The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Role of autophagy in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a leading contributor to low back pain. The intervertebral disc (IVD) is composed of three tissue types: the central gelatinous nucleus pulposus (NP) tissue, the surrounding annulus fibrosus (AF) tissue, and the inferior and superior cartilage endplates. The IVD microenvironment is hypoxic, acidic, hyperosmotic, and low in nutrients because it is mostly avascular. The cellular processes that underlie IDD initiation and progression are still poorly understood. Specifically, a lack of understanding regarding NP cell metabolism and physiology hinders the development of effective therapeutics to treat IDD patients. Autophagy is a vital intracellular degradation process that removes damaged organelles, misfolded proteins, and intracellular pathogens and recycles the degraded components for cellular energy and function. NP cells have adapted to survive within their harsh tissue microenvironment using processes that are largely unknown, and we postulate autophagy is one of these undiscovered mechanisms. In this review, we describe unique features of the IVD tissue, review how physiological stressors impact autophagy in NP cells in vitro, survey the current understanding of autophagy regulation in the IVD, and assess the relationship between autophagy and IDD. Published studies confirm autophagy markers are present in IVD tissue, and IVD cells can regulate autophagy in response to cellular stressors in vitro. However, data are still lacking to determine the exact mechanisms regulating autophagy in IVD cells. More in-depth research is needed to establish whether autophagy is necessary to maintain IVD cell health and validate autophagy as a relevant therapeutic target for treating IDD.

GRK2 regulates group 2 innate lymphoid cell mobilization in sepsis.

BACKGROUND: Sepsis induces group 2 innate lymphoid cell (ILC2) expansion in the lung. However, the origin of these lung-recruited ILC2 and the mechanism of ILC2 expansion are unclear. This study aims to determine the origin of lung-recruited ILC2 and its underlying mechanism in sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP) model in wild-type, IL-33-deficient and ST2-deficient mice. The frequency, cell number and C-X-C chemokine receptor 4 (CXCR4) expression of ILC2 in bone marrow (BM), blood and lung were measured by flow cytometry. In the in vitro studies, purified ILC2 progenitor (ILC2p) were challenged with IL-33 or G protein-coupled receptor kinase 2 (GRK2) inhibitor, the CXCR4 expression and GRK2 activity were detected by confocal microscopy or flow cytometry. RESULTS: We show that IL-33 acts through its receptor, ST2, on BM ILC2p to induce GRK2 expression and subsequent downregulation of cell surface expression of CXCR4, which results in decreasing retention of ILC2p in the BM and promoting expansion of ILC2 in the lung. Importantly, we demonstrate that reduced IL-33 level in aging mice contributes to impaired ILC2 mobilization from BM and accumulation in the lung following sepsis. CONCLUSION: This study identifies a novel pathway in regulating ILC2p mobilization and expansion during sepsis and indicates BM as the main source of ILC2 in the lung following sepsis.

Maresin 1 protects the liver against ischemia/reperfusion injury via the ALXR/Akt signaling pathway.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury can be a major complication following liver surgery contributing to post-operative liver dysfunction. Maresin 1 (MaR1), a pro-resolving lipid mediator, has been shown to suppress I/R injury. However, the mechanisms that account for the protective effects of MaR1 in I/R injury remain unknown. METHODS: WT (C57BL/6J) mice were subjected to partial hepatic warm ischemia for 60mins followed by reperfusion. Mice were treated with MaR1 (5-20 ng/mouse), Boc2 (Lipoxin A4 receptor antagonist), LY294002 (Akt inhibitor) or corresponding controls just prior to liver I/R or at the beginning of reperfusion. Blood and liver samples were collected at 6 h post-reperfusion. Serum aminotransferase, histopathologic changes, inflammatory cytokines, and oxidative stress were analyzed to evaluate liver injury. Signaling pathways were also investigated in vitro using primary mouse hepatocyte (HC) cultures to identify underlying mechanisms for MaR1 in liver I/R injury. RESULTS: MaR1 treatment significantly reduced ALT and AST levels, diminished necrotic areas, suppressed inflammatory responses, attenuated oxidative stress and decreased hepatocyte apoptosis in liver after I/R. Akt signaling was significantly increased in the MaR1-treated liver I/R group compared with controls. The protective effect of MaR1 was abrogated by pretreatment with Boc2, which together with MaR1-induced Akt activation. MaR1-mediated liver protection was reversed by inhibition of Akt. CONCLUSIONS: MaR1 protects the liver against hepatic I/R injury via an ALXR/Akt signaling pathway. MaR1 may represent a novel therapeutic agent to mitigate the detrimental effects of I/R-induced liver injury.

