In Defa4Cre;APCf/f mice, no adenoma formation was detected. By contrast, Defa4Cre;APCf/f;Rosa26NICD mice developed robust adenomas, indicating that Notch activation leads to PC dedifferentiation into stem/progenitor cells susceptible to APCmediated transformation. Several intestinal injury models have been used to study stem cell reversion. Following irradiation in Defa4Cre;Rosa26YFP mice, a rare subset of YFP+/ EdU+ cells were detected indicating re-entry into the cell cycle. Robust, albeit variable, Tom+ crypt/ villus lineage tracing was detected following DXR treatment in Defa4Cre;Rosa26Tomato mice. However, in Notch LOF Defa4Cre;Adam10f/f;Rosa26Tomato mice, no ADAM10-deficient lineage tracing was observed after DXR. Conclusions. In the adult small intestine, Defa4Cre-expressing cells are mature PCs and do not lineage trace. However, constitutive Notch activation in Defa4Cre cells provides permissive conditions for PCs to dedifferentiate and regain stemness and these cells are susceptible to transformation. Following intestinal injury, Defa4Creexpressing cells can also undergo stem cell reversion and can contribute to intestinal regeneration.
Su2090 SUCCESSFUL TREATMENT OF MURINE COLITIS AND ACUTE GvHD BY GUT-LOCALIZED PD-L1 EXPRESSION Ghania Chikh, Shauna Dauphinee, Connor McCarthy, Jeremy Dupal-Chicoine, Natalie Tam, Eric Hsu, Anthony Cheung Immunoregulatory T cell costimulation by programmed death 1 (PD- 1) receptor and PD ligand 1 (PD-L1) can provide a negative signal to inhibit T cell proliferation, mediate tolerance and prevent inflammation. Therefore, we hypothesize that modulation of PD-L1 levels in the gastrointestinal tract (GIT) provides a novel mechanism to control intestinal inflammation in colitis and acute GvHD which are caused by overactive cytotoxic T cells. We have developed a proprietary modified oligomeric chitosan, dually-derivitized chitosan (DDchitosan), capable of packaging plasmid DNA into nanoparticles for in vivo delivery to mucosal tissues of the gastrointestinal tract. Codon optimized gene sequences of human membrane bound full-length PD-L1 or soluble PD-L1 protein (extracellular region fused to non-lytic human IgG1-Fc) were sub-cloned into the pVax expression plasmid. PD-L1 and PD-L1-Fc plasmids were formulated in DD-chitosan resulting in nanoparticles. Protein expression and potency was confirmed following in vitro transfection. PD-L1-Fc was purified with Protein G affinity chromatography, quantified by ELISA and the purity confirmed by Coomassie stain. Full length membrane bound PD-L1 was quantified in cell lysate. In vitro potency of the PD-L1-Fc construct was assessed based on inhibition of T cell activation using T cells isolated from mouse splenocytes. In vitro potency of the membrane bound PD-L1 was assessed by PD-1 receptor binding assay using NIH/3T3 cells transfected to express PD-L1. NIH/3T3 cells expressing PD-L1 were incubated with or without PD-1 at various concentrations and binding of anti-PD-L1 or anti-PD-1 fluorescent antibodies was assed using flow cytometry. In vivo expression of PD-L1-Fc and PD-L1 in the colon was confirmed using RT-qPCR following intracolonic instillation of the polyplex to C57Bl/6 mice. Using T cell transfer model of colitis, we have shown statistically significant improvement in weight loss, survival and clinical signs with mice treated with PD-L1-Fc-PP and PD-L1-PP compared to mice treated with controls. Target engagement assessment showed a significant increase in FOXP3 expression by CD4+CD25+FOXP3+ regulatory T cells in the mesenteric lymph node and splenocytes of mice treated with PD-L1-PP compared to plasmid control. Striking therapeutic response was also observed in an acute GvHD mouse model. Treatment with PD-L1-Fc-PP and PD-L1PP demonstrated significant survival, weight and clinical signs benefit over controls. The data presented herein suggests that gut-localized delivery of PDL1 using our novel DD-chitosan carrier improves clinical manifestations of disease in a mouse model of GvHD and colitis. These findings support further development of this novel gene therapy strategy of gut-localized delivery of immune checkpoint proteins to treat Tcell mediated immune disorders of the GIT.
