Dependent Secretion Research Articles

Novel Endothelial Cell Targeted Peptide Nanoformulation for Inhibiting von Willebrand Factor Secretion to Reduce Thrombotic Complications in Sepsis

RationaleMicrovascular thrombosis and disseminated intravascular coagulation (DIC) are primary determinants of ischemia, organ failure and mortality/morbidity in sepsis. Interestingly, elevated plasma level of von Willebrand factor (vWF), the glue to which platelets adhere to form a clot, is associated with reduced survival and poor outcome in septic patients. Previously, we demonstrated that endothelial cell (EC) Gα12 is required for soluble N‐ethylmaleimide‐sensitive‐fusion factor attachment protein α (αSNAP)‐dependent vWF secretion from Weibel Palade bodies. Here, we report that sterically stabilized simple micelles (SSM) containing cell‐permeable myristoylated αSNAP binding domain (Myr‐SBD) peptide based on the unique N‐terminal Gα12 sequence that binds to αSNAP, inhibits vWF secretion.MethodsStructural complex of αSNAP/SBD was determined by blind‐ and data‐driven docking using AutoDock and HADDOCK, respectively. After incorporating Myr‐SBD into SSM (Myr‐SBD‐SSM) by film hydration method, micelle size and shape were confirmed by dynamic light scattering and transmission electron microscopy (TEM), respectively. Intracellular delivery of Myr‐SBD‐SSM was evaluated by fluorescence microscopy and fluorometry in cultured ECs. Cecal ligation and puncture (CLP) in C57BL/6 mice (14–16 wks, 25 g) was used to induce severe inflammation, microvascular thrombosis and DIC, and blood was collected 6 h and 24 h post CLP. The effect of Myr‐SBD‐SSM on plasma vWF, platelet count and inflammatory cytokines was assessed following addition of 0, 2 or 4 μmol Myr‐SBD/kg given i.v. at the time of surgery. Bleeding was assessed by tail‐snip assay. Glomerular fibrin deposition was scored by IHC analysis at 24 h post CLP.ResultsThe αSNAP/SBD structure showed the potential interaction between Arg of SBD peptide and Glu139 of αSNAP by H‐bond. Spherical shape of Myr‐SBD‐SSM (14.1 ± 2.7 nm in diameter) was confirmed by negative staining TEM. Addition of Myr‐SBD‐SSM to ECs resulted in the intracellular accumulation of Myr‐SBD which blocked thrombin‐induced vWF secretion. Intravenous delivery of Myr‐SBD‐SSM to mice at the time of CLP blunted the increase in plasma vWF and associated microvascular thrombosis in the kidney glomerulus. Importantly, Myr‐SBD‐SSM treatment resulted in increased platelet and WBC counts 6 h after surgery and improved survival compared to untreated or scrambled peptide treated mice with severe vascular inflammation suggesting Myr‐SBD‐SSM reduced clotting, platelet consumption and systemic inflammation in intraperitoneal sepsis. Moreover, Myr‐SBD‐SSM treatment did not increase tail‐snip bleeding time suggesting it did not prevent hemostasis.ConclusionOur studies demonstrate that Myr‐SBD‐SSM nanoformulation reduces vWF secretion and attenuates the thrombotic complications of severe inflammation.Support or Funding InformationThis work was supported by MPI R01 HL125356 (RDM, XD), Michael Reese Research and Education Foundation (RDM) and American Heart Association (MB, LR).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A

The protein structure and function paradigm is a foundational tenet of biomolecular science that underlies many infectious diseases. Hemolysin A (HpmA), a hemolytic protein produced by Proteus mirabilis, was used as a model to investigate the protein structure‐function paradigm. HpmA is a member of the two‐partner secretion (TPS) pathway, which is used by gram‐negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A‐component (HpmA) is translocated, folded, and activated by its cognate B‐component in the absence of high‐energy bond and electrochemical gradient dependency. All TpsA components are relatively large and can be further divided into two domains, the two‐partner secretion domain and the functional domain. Universally, all known TPS domains harbor a right‐handed, parallel b‐helix architecture, which has been shown critical for cognate TpsB‐dependent recognition and secretion. Conversely, functional domains provide TpsA diversification including, cytolysis, host cell adhesion, contact‐growth inhibition, and iron sequestration. A truncated version of hemolysin A (HpmA265) was implemented to define the contributions of the TPS domain toward TpsA structure and function. Recently, our group has further dissected HpmA265 into three sequentially folded structural units termed the polar core, non‐polar core, and carboxy‐terminal subdomains. This research project aims to expand upon our recent results and structurally map the role of the nonpolar core during TPS domain dependent secretion, folding, and function. Specifically, residues within the non‐polar core subdomain have been selectively targeted and modified. The structural and functional effects of these site‐selective modifications have been evaluated via chemical denaturation, protease sensitivity, and template‐assisted hemolytic activity. Site‐selective alterations within the non‐polar core subdomain shift four‐state b‐helix unfolding to a three‐state model, increased protease sensitivity, and decrease template‐assisted hemolysis. In contrast, site‐selective alterations on the exterior of the non‐polar core subdomain maintain four‐state unfolding and protease sensitivity, and increase template‐assisted hemolysis. Collectively, these results point toward TPS b‐helix stability as a requisite for robust template‐assisted hemolysis. Conservation of the TPS domain across gram‐negative bacteria makes development of antibiotics targeting b‐helix structure and function attractive.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unraveling the mechanisms of calcium-dependent secretion.

