Lisa Waggoner

Loss of IL‐4Rα–mediated PI3K signaling accelerates the progression of IgE/mast cell–mediated reactions

Clinical and experimental evidence indicate that polymorphisms within the interleukin 4 (IL‐4) receptor (IL‐4R) chain are sufficient for altered strength of IL‐4/IL‐13 signaling, leading to an exaggerated allergic inflammatory response and increase susceptibility to allergic phenotypes. In the present study, we show that ablation of IL‐4Rα–induced phosphatidylinositol 3‐kinase (PI3K) activating signal by germline point mutation within the IL‐4Rα motif (Y500F) did not alter susceptibility to IgE‐mediated, food‐induced experimental anaphylaxis. Moreover, diarrhea occurrence, antigen‐specific IgE and intestinal mastocytosis were comparable between WT and IL‐4RαY500F mice. However, mice unable to stimulate IL‐4Rα–mediated PI3K signaling had accelerated disease progression. Notably, the accelerated anaphylactic response was associated with more rapid histamine‐induced hypovolemia. Mechanistic in vitro and in vivo analyses revealed that endothelial IL‐4Rα PI3K signaling negatively regulates the histamine‐induced endothelial leak response. These results define an unanticipated role for IL‐4Rα–mediated PI3K signaling in negative regulation of IgE‐mediated anaphylactic reactions.

Lipopolysaccharide suppresses IgE-mast cell-mediated reactions

Clinical and experimental analyses have identified a central role for IgE/FcεRI/mast cells in promoting IgE‐mediated anaphylaxis. Recent data from human studies suggest that bacterial infections can alter susceptibility to anaphylaxis.

Solute carrier family 9, subfamily A, member 3 (SLC9A3)/sodium-hydrogen exchanger member 3 (NHE3) dysregulation and dilated intercellular spaces in patients with eosinophilic esophagitis

Background: Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue, including eosinophil‐rich inflammation, basal zone hyperplasia, and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of EoE remain largely unexplored. Objective: We sought to investigate the involvement of solute carrier family 9, subfamily A, member 3 (SLC9A3) in esophageal epithelial intracellular pH (pHi) and DIS formation and the histopathologic features of EoE. Methods: We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3‐dimensional model system (EPC2‐ALI). Molecular and cellular analyses and ion transport assays were used to evaluate the expression and function of SLC9A3. Results: We identified altered expression of gene networks associated with regulation of pHi and acid‐protective mechanisms in esophageal biopsy specimens from pediatric patients with EoE (healthy subjects, n = 6; patients with EoE, n = 10). The most dysregulated gene central to regulating pHi was SLC9A3. SLC9A3 expression was increased within the basal layer of esophageal biopsy specimens from patients with EoE, and expression positively correlated with disease severity (eosinophils/high‐power field) and DIS (healthy subjects, n = 10; patients with EoE, n = 10). Analyses of esophageal epithelial cells revealed IL‐13–induced, signal transducer and activator of transcription 6–dependent SLC9A3 expression and Na+‐dependent proton secretion and that SLC9A3 activity correlated positively with DIS formation. Finally, we showed that IL‐13–mediated, Na+‐dependent proton secretion was the primary intracellular acid‐protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL‐13–induced DIS formation. Conclusions: SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE. GRAPHICAL ABSTRACT Figure. No caption available.

Myeloid-derived NF-κB negative regulation of PU.1 and c/EBP-β-driven pro-inflammatory cytokine production restrains LPS-induced shock.