Return of Individual Research Results: A Guide for Biomedical Researchers Utilizing Human Biospecimens.

The recent movement toward returning individual research results to study subjects/participants generates ethical and legal challenges for laboratories performing research on human biospecimens. The concept of an individual's interest in knowing the results of testing on their tissue is pitted against individual and systemic risks and an established legal framework regulating the performance of laboratory testing for medical care purposes. This article discusses the rationale for returning individual research results to subjects, the potential risks associated with returning these results, and the legal framework in the United States that governs testing of identifiable human biospecimens. On the basis of these considerations, this article provides recommendations for investigators to consider when planning and executing human biospecimen research, with the objective of appropriately balancing the interests of research subjects, the need for ensuring integrity of the research process, and compliance with US laws and regulations.

Immune-Responsive Gene 1/Itaconate Activates Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocytes to Protect Against Liver Ischemia-Reperfusion Injury.

BACKGROUND AND AIMS: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown. APPROACH AND RESULTS: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury. CONCLUSIONS: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.

Platelet Extracellular Vesicles Drive Inflammasome-IL-1ß-Dependent Lung Injury in Sickle Cell Disease.

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods:In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1ß and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1ß pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that platelet-inflammasome-dependent shedding of IL-1ß and caspase-1-carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.

MetAP2 inhibitor treatment of high-fat and -fructose-fed dogs: impact on the response to oral glucose ingestion and a hyperinsulinemic hyperglycemic clamp.

We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.

The kinetics of glucagon action on the liver during insulin-induced hypoglycemia.

Glucagon's effect on hepatic glucose production (HGP), under hyperglycemic conditions, is time dependent such that after an initial burst of HGP, it slowly wanes. It is not known whether this is also the case under hypoglycemic conditions, where an increase in HGP is essential. This question was addressed using adrenalectomized dogs to avoid the confounding effects of other counterregulatory hormones. During the study, infusions of epinephrine and cortisol were given to maintain basal levels. Somatostatin and insulin (800 µU·kg-1·min-1) were infused to induce hypoglycemia. After 30 min, glucagon was infused at a basal rate (1 ng·kg-1·min-1, baGGN group, n = 5 dogs) or a rate eightfold basal (8 ng·kg-1·min-1, hiGGN group, n = 5 dogs) for 4 h. Glucose was infused to match the arterial glucose levels between groups (≈50 mg/dL). Our data showed that glucagon has a biphasic effect on the liver despite hypoglycemia. Hyperglucagonemia stimulated a rapid, transient peak in HGP (4-fold basal production) over ~60 min, which was followed by a slow reduction in HGP to a rate 1.5-fold basal. During the last 2 h of the experiment, hiGGN stimulated glucose production at a rate fivefold greater than baGGN (2.5 vs. 0.5 mg·kg-1·min-1, respectively), indicating a sustained effect of the hormone. Of note, the hypoglycemia-induced rises in norepinephrine and glycerol were smaller in hiGGN compared with the baGGN group despite identical hypoglycemia. This finding suggests that there is reciprocity between glucagon and the sympathetic nervous system such that when glucagon is increased, the sympathetic nervous response to hypoglycemia is downregulated.

Hepatocyte high-mobility group box 1 protects against steatosis and cellular stress during high fat diet feeding.