Ca2+-dependent secretion is a process by which important signaling molecules that are produced within a cell-including proteins and neurotransmitters-are expelled to the extracellular environment. The cellular mechanism that underlies secretion is referred to as exocytosis. Many years of work have revealed that exocytosis in neurons and neuroendocrine cells is tightly coupled to Ca2+ and orchestrated by a series of protein-protein/protein-lipid interactions. Here, we highlight landmark discoveries that have informed our current understanding of the process. We focus principally on reductionist studies performed using powerful model secretory systems and cell-free reconstitution assays. In recent years, molecular cloning and genetics have implicated the involvement of a sizeable number of proteins in exocytosis. We expect reductionist approaches will be central to attempts to resolve their roles. The Journal of General Physiology will continue to be an outlet for much of this work, befitting its tradition of publishing strongly mechanistic, basic research.

Dead space ventilation promotes alveolar hypocapnia reducing surfactant secretion by altering mitochondrial function

BackgroundIn acute respiratory distress syndrome (ARDS), pulmonary perfusion failure increases physiologic dead space ventilation (VD/VT), leading to a decline of the alveolar CO2 concentration [CO2]iA. Although it has been shown...

CAPS1 promotes colorectal cancer metastasis via Snail mediated epithelial mesenchymal transformation.

Colorectal cancer (CRC) is a common gastrointestinal cancer with high mortality rate mostly due to metastasis. Ca2+-dependent activator protein for secretion 1 (CAPS1) was originally identified as a soluble factor that reconstitutes Ca2+-dependent secretion. In this study, we discovered a novel role of CAPS1 in CRC metastasis. CAPS1 is frequently up-regulated in CRC tissues. Increased CAPS1 expression is associated with frequent metastasis and poor prognosis of CRC patients. Overexpression of CAPS1 promotes CRC cell migration and invasion in vitro, as well as liver metastasis in vivo, without affecting cell proliferation. CAPS1 induces epithelial-mesenchymal transition (EMT), including decreased E-cadherin and ZO-1, epithelial marker expression, and increased N-cadherin and Snail, mesenchymal marker expression. Snail knockdown reversed CAPS1-induced EMT, cell migration and invasion. This result indicates that Snail is required for CAPS1-mediated EMT process and metastasis in CRC. Furthermore, CAPS1 can bind with Septin2 and p85 (subunit of PI3K). LY294002 and wortmanin, PI3K/Akt inhibitors, can abolish CAPS1-induced increase of Akt/GSK3β activity, as well as increase of Snail protein level. Taken together, CAPS1 promotes colorectal cancer metastasis through PI3K/Akt/GSK3β/Snail signal pathway-mediated EMT process.

Calcium Channels Gate Calcium-Independent But Voltage-Dependent Secretion in Mammalian Cells

The somata of primary sensory neurons, including dorsal root ganglion (DRG) neurons, release neurotransmitters and neuropeptides. Following physiological action potentials, in addition to Ca2+-dependent secretion, we have discovered and studied Ca2+-independent but voltage-dependent secretion (CiVDS) in somata of freshly isolated DRG neurons. Major open question of CiVDS is the molecular mechanism, including 3 components: fusion-pore machinery, voltage-sensor and the linker of fusion-pore and voltage-sensor. Here we report, by using exocytosis assays of membrane capacitance and single vesicle imaging, CiVDS is jointly contributed by (1) SNARE protein SNAP-25 for fusion pore, (2) voltage gated Ca channel (VGCC) Cav2.2 for Ca sensor; (3) and the “synprint”, which is Cav2.2 intracellular loop718-963aa, as the linker between fusion pore and voltage sensor; (4) CiVDS presents in both somata and axons. Following automatic knockdown of CiVDS by 3d-culture of DRG neurons, CiVDS is rescued by overexpressing any component of SNAP-25 or Cav2.2. On other hand, CiVDS is inhibited by blockers against SNAP-25, Cav2.2 and synprint. CiVDS is blocked by Cav2.2-RNAi-KD in DRG pre-transfected in vivo (Chai et al, Neuron, 2017). Importantly, our recent new evidences show that CiVDS presents and functions also robustly in other essential cells, implicating that CiVDS may impact mammalian physiology and disease much more than previously thought.