Su2088 A NOVEL ORAI1 CALCIUM CHANNEL INHIBITOR AS ORAL THERAPY FOR COLITIS Jonathan Skupsky, Shivashankar Othy, Andriy Yeromin, Tobias Dong, Andrew Newman, Milton Greenberg, Michael Cahalan The Orai1 calcium channel is an exciting new pharmacological target for treatment of Inflammatory Bowel Disease (IBD), because it is upstream in the inflammatory cascade and absolutely necessary for appropriate immune responses. Calcium-selective Orai1 channels (formerly called CRAC channels) become activated when T cells recognize antigen and allow calcium influx to the cytoplasm leading to increased NF-κB and NFAT-mediated gene expression driving inflammation. Orai1 blockers have shown promising results for multiple sclerosis and psoriasis with limited side-effects. To target this pathway for the treatment of IBD, we have developed an Orai1 inhibitor called VV2003. The drug was designed for oral administration as a next-generation alternative to mesalamine. In-vitro studies demonstrate that the drug has minimal toxicity and that it potently suppresses T cell proliferation and calcium signaling. Permeability studies show that VV2003 has limited ability to cross the GI mucosa restricting its anti-inflammatory effects to sites of ulceration and active colitis. Since Orai1 expression is limited, we expect to see little if any systemic side effects. We tested the drug in the preclinical adoptive transfer model for colitis. In this model, immunodeficient Rag knock-out mice are hosts for adoptive transfer of naïve CD4+ T cells. In the absence of regulatory T cells, the transferred cells expand and differentiate in the colon causing disease. Without treatment, these mice develop signs of colitis including weight loss, altered crypt architecture and infiltration of the lamina propria by week four. When given a daily oral gavage of 20 mg/kg VV2003 starting week two, treated mice showed a significant increase in weight over controls by week six and improved histological scores. These preliminary studies demonstrate that VV2003 has potential to become a novel nextgeneration oral therapy for colitis because it effectively limits leukocyte function in-vitro and in-vivo with minimal systemic absorption and minimal expected side-effects.
Su2091 HEDGEHOG SIGNALING REGULATES PDL-1 EXPRESSION IN GASTRIC CANCER CELLS TO INDUCE TUMOR PROLIFERATION Jayati Chakrabarti, Loryn L. Holokai, Nina Bertaux-Skeirik, LiJyun Syu, Taylor Broda, Julie Chang, Emma L. Teal, James Wells, Andrzej A. Dlugosz, Yana Zavros
Su2089 Background: The Hedgehog (Hh) signaling pathway is often reactivated in various types of cancers including gastric, and hence raises the interest of targeting the Hh pathway as a potential therapeutic target for the treatment of gastric cancer. Tumor cells expressing programmed cell death ligand1 (PDL-1) interact with PD-1 on CD8+ cytotoxic T lymphocytes (CTLs). This interaction inhibits CTL effector function, subsequently leading to immune evasion and cancer cell proliferation. Regulation of PDL-1 may be a promising therapeutic strategy. However, the mechanism regulating PDL-1 expression remains unclear in gastric cancer. Hypothesis: Hh-induced PDL-1 inactivates effector T cell function and allows gastric cancer cell proliferation. Methods: Gastric organoids were generated from an induced pluripotent stem cell (iPSC) line from a patient with (GC HGO) and without (HGO) hereditary diffuse gastric cancer. Mouse gastric-derived organoids were generated from cancer tissue of a triple-transgenic model engineered to express an activated GLI2 allele, GLI2A, in Lgr5expressing stem cells in adult mice when treated with doxycycline (iLgr5-GLI2A mTGOs). Organoids were also derived from normal mouse gastric tissue (mGOs). Conditioned media
HEPATOCYTE PPARγ MAY REGULATE INTESTINAL ABSORPTION AND/OR HEPATIC CLEARANCE OF TRIACYLGLYCEROLS, TO PREVENT POSTPRANDIAL DYSLIPIDEMIA. Jose Cordoba Chacon Aged, 40 week-old, congenital hepatocyte PPARγ knockout (HepPPARγKO) mice show elevated plasma triacylglycerols (TAG) levels. It is thought that elevated TAG levels are consequence of impairment in the clearance of VLDL and chylomicrons. In order to assess if loss of hepatocyte PPARγ directly promotes dyslipidemia in adult mice, we have generated an adult-onset hepatocyte-specific PPARγ knockdown (aHepPPARγkd) mouse model. aHepPPARγkd mice are generated by injecting 10 week-old PPARγ floxed (PPARγfl/fl) mice with a single dose of adeno-associated virus serotype 8 (AAV8) that express a Cre recombinase driven by a thyroxine binding globulin promoter (TBGp, hepatocyte-specific promoter)
S-1311
DDW Abstracts
DDW Abstracts
Su2087