BACKGROUND: Circulating high-mobility group box 1 (HMGB1) plays important roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Intracellular HMGB1 is critical for the biology of hepatocytes. However, the intracellular role of HMGB1 in hepatocellular steatosis is unknown. Therefore, we aimed to investigate the role of hepatocyte-specific HMGB1 (HC-HMGB1) in development of hepatic steatosis. METHODS: Wild type (WT) C57BL/6 and HC-HMGB1-/- mice were fed high-fat diet (HFD) or low-fat diet (LFD) for up to 16 weeks. RESULTS: As expected, HMGB1 translocated from nuclear into cytoplasm and released into circulation after HFD treatment. HC-HMGB1 deficiency significantly reduced circulating HMGB1, suggesting that hepatocyte is a major source of circulating HMGB1 during NAFLD. Unexpectedly, HC-HMGB1 deficiency promoted rapid weight gain with enhanced hepatic fat deposition compared with WT at as early as 4 weeks after HFD treatment. Furthermore, there was no difference between WT and HC-HMGB1-/- mice in glucose tolerance, energy expenditure, liver damage or systemic inflammation. Interestingly, hepatic gene expression related to free fatty acid (FFA) ß-oxidation was significantly down-regulated in HC-HMGB1-/- mice compared with WT, and endoplasmic reticulum (ER) stress markers were significantly higher in livers of HC-HMGB1-/- mice. In vitro experiments using primary mouse hepatocytes showed absence of HMGB1 increased FFA-induced intracellular lipid accumulation, accompanied by increased ER-stress, significant downregulation of FFA ß-oxidation, and reduced oxidative phosphorylation. CONCLUSIONS: Our findings suggest that hepatocyte HMGB1 protects against dysregulated lipid metabolism via maintenance of ß-oxidation and prevention of ER stress. This represents a novel mechanism for HMGB1-regulation of hepatocellular steatosis, and suggests that stabilizing HMGB1 in hepatocytes may be effective strategies for prevention and treatment of NAFLD.

Caspase1/11 signaling affects muscle regeneration and recovery following ischemia, and can be modulated by chloroquine.

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.

RAGE-induced ILC2 expansion in acute lung injury due to haemorrhagic shock.

BACKGROUND: Type 2 immune dysfunction contributes to acute lung injury and lethality following haemorrhagic shock (HS) and trauma. Group 2 innate lymphoid cells (ILC2s) play a significant role in the regulation of type 2 immune responses. However, the role of ILC2 in post-HS acute lung injury and the underlying mechanism has not yet been elucidated. OBJECTIVE: To investigate the regulatory role of ILC2s in HS-induced acute lung injury and the underlying mechanism in patients and animal model. METHODS: Circulating markers of type 2 immune responses in patients with HS and healthy controls were characterised. Using a murine model of HS, the role of high-mobility group box 1 (HMGB1)-receptor for advanced glycation end products (RAGE) signalling in regulation of ILC2 proliferation, survival and function was determined. And the role of ILC2 in inducing type 2 immune dysfunction was assessed as well. RESULTS: The number of ILC2s was significantly increased in the circulation of patients with HS that was correlated with the increase in the markers of type 2 immune responses in the patients. Animal studies showed that HMGB1 acted via RAGE to induce ILC2 accumulation in the lungs by promoting ILC2 proliferation and decreasing ILC2 death. The expansion of ILC2s resulted in type 2 cytokines secretion and eosinophil infiltration in the lungs, both of which contributed to lung injury after HS. CONCLUSIONS: These results indicate that HMGB1-RAGE signalling plays a critical role in regulating ILC2 biological function that aggravates type 2 lung inflammation following HS.

Cutting Edge: The Heat Shock Protein gp96 Activates Inflammasome-Signaling Platforms in APCs.

Several heat shock proteins (HSPs) prime immune responses, which are, in part, a result of activation of APCs. APCs respond to these immunogenic HSPs by upregulating costimulatory molecules and secreting cytokines, including IL-1ß. These HSP-mediated responses are central mediators in pathological conditions ranging from cancer, sterile inflammation associated with trauma, and rheumatoid arthritis. We tested in this study the requirement of inflammasomes in the release of IL-1ß by one immunogenic HSP, gp96. Our results show that murine APCs activate NLRP3 inflammasomes in response to gp96 by K+ efflux. This is shown to initiate inflammatory conditions in vivo in the absence of additional known inflammasome activators or infection. These results document a novel mechanism by which proteins of endogenous origin, the HSPs, can modulate an inflammatory response following their release from aberrant cells.

Effect of portal glucose sensing on incretin hormone secretion in a canine model.