ADPKD cell proliferation and Cl--dependent fluid secretion.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by bilateral fluid-filled cysts, renal inflammation and extensive fibrosis, leading to the progressive decline in kidney function. Renal cyst formation begins in utero from aberrant proliferation of tubule epithelial cells; however, the mechanisms for cystogenesis remain unclear. Cell proliferation and Cl--dependent fluid secretion, which drives the accumulation of cyst fluid, are responsible for inexorable growth of cysts and the remarkable appearance of massively enlarged ADPKD kidneys. Investigators have used in vitro assays to explore cellular and molecular mechanisms involved in ADPKD cyst epithelial cell proliferation and Cl--dependent fluid secretion in experimentally controlled environments. These assays have been used to evaluate potential therapeutic approaches to inhibit cellular pathways involved in cyst growth. This chapter discusses methods for measuring ADPKD cell proliferation, transepithelial Cl- secretion, and net fluid transport across cyst epithelial cell monolayers.

Лактотрофия младенцев в ракурсе дигестиологии (актовая речь)

The relevance of milk-feeding of babies, the importance of not only the composition and properties of breast milk, but also the technology of enzymatic degradation of its nutrients are cosidered In the Assembly speech. First, it is produced according to the type of its own digestion by hydrolases of the digestive glands and the small intestine of the infant, the complex of which is formed antenatally and called the starting multienzyme digestive potential in the newborn child. Its quantitative characterization is possible by determining the main digestive hydrolases in the amniotic fluid, in the blood of the umbilical cord and in the gastric aspirate of the infant. The expression of this potential in the point system is proposed. With incomplete gestation, the digestive potential is reduced. Secondly, hydrolysis of breast milk nutrients is carried out by its own hydrolases in the autolytic type of digestion. The dynamics of secretion of hydrolases by the mammary glands of healthy women during one year of lactation was studied. The quantitative dependence of the hydrolase secretion on its value in the first lactation month is described with a general tendency to a decrease in the hydrolase content in milk with an increase in the monthly lactation period, and the dependence of the secretion of enzymes by the mammary glands from the age of the pregnant women. The dynamic convergence of the multienzyme digestive potentials of the child's digestion system and the mother's milk it takes is relevant in lactotrophy. Based on the results of peptidic analysis of auto-and inragastral proteolysis of breast milk, milk hydrolases are not only considered as trophotropic participants in the degradation of its nutrients, but also in formation of by milk proteases functionally multipotent regulatory peptides with a characteristic change in their number and content in the dynamics of infant feeding and timing of gestation.

Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.

Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

Rhodomyrtone decreases Staphylococcus aureus SigB activity during exponentially growing phase and inhibits haemolytic activity within membrane vesicles

Sigma factor B (SigB) controls the expression of Staphylococcus aureus genes including virulence factors and plays a role in the bacterial secretion system through membrane vesicle production. Inhibition of SigB could attenuate SigB dependent virulence and secretion system. The objective of this study was to determine the effects of rhodomyrtone on SigB and virulence factors related to SigB. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of rhodomyrtone against 67 clinical methicillin-resistant S. aureus isolates were 0.25–8 μg/ml, which were similar to those of vancomycin. Using luciferase gene fused to SigB dependent promoters of asp23, five time reduction in SigB activity was observed when the bacteria were treated with rhodomyrtone for 3 h. Rhodomyrtone significantly reduced SigB activity in a concentration dependent manner in exponentially growing cells (P < 0.05). In addition, sigB mutant was more sensitive towards increasing concentrations of rhodomyrtone than the wild type and yabJ-spoVG mutant. Rhodomyrtone at 0.625 μg/ml reduced the growth of sigB mutant by approximately 99%, compared with the yabJ-spoVG mutant and the wild type. Membrane vesicles were significantly reduced in the bacterial cells when treated with 0.5 × MIC rhodomyrtone (P < 0.05). Decreased haemolytic activity was detected within rhodomyrtone-treated membrane vesicles. The results indicated that rhodomyrtone inhibited S. aureus SigB activity during exponentially growing phase and inhibited haemolytic activity within membrane vesicles.