Sepsis is a life-threatening event predominantly caused by Gram-negative bacteria. Bacterial infection causes a pronounced macrophage (MΦ) and dendritic cell activation that leads to excessive pro-inflammatory cytokine IL-1β, IL-6 and TNF-α production (cytokine storm), resulting in endotoxic shock. Previous experimental studies have revealed that inhibiting NF-κB signaling ameliorates disease symptoms; however, the contribution of myeloid p65 in endotoxic shock remains elusive. In this study, we demonstrate increased mortality in mice lacking p65 in the myeloid lineage (p65Δmye) compared with wild type mice upon ultra-pure LPS challenge. We show that increased susceptibility to LPS-induced shock was associated with elevated serum level of IL-1β and IL-6. Mechanistic analyses revealed that LPS-induced pro-inflammatory cytokine production was ameliorated in p65-deficient bone marrow-derived MΦs; however, p65-deficient ‘activated’ peritoneal MΦs exhibited elevated IL-1β and IL-6. We show that the elevated pro-inflammatory cytokine secretion was due, in part, to increased accumulation of IL-1β mRNA and protein in activated inflammatory MΦs. The increased IL-1β was linked with heightened binding of PU.1 and CCAAT/enhancer binding protein-β to Il1b and Il6 promoters in activated inflammatory MΦs. Our data provide insight into a role for NF-κB in the negative regulation of pro-inflammatory cytokines in myeloid cells.

The vascular endothelial specific IL-4 receptor alpha–ABL1 kinase signaling axis regulates the severity of IgE-mediated anaphylactic reactions

Background Severe IgE‐mediated, food‐induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life‐threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. Objective We sought to define the interaction and requirement of IL‐4 and vascular endothelial (VE) IL‐4 receptor &agr; chain (IL‐4R&agr;) signaling in histamine‐abelson murine leukemia viral oncogene homology 1 (ABL1)–mediated VE dysfunction and fluid extravasation in the severity of IgE‐mediated anaphylactic reactions in mice. Methods Mice deficient in VE IL‐4R&agr; and models of passive and active oral antigen– and IgE‐induced anaphylaxis were used to define the requirements of the VE IL‐4R&agr; and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. Results IL‐4 exacerbation of histamine‐induced hypovolemic shock in mice was dependent on VE expression of IL‐4R&agr;. IL‐4– and histamine‐induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1‐dependent. Development of severe IgE‐mediated hypovolemia and shock required VE‐restricted ABL1 expression. Treatment of mice with a history of food‐induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE‐mediated anaphylaxis. Conclusion IL‐4 amplifies IgE‐ and histamine‐induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL‐4R&agr;/ABL1–dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE‐mediated reactions. Graphical abstract Figure. No Caption available.

P-293 Loss of Star-related Lipid Transfer Domain Protein (StarD7) Induces Intestinal Epithelial Mitochondrial and Barrier Dysfunction and Increases Susceptibility to Colitis

Background: The inflammatory bowel diseases (IBD) Crohns disease (CD) and ulcerative colitis (UC) are chronic relapsing gastrointestinal (GI) inflammatory diseases that affect 1 to 1.5 million Americans and cause substantial morbidity and decreased quality of life. We have recently identified dysregulated expression of the protein, Star-related lipid transfer domain protein (StarD7) in colonic biopsy samples of inflamed UC patients. StarD7 is a steroidogenic acute regulatory protein-related lipid transfer domain containing protein that regulates intracellular non-vesicular trafficking of phosphatidylcholine (PC) to mitochondrial membranes and the nucleus and regulates cell proliferation, migration and differentiation. Methods: We generated intestinal epithelial-specific and global StarD7-deficient mice and employed Dextran sodium sulphate (DSS)-induced and IL-10-deficient models of colitis to define the importance of StarD7 in the susceptability to colitis phenotypes. We utilized lentiviral StarD7 knockdown intestinal epithelial cell lines to define the contribution of this molecule to intestinal epithelial mitochondrial function, barrier properties and pro-inflammatory cytokine production. Results: We generated intestinal epithelial-specific and global StarD7-deficient mice. We show that ShhCreStarD7fl/fl mice have altered homeostatic intestinal epithelial barrier dysfunction compared with WT mice (Resistance RT 167.1 ± 8.1 versus 125.5 ± 5.6 W·cm2; mean ± SEM, P < 0.05; FITC-Dextran 4kDa Flux 2.7 ± 0.2 versus 4.4 ± 0.3 pg·mL·min−1; mean ± SEM, P < 0.01; WT versus ShhCreStarD7fl/fl, respectively). Notably, the dysregulation of intestinal epithelial barrier function was associated with altered TJ protein expression including Claudin-4 and claudin-5 and increased susceptibility to Dextran sodium sulphate (DSS)-induced colitis (histological score: 3.5 ± 0.9 versus 8.6 ± 1.0; mean ± SEM, P < 0.05; WT versus ShhCreStarD7fl/fl, respectively). In a series of in vitro experiments employing intestinal epithelial cell lines and siRNA StarD7 knockdown technology we identified novel critical functions for StarD7 in maintenance of (1) intestinal epithelial homeostatic mitochondrial function and (2) PPAR:RXR transcription factor function which culminates in preservation of TJ protein expression and intestinal epithelial barrier function. Moreover, we show that StarD7 knockdown in human intestinal epithelial cells promotes mitochondrial and ER stress which was associated with increased pro-inflammatory cytokine (IL-25, IL-33 and TSLP) production. Indeed, rescue of mitochondrial stress by pharmacological agonist rescued intestinal TJ expression and barrier function in vitro and in vivo. Conclusions: These data suggest that the PC-lipid transfer protein Stard7 is critical for intestinal epithelial mitchondrial function and maintenance of immune homeostasis.