It is unknown whether activation of hepato-portal vein (PV) glucose sensors plays a role in incretin hormone amplification of oral glucose-stimulated insulin secretion (GSIS). In previous studies, PV glucose infusion increased GSIS through unknown mechanisms, perhaps neural stimulation of pancreatic ß-cells and/or stimulation of gut incretin hormone release. Thus, there could be a difference in the incretin effect when comparing GSIS with portal rather than leg vein (LV) glucose infusion. Plasma insulin and incretin hormones were studied in six overnight-fasted dogs. An oral glucose tolerance test (OGTT) was administered, and then 1 and 2 wk later the arterial plasma glucose profile from the OGTT was mimicked by infusing glucose into either the PV or a LV. The arterial glucose levels were nearly identical between groups (AUCs within 1% of each other). Oral glucose administration increased arterial GLP-1 and GIP levels by more than sixfold, whereas they were not elevated by PV or LV glucose infusion. Oral glucose delivery was associated with only a small incretin effect (arterial insulin and C-peptide were 21 ± 23 and 24 ± 17% greater, respectively, during the 1st hour with oral compared with PV glucose and 14 ± 37 and 13 ± 35% greater, respectively, in oral versus LV; PV versus LV responses were not significantly different from each other). Thus, following an OGTT incretin hormone release did not depend on activation of PV glucose sensors, and the insulin response was not greater with PV compared with LV glucose infusion in the dog. The small incretin effect points to species peculiarities, which is perhaps related to diet.

MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.

Peripherally delivered hepatopreferential insulin analog insulin-406 mimics the hypoglycaemia-sparing effect of portal vein human insulin infusion in dogs.

AIMS: We previously quantified the hypoglycaemia-sparing effect of portal vs peripheral human insulin delivery. The current investigation aimed to determine whether a bioequivalent peripheral vein infusion of a hepatopreferential insulin analog, insulin-406, could similarly protect against hypoglycaemia. MATERIALS AND METHODS: Dogs received human insulin infusions into either the hepatic portal vein (PoHI, n = 7) or a peripheral vein (PeHI, n = 7) for 180 minutes at four-fold the basal secretion rate (6.6 pmol/kg/min) in a previous study. Insulin-406 (Pe406, n = 7) was peripherally infused at 6.0 pmol/kg/min, a rate determined to decrease plasma glucose by the same amount as with PoHI infusion during the first 60 minutes. Glucagon was fixed at basal concentrations, mimicking the diminished α-cell response seen in type 1 diabetes. RESULTS: Glucose dropped quickly with PeHI infusion, reaching 41 ± 3 mg/dL at 60 minutes, but more slowly with PoHI and Pe406 infusion (67 ± 2 and 72 ± 4 mg/dL, respectively; P < 0.01 vs PeHI for both). The hypoglycaemic nadir (c. 40 mg/dL) occurred at 60 minutes with PeHI infusion vs 120 minutes with PoHI and Pe406 infusion. ΔAUCepinephrine during the 180-minute insulin infusion period was two-fold higher with PeHI infusion compared with PoHI and Pe406 infusion. Glucose production (mg/kg/min) was least suppressed with PeHI infusion (Δ = 0.79 ± 0.33) and equally suppressed with PoHI and Pe406 infusion (Δ = 1.16 ± 0.21 and 1.18 ± 0.17, respectively; P = NS). Peak glucose utilization (mg/kg/min) was highest with PeHI infusion (4.94 ± 0.17) and less with PoHI and Pe406 infusion (3.58 ± 0.58 and 3.26 ± 0.08, respectively; P < 0.05 vs Pe for both). CONCLUSIONS: Peripheral infusion of hepatopreferential insulin can achieve a metabolic profile that closely mimics portal insulin delivery, which reduces the risk of hypoglycaemia compared with peripheral insulin infusion.

Aging-Impaired Filamentous Actin Polymerization Signaling Reduces Alveolar Macrophage Phagocytosis of Bacteria.

In elderly patients, bacterial infection often causes severe complications and sepsis. Compared to younger patients, older patients are more susceptible to sepsis caused by respiratory infection. Macrophage (Mϕ) phagocytosis of bacteria plays a critical role in the clearance of pathogens and the initiation of immune responses. It has been suggested that Mϕ exhibit age-related functional alterations, including reduced chemotaxis, phagocytosis, antibacterial defense, and the ability to generate reactive oxygen species. However, the mechanisms behind these changes remain unclear. The present study sought to determine changes in bacterial phagocytosis in aging alveolar Mϕ (AMϕ) and the underlying mechanisms. We show that bacteria initiate cytoskeleton remodeling in AMϕ through interaction with macrophage receptor with collagenous structure (MARCO), a bacterial scavenger receptor. This remodeling, in turn, promotes enhanced cell surface expression of MARCO and bacterial phagocytosis. We further demonstrate that Rac1-GTP mediates MARCO signaling and activates actin-related protein-2/3 complex, an F-actin nucleator, thereby inducing F-actin polymerization, filopodia formation, and increased cell surface expression of MARCO, all of which are essential for the execution of bacteria phagocytosis. However, AMϕ isolated from aging mice exhibit suppressed Rac1 mRNA and protein expression, which resulted in decreases in Rac1-GTP levels and actin-related protein-2/3 activation, as well as subsequent attenuation of F-actin polymerization, filopodia formation, and cell surface expression of MARCO. As a result, bacterial phagocytosis in aging AMϕ is decreased. This study highlights a previously unidentified mechanism by which aging impairs Mϕ phagocytosis of bacteria. Targeting these pathways may improve outcomes of bacterial infection in elderly patients.

cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING.

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS-/-), and STING-deficient (STINGgt/gt) mice to warm liver I/R injury and that found cGAS-/- mice had significantly increased liver injury compared with WT or STINGgt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS-/- mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS-/- hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

Redox-dependent regulation of hepatocyte absent in melanoma 2 inflammasome activation in sterile liver injury in mice.

Sterile liver inflammation, such as liver ischemia-reperfusion, hemorrhagic shock after trauma, and drug-induced liver injury, is initiated and regulated by endogenous mediators including DNA and reactive oxygen species. Here, we identify a mechanism for redox-mediated regulation of absent in melanoma 2 (AIM2) inflammasome activation in hepatocytes after redox stress in mice, which occurs through interaction with cytosolic high mobility group box 1 (HMGB1). We show that in liver during hemorrhagic shock in mice and in hepatocytes after hypoxia with reoxygenation, cytosolic HMGB1 associates with AIM2 and is required for activation of caspase-1 in response to cytosolic DNA. Activation of caspase-1 through AIM2 leads to subsequent hepatoprotective responses such as autophagy. HMGB1 binds to AIM2 at a non-DNA-binding site on the hematopoietic interferon-inducible nuclear antigen domain of AIM2 to facilitate inflammasome and caspase-1 activation in hepatocytes. Furthermore, binding of HMGB1 to AIM2 is stronger with fully reduced all-thiol HMGB1 than with partially oxidized disulfide-HMGB1, and binding strength corresponds to caspase-1 activation. These data suggest that HMGB1 redox status regulates AIM2 inflammasome activation. CONCLUSION: These findings suggest a novel and important mechanism for regulation of AIM2 inflammasome activation in hepatocytes during redox stress and may suggest broader implications for how this and other inflammasomes are activated and how their activation is regulated during cell stress, as well as the mechanisms of inflammasome regulation in nonimmune cell types. (Hepatology 2017;65:253-268).

Inflammasome and autophagy regulation - a two-way street.

Inflammation plays a significant role in protecting hosts against pathogens. Inflammation induced by non-infectious, endogenous agents can be detrimental, and if excessive can result in organ and tissue damage. The inflammasome is a major innate immune pathway that can be activated via both exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). Inflammasome activation involves formation and oligomerization of a protein complex including a NOD-like receptor (NLR), an adaptor protein (ASC) and procaspase-1. This then allows cleavage and activation of caspase-1, followed by downstream cleavage and release of proinflammatory cytokines, IL-1ß and IL-18, from innate immune cells. Hyperinflammation caused by unrestrained inflammasome activation is linked with multiple inflammatory diseases, including inflammatory bowel disease, Alzheimer's disease and multiple sclerosis. So there is an understandable rush to understand mechanisms that regulate such potent inflammatory pathways. Autophagy has now been identified as a main regulator of inflammasomes. Autophagy is a vital intracellular process involved in cellular homeostasis, recycling and removal of damaged organelles (e.g. mitochondria) and intracellular pathogens. Autophagy is regulated by proteins that are important in endosomal/phagosomal pathways, as well as by specific autophagy proteins coded for by autophagy-related genes. Cytosolic components are surrounded and contained by a double-membraned vesicle, which then fuses with lysosomes to enable degradation of the contents. Autophagic removal of intracellular DAMPs, inflammasome components or cytokines can reduce inflammasome activation. Similarly, inflammasomes can regulate the autophagic process, allowing for a two-way, mutual regulation of inflammation that may hold the key for treatment of multiple diseases.