Alternative to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type A.

Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SE detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility, and causes ethical concerns regarding the use of experimental animals. The Lautenberg Chemical Safety Act encourages the development and adoption of alternatives to testing on animals for chemical toxicity methodologies. In this study, we utilized the superantigenic effect of SE type A (SEA) and used an ex vivo bioassay as an alternative to live animal testing. We found that interleukin-2 (IL-2) secreted by splenocyte can be utilized for quantifiable detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we separated SEA from spiked food products by employing immunomagnetic beads that were coated with an anti-SEA antibody. This ex vivo method has achieved the detection of 1 ng mL−1 of SEA, which is 107 times more sensitive than the existing live animal testing methods. However, this ex vivo bioassay requires sacrificing of mice. To overcome this limitation, we established a cell based in vitro assay using CCRF-CEM, a human CD4+ T-cell line, for the quantitative detection of SEA. Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA, compared with the related SE toxin subtypes B, D, and E or heat inactivated SEA, which produce no secretion of IL-2. This is the first demonstration of an alternative assay that completely eliminates the use of animals for quantitative detection of active SEA.

Fine-tuning pyroptosis with ESCRT-III

Immunology Pyroptosis is an inflammatory form of cell death induced by select caspases downstream of inflammasome complexes. These caspases cleave gasdermin D (GSDMD), whose N-terminal fragments quickly form large permeability pores that induce cell death. However, a large percentage of cells with active inflammasomes are resistant to pyroptosis. Ruhl et al. found that the membrane-remodeling ESCRT-III machinery was recruited to the plasma membrane upon GSDMD activation. ESCRT-III–dependent membrane repair limited proinflammatory cytokine secretion and pyroptosis after activation of inflammasomes. Science , this issue p. [956][1] [1]: /lookup/doi/10.1126/science.aar7607

Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation

ObjectivesTNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked...

Cellular Clearance and Biological Activity of Calciprotein Particles Depend on Their Maturation State and Crystallinity.

Background: The liver-derived plasma protein fetuin-A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes saturated mineral solutions by forming colloidal protein-mineral complexes called calciprotein particles (CPP). CPP are initially spherical, amorphous and soft, and are referred to as primary CPP. These particles spontaneously convert into secondary CPP, which are larger, oblongate, more crystalline, and less soluble. CPP mediate excess mineral transport and clearance from circulation.Methods: We studied by intravital two-photon microscopy the clearance of primary vs. secondary CPP by injecting i.v. synthetic fluorescent CPP in mice. We analyzed CPP organ distribution and identified CPP endocytosing cells by immunofluorescence. Cellular clearance was studied using bone marrow-derived mouse wildtype and scavenger receptor A (SRA)-deficient macrophages, as well as human umbilical cord endothelial cells (HUVEC), monocyte-derived macrophages (hMDM), and human aortic endothelial cells (haEC). We employed mouse wildtype and mutant immortalized macrophages to analyze CPP-induced inflammasome activation and cytokine secretion.Results: In live mice, only primary CPP were rapidly cleared by liver sinusoidal endothelial cells (LSEC), whereas primary and secondary CPP were cleared by Kupffer cells. Scavenger receptor A (SRA)-deficient bone marrow macrophages endocytosed secondary CPP less well than did wildtype macrophages. In contrast, primary CPP endocytosis did not depend on the presence of SRA, suggesting involvement of an alternative clearance pathway. CPP triggered TLR4 dependent TNFα and IL-1β secretion in cultured macrophages. Calcium content-matched primary CPP caused twice more IL-1β secretion than did secondary CPP, which was associated with increased calcium-dependent inflammasome activation, suggesting that intracellular CPP dissolution and calcium overload may cause this inflammation.Conclusions: Secondary CPP are endocytosed by macrophages in liver and spleen via SRA. In contrast, our results suggest that primary CPP are cleared by LSEC via an alternative pathway. CPP induced TLR4-dependent TNFα and inflammasome-dependent IL-1β secretion in macrophages suggesting that inflammation and calcification may be considered consequences of prolonged CPP presence and clearance.

Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing.

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.

An Emerging Role of Endometrial Inflammasome in Reproduction: New Therapeutic Approaches.

