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Impaired autophagy increases susceptibility to endotoxin-induced chronic pancreatitis.

ABSTRACT: Chronic pancreatitis (CP) is associated with elevated plasma levels of bacterial lipopolysaccharide (LPS) and we have demonstrated reduced acinar cell autophagy in human CP tissue. Therefore, we investigated the role of autophagy in experimental endotoxin-induced pancreatic injury and aimed to identify LPS in human CP tissue. Pancreatic Atg7-deficient mice were injected with a single sub-lethal dose of LPS. Expression of autophagy, apoptosis, necroptosis, and inflammatory markers was determined 3 and 24?h later utilizing immunoblotting and immunofluorescence. The presence of LPS in pancreatic tissue from mice and from patients and healthy controls was determined using immunohistochemistry, immunoblots, and chromogenic assay. Mice lacking pancreatic autophagy exhibited local signs of inflammation and were particularly sensitive to the toxic effect of LPS injection as compared to control mice. In response to LPS, Atg7?pan mice exhibited enhanced vacuolization of pancreatic acinar cells, increase in TLR4 expression coupled to enhanced expression of NF-??, JNK, and pro-inflammatory cytokines by acinar cells and enhanced infiltration by myeloid cells (but not Atg7F/F controls). Cell death was enhanced in Atg7?pan pancreata, but only necroptosis and trypsin activation was further amplified following LPS injection along with elevated pancreatic LPS. The presence of LPS was identified in the pancreata from all 14 CP patients examined but was absent in the pancreata from all 10 normal controls. Altogether, these results support a potential role for metabolic endotoxemia in the pathogenesis of CP. Moreover, the evidence also supports the notion that autophagy plays a major cytoprotective and anti-inflammatory role in the pancreas, and blunting metabolic endotoxemia-induced CP.

SUBMITTER: Xia L

PROVIDER: S-EPMC7578033 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Impaired autophagy increases susceptibility to endotoxin-induced chronic pancreatitis.

Xia L L Xu Z Z Zhou X X Bergmann F F Grabe N N Büchler M W MW Neoptolemos J P JP Hackert T T Kroemer G G Fortunato F F

Cell death & disease 20201021 10

Chronic pancreatitis (CP) is associated with elevated plasma levels of bacterial lipopolysaccharide (LPS) and we have demonstrated reduced acinar cell autophagy in human CP tissue. Therefore, we investigated the role of autophagy in experimental endotoxin-induced pancreatic injury and aimed to identify LPS in human CP tissue. Pancreatic Atg7-deficient mice were injected with a single sub-lethal dose of LPS. Expression of autophagy, apoptosis, necroptosis, and inflammatory markers was determined ...[more]

PMID: 33087696

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Project description:Background & aimsPancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy.MethodsWe obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays.ResultsHuman pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen.ConclusionsIn studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Project description:Proteasome degradation is an integral part of cellular growth and function. Proteasomal intervention may mitigate adverse myocardial remodeling, but is associated with the onset of heart failure. Previously, we have demonstrated that increasing abundance of cardiac Lmp2 and its incorporation into proteasome complexes is an endogenous mechanism for proteasome regulation during hypertrophic remodeling of the heart induced by chronic ß-adrenoreceptor stimulation. Here, we investigated whether Lmp2 is required for myocardial remodeling not driven by inflammation and show that Lmp2 is a tipping element for growth and function in the heart but not for proteasome insufficiency. While it has no apparent impact under unchallenged conditions, myocardial remodeling without Lmp2 exacerbates hypertrophy and restricts cardiac function. Under chronic ß-adrenoreceptor stimulation, as seen in the development of cardiovascular disease and the manifestation of heart failure, genetic ablation of Lmp2 in mice caused augmented concentric hypertrophy of the left ventricle. While the heart rate was similarly elevated as in wildtype, myocardial contractility was not maintained without Lmp2, and apparently uncoupled of the ß-adrenergic response. Normalized to the exacerbated myocardial mass, contractility was reduced by 41% of the pretreatment level, but would appear preserved at absolute level. The lack of Lmp2 interfered with elevated 26S proteasome activities during early cardiac remodeling reported previously, but did not cause bulk proteasome insufficiency, suggesting the Lmp2 containing proteasome subpopulation is required for a selected group of proteins to be degraded. In the myocardial interstitium, augmented collagen deposition suggested matrix stiffening in the absence of Lmp2. Indeed, echocardiography of left ventricular peak relaxation velocity (circumferential strain rate) was reduced in this treatment group. Overall, targeting Lmp2 in a condition mimicking chronic ß-adrenoreceptor stimulation exhibited the onset of heart failure. Anticancer therapy inhibiting proteasome activity, including Lmp2, is associated with adverse cardiac events, in particular heart failure. Sparing Lmp2 may be an avenue to reduce adverse cardiac events when chronic sympathetic nervous system activation cannot be excluded.

Dataset Information

Impaired autophagy increases susceptibility to endotoxin-induced chronic pancreatitis.

Publications

Impaired autophagy increases susceptibility to endotoxin-induced chronic pancreatitis.

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