STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, in... more STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130 757F/F mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3 flox ; gp130 757F/F LysMcre/STAT3 flox). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130 757F/F mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin that comprises myeloid progenitor cells and immature myeloid cells 1. They expand during inflammation, infection and cancer and are potent suppressors of various T-cell functions 2,3. In addition, MDSCs regulate innate immune responses and non-immunological processes 4,5. In mice, MDSCs consist of two main subsets: monocytic (M-) MDSCs, which have a CD11b + Ly6G – Ly6C high CD49 + phenotype, and granulocytic (G-)MDSCs, which have a CD11b + Ly6G + Ly6C low CD49 – phenotype 6,7. Most of the factors that induce MDSC expansion and activation trigger signalling pathways in MDSCs that converge on Janus kinase (JAK) protein family members, signal transducer and activator of transcription (STAT), extracellular signal regulated kinase (ERK) and nuclear factor-κ b (NFκ B), which are signalling molecules that are involved in cell survival, proliferation and differentiation 8,9. The M-MDSC subset shows upregulated expression of STAT1, whereas G-MDSCs are characterized by increased activity of STAT3. Abnormal and persistent activation of STAT3 in myeloid progenitor cells prevents their differentiation into mature myeloid cells and thereby promotes MDSC expansion. Activation of both MDSC subsets in pathological conditions results in increased levels of arginase 1, which is an important immune suppressive factor 4. In addition, MDSCs induce a T helper cell type 2 (Th2) phenotype by producing the Th2 cell cytokine interleukin (IL)-10 10. Emerging evidence suggests that MDSCs play a regulatory role in inflammatory bowel diseases (IBD), in which an abnormal immune response against the microorganisms of the intestinal flora and a breakdown in diverse regulatory pathways is responsible for the chronic inflammatory pathology in genetically susceptible individuals 11,12. The adoptive transfer of MDSCs in different animal models of IBD ameliorates colitis, and suggest that MDSCs may be used as the basis for a novel adoptive cell therapy in IBD 13–17. However,
Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, theref... more Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.
American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015
We tested whether the Th2 agonist and allergenic ligand IL-33, was associated with eosinophilic e... more We tested whether the Th2 agonist and allergenic ligand IL-33, was associated with eosinophilic esophagitis (EoE) development in a paediatric cohort, and whether IL-33 protein could induce disease symptoms in mice. Biopsies from eosinophilic esophagitis patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast and epithelial cell markers, near blood vessels. Circulating levels of IL-33 were not increased. The time-course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model, and chronicity of IL-33 expression was demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 week, and esophageal pathology evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and up-regulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, were increased after IL-33 application, along with ILC2, Th1/17 and M2 macrophage marker genes. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with paediatric EoE development, and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.
Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactiv... more Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastr...
STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, in... more STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130(757F/F) mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3(flox); gp130(757F/F) LysMcre/STAT3(flox)). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130(757F/F) mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines.
Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system th... more Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa. Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting. The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach. The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.
STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mu... more STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well-placed to control inflammatory and oncogenic outcomes in the context of H. pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3-dependent mechanisms that might be exploited as drug targets.
STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, in... more STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130 757F/F mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3 flox ; gp130 757F/F LysMcre/STAT3 flox). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130 757F/F mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin that comprises myeloid progenitor cells and immature myeloid cells 1. They expand during inflammation, infection and cancer and are potent suppressors of various T-cell functions 2,3. In addition, MDSCs regulate innate immune responses and non-immunological processes 4,5. In mice, MDSCs consist of two main subsets: monocytic (M-) MDSCs, which have a CD11b + Ly6G – Ly6C high CD49 + phenotype, and granulocytic (G-)MDSCs, which have a CD11b + Ly6G + Ly6C low CD49 – phenotype 6,7. Most of the factors that induce MDSC expansion and activation trigger signalling pathways in MDSCs that converge on Janus kinase (JAK) protein family members, signal transducer and activator of transcription (STAT), extracellular signal regulated kinase (ERK) and nuclear factor-κ b (NFκ B), which are signalling molecules that are involved in cell survival, proliferation and differentiation 8,9. The M-MDSC subset shows upregulated expression of STAT1, whereas G-MDSCs are characterized by increased activity of STAT3. Abnormal and persistent activation of STAT3 in myeloid progenitor cells prevents their differentiation into mature myeloid cells and thereby promotes MDSC expansion. Activation of both MDSC subsets in pathological conditions results in increased levels of arginase 1, which is an important immune suppressive factor 4. In addition, MDSCs induce a T helper cell type 2 (Th2) phenotype by producing the Th2 cell cytokine interleukin (IL)-10 10. Emerging evidence suggests that MDSCs play a regulatory role in inflammatory bowel diseases (IBD), in which an abnormal immune response against the microorganisms of the intestinal flora and a breakdown in diverse regulatory pathways is responsible for the chronic inflammatory pathology in genetically susceptible individuals 11,12. The adoptive transfer of MDSCs in different animal models of IBD ameliorates colitis, and suggest that MDSCs may be used as the basis for a novel adoptive cell therapy in IBD 13–17. However,
Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, theref... more Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.
American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015
We tested whether the Th2 agonist and allergenic ligand IL-33, was associated with eosinophilic e... more We tested whether the Th2 agonist and allergenic ligand IL-33, was associated with eosinophilic esophagitis (EoE) development in a paediatric cohort, and whether IL-33 protein could induce disease symptoms in mice. Biopsies from eosinophilic esophagitis patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast and epithelial cell markers, near blood vessels. Circulating levels of IL-33 were not increased. The time-course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model, and chronicity of IL-33 expression was demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 week, and esophageal pathology evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and up-regulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, were increased after IL-33 application, along with ILC2, Th1/17 and M2 macrophage marker genes. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with paediatric EoE development, and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.
Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactiv... more Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastr...
STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, in... more STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130(757F/F) mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3(flox); gp130(757F/F) LysMcre/STAT3(flox)). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130(757F/F) mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines.
Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system th... more Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa. Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting. The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach. The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.
STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mu... more STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well-placed to control inflammatory and oncogenic outcomes in the context of H. pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3-dependent mechanisms that might be exploited as drug targets.
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