One of the common complications of pregnancy is spontaneous pregnancy loss which occurs in an estimated 5- 15% of pregnancies. Of all women 1%-5% suffer from Recurrent Pregnancy Loss (RPL). Despite the fact that RPL has been associated to various anatomic, hormonal, immune, hematologic, and genetic defects, in 30% of the patients, screening tests included in the RPL workup may have negative results. Recently, we demonstrated a significant increased activation of endometrial NALP-3 inflammasome, and a caspase-1 dependent secretion of IL-18 and IL-1β in the endometrial tissues obtained from RPL women compared with a fertile women group. The inflammasome has emerged as a key player in innate immunity and inflammation. An abnormal inflammasome activation, in absence of detectable infectious causes, might be one of the molecular mechanisms involved in establishing an unreceptive endometrium, potentially leading to early fetal loss. Upon activation, this multiprotein complex makes possible the caspase- 1-mediated proteolytic processing of proinflammatory cytokines generating their respective mature secretory forms. The understanding of molecular modulation of inflammasome associated pathways is critical for drug design, development and delivery. To date many promising inhibitors of inflammasome complex activation have been described, such as MCC950, β-Hydroxybutyrate or Micro RNAs that affect NALP3 expression and activation. Furthermore, several herbal extracts and its bioactive constituents have shown to be effective in inflammatory response mediated by NLRP3 inflammasome activation. Nevertheless all these molecules represent a significant progress toward developing therapies that target IL-18 and IL-1β secretion in a variety of diseases.

Phenylalanine and tryptophan stimulate gastrin and somatostatin secretion and H+-K+-ATPase activity in pigs through calcium-sensing receptor

In rodents and humans, aromatic amino acids increase gut hormone secretion and H+-K+-ATPase activity by modulating calcium-sensing receptor (CaSR). However, the role of CaSR and its related signaling molecules in amino acid-induced gut hormone secretion in swine has not been investigated. Here, we examined whether a CaSR-dependent pathway modulated gastrin and somatostatin (SS) secretion and H+-K+-ATPase activity in pigs. Perfusion of pig stomach tissues in the presence of extracellular 80 mM l-phenylalanine (Phe) or 20 mM l-tryptophan (Trp) and a CaSR agonist cinacalcet triggered gastrin and SS secretion and H+-K+-ATPase activity (P < 0.05) and increased CaSR expression (P < 0.05). This effect of Phe and Trp was dependent on Ca2+ (P < 0.05) and was abolished after treatment with NPS 2143, an inhibitor of CaSR, and 2-aminoethyl diphenyl borinate, an inhibitor of CaSR downstream signaling molecule inositol 1,4,5-triphosphate receptor (IP3R). These findings indicate that Phe and Trp induce Ca2+-dependent gastrin and SS secretion and H+-K+-ATPase activity through CaSR and its downstream signaling molecule IP3R.

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

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. 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. We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an invitro 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. 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. SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE.

P-21 - Redox-activated mitochondrial iPLA2γ participates in fatty acid-stimulated insulin secretion

Mitochondrial calcium-independent phospholipase isoform γ (iPLA2γ/PNPLA8) has been shown to augment G-protein coupled receptor 40- (GPR40-) dependent insulin secretion in β-cell model insulinoma INS1-E cells [1]. Fatty acid β-oxidation produces the intramitochondrial redox signal by the elevated superoxide formation on the ETF:QOR. Superoxide is dismuted to H2O2 by MnSOD and H2O2 directly stimulates iPLA2γ, which cleaves fatty acids (FAs) from phospholipids of mitochondrial membranes. FAs literally radiate to the plasma membrane and serve as GPR40 agonists. We intended to confirm and study this mechanism in vivo comparing FA-stimulated insulin secretion (FASIS) in iPLA2γ-knockout to wild type mice. Our results support the promoting role of iPLA2γ in insulin secretion in vivo.

Cutting Edge: Mitochondrial Assembly of the NLRP3 Inflammasome Complex Is Initiated at Priming.

The NLRP3 inflammasome is activated in response to microbial and danger signals, resulting in caspase-1-dependent secretion of the proinflammatory cytokines IL-1β and IL-18. Canonical NLRP3 inflammasome activation is a two-step process requiring both priming and activation signals. During inflammasome activation, NLRP3 associates with mitochondria; however, the role for this interaction is unclear. In this article, we show that mouse NLRP3 and caspase-1 independently interact with the mitochondrial lipid cardiolipin, which is externalized to the outer mitochondrial membrane at priming in response to reactive oxygen species. An NLRP3 activation signal is then required for the calcium-dependent association of the adaptor molecule ASC with NLRP3 on the mitochondrial surface, resulting in inflammasome complex assembly and activation. These findings demonstrate a novel lipid interaction for caspase-1 and identify a role for mitochondria as supramolecular organizing centers in the assembly and activation of the NLRP3 inflammasome.