515 microRNA-375-KLF5 Regulation of Intestinal Epithelial CFTR Function

Background: Matrix Metalloproteinase (MMP)-induced extracellular matrix remodeling modulates intestinal inflammation. MMP-12 is a human macrophage elastase capable of degrading basement membrane (BM). Defective intestinal barrier leading to increased intestinal permeability is an important pathogenic factor for intestinal inflammation. The role of MMP-12 in intestinal barrier function and intestinal inflammation remains unclear. We hypothesize that MMP-12 induces degradation of BM and helps macrophage transmigration thereby compromising intestinal barrier and augmenting intestinal inflammation. Aim: The aim of this study was to investigate the role of MMP-12 in intestinal epithelial tight junction (TJ) permeability and macrophage transmigration in experimental dextran sodium sulfate (DSS) colitis and in vitro Caco-2 cell model. Methods: Nine weeks-old wild type (WT) and MMP-12-/mice were administered 3% DSS in drinking water for 7 days. An in vivo colonic recycling perfusion and in vitro epithelial cell model was used to study epithelial permeability. Results: DSS treatment resulted in a marked increase in MMP-12 protein expression in WT colon. DSS administration significantly increased the colonic permeability in WT but not MMP-12-/mice (p<0.01). The loss of body weight, disease activity index, and histological lesion score of colitis was significantly attenuated in MMP-12-/DSS group compared to WT DSS group (p<0.01). In immunohistochemical study, the BM laminin was significantly lost in WT DSS colon but not in MMP-12-/DSS colon. The epithelial infiltration of macrophages in DSS colitis, as assessed by macrophage marker CD68 staining, was found to be significantly lower in MMP-12-/mice than WT mice. To further investigate the role of MMP-12 in intestinal TJ permeability, human intestinal epithelial Caco-2 cells were co-cultured with phorbol myristate acetate activated, MMP-12 secreting human macrophage U937 cells. The co-culture resulted into progressive and significant decrease in Caco-2 transepithelial resistance (TER) and concomitant increase in paracellular inulin flux, indicating loss of Caco-2 TJ barrier. Furthermore, siRNA-induced knock down of MMP-12 expression in U937 macrophage cells attenuated loss of Caco-2 TER and increase in inulin flux after U937 co-culture. Also, siRNA-induced knock down of MMP-12 significantly prevented U937 macrophage transmigration across the Caco-2 cells. Conclusion:The clinical severity, colonic permeability, and macrophage infiltration in colitis was attenuated in MMP-12-/mice. Macrophage derived MMP-12 increased intestinal epithelial permeability and enables macrophage transmigration in a cell culture model. These data suggest that MMP-12-induced macrophage transmigration and loss of intestinal epithelial TJ barrier contributes to the development of colitis.