sEH-derived metabolites of linoleic acid drive pathologic inflammation while impairing key innate immune cell function in burn injury.

Fatty acid composition in the Western diet has shifted from saturated to polyunsaturated fatty acids (PUFAs), and specifically to linoleic acid (LA, 18:2), which has gradually increased in the diet over the past 50 y to become the most abundant dietary fatty acid in human adipose tissue. PUFA-derived oxylipins regulate a variety of biological functions. The cytochrome P450 (CYP450)­formed epoxy fatty acid metabolites of LA (EpOMEs) are hydrolyzed by the soluble epoxide hydrolase enzyme (sEH) to dihydroxyoctadecenoic acids (DiHOMEs). DiHOMEs are considered cardioprotective at low concentrations but at higher levels have been implicated as vascular permeability and cytotoxic agents and are associated with acute respiratory distress syndrome in severe COVID-19 patients. High EpOME levels have also correlated with sepsis-related fatalities; however, those studies failed to monitor DiHOME levels. Considering the overlap of burn pathophysiology with these pathologies, the role of DiHOMEs in the immune response to burn injury was investigated. 12,13-DiHOME was found to facilitate the maturation and activation of stimulated neutrophils, while impeding monocyte and macrophage functionality and cytokine generation. In addition, DiHOME serum concentrations were significantly elevated in burn-injured mice and these increases were ablated by administration of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a sEH inhibitor. TPPU also reduced necrosis of innate and adaptive immune cells in burned mice, in a dose-dependent manner. The findings suggest DiHOMEs are a key driver of immune cell dysfunction in severe burn injury through hyperinflammatory neutrophilic and impaired monocytic actions, and inhibition of sEH might be a promising therapeutic strategy to mitigate deleterious outcomes in burn patients.

Microvesicles from stored red blood cells induce P-selectin and von Willebrand factor release from endothelial cells via a protein kinase C-dependent mechanism.

INTRODUCTION: The use of packed red blood cells (pRBCs) for resuscitation is limited by the red blood cell storage lesion, a series of biochemical and physiological changes that occur during the storage and aging of blood. Microvesicles (MVs) shed from pRBCs during this process are one component of the red blood cell storage lesion and lead to acute lung injury and pulmonary vascular microthrombi. We hypothesized that MVs from stored pRBCs lead to the release of P-selectin and von Willebrand factor (vWF) from endothelial cells and that this mechanism is mediated via activation of protein kinase C (PKC) or protein kinase A (PKA). METHODS: Leukoreduced, platelet-poor murine pRBCs were isolated from C57BL/6 8-12 week-old male mice via cardiac puncture, prepared via centrifugation using a Ficoll gradient, and stored for up to 14 days, the equivalent of 42 days of storage in humans. MVs were isolated from the stored pRBC units via sequential high-speed centrifugation. Murine lung endothelial cells (MLECs) were cultured and grown to confluence, then treated with MVs and either calphostin C, a PKC inhibitor (10 µg/mL), or PKI 14-22 amide, a PKA inhibitor (10 µM). The supernatant was collected after 1 h. P-selectin and vWF A2 concentrations were quantified via ELISA. Immunofluorescent staining for vWF was performed on MLECs. Statistical analysis was performed via unpaired t-test or ANOVA as indicated and reported as mean ± SD. Concentration is reported as pg/mL. RESULTS: MLECs treated with MVs isolated from stored pRBCs demonstrated increased release of P-selectin and vWF A2 in a dose-dependent fashion. MLECs treated with MVs prepared from stored as compared to fresh pRBCs demonstrated increased release of P-selectin (3751 ± 726 vs 359 ± 64 pg/mL, p < 0.0001) and vWF A2 (3141 ± 355 vs 977 ± 75 pg/mL, p < 0.0001) with increasing duration of storage. The treatment of MVs with calphostin C decreased the amount of P-selectin (1471 ± 444 vs 3751 ± 726 pg/mL, p < 0.0001) and VWF A2 (2401 ± 289 vs 3141 ± 355 pg/mL, p = 0.0017) released into the supernatant by MLECs compared to MVs alone. The treatment of MVs with PKI 14-22 increased the amount of P-selectin released compared to MVs alone (1999 ± 67 vs 1601 ± 135 pg/mL, p = 0.0018). CONCLUSIONS: MVs from stored pRBCs stimulate the release of P-selectin and VWF A2 from endothelial cells. The effect of MVs increases with both dose of MVs and age of stored pRBCs from which they are formed. This mechanism is dependent on activation of PKC and inhibition of this enzyme represents a potentially significant strategy to modulate the inflammatory response to resuscitation with stored pRBCs.

A Prospective Analysis of the SVS WIfI Classification System to Stratify Immediate and 1-Year Patient Outcomes.

The purpose of this study was to prospectively enroll patients that presented to the emergency department with a lower extremity infection, stratify risk and record outcomes. Risk stratification was performed based on the Society of Vascular Surgery Wound, foot Infection, and Ischemia (WIfI) classification system. This study aimed to establish the efficacy and validity of this classification in predicting patient outcomes during immediate hospitalization and throughout a 1 year follow up. A total of 152 patients were enrolled in the study and of these, 116 met the inclusion criteria and had at least 1 year of follow up for analysis. Each patient was assigned a WIfI score based on wound, ischemia, and foot infection severity according to the classification guidelines. Patient demographics as well as all podiatric and vascular procedures were recorded. The major end points of the study were rates of proximal amputation, time to wound healing, surgical procedures, surgical dehiscence, readmission rates, and mortality. A difference in rates of healing (p = .04), surgical dehiscence (p < .01), and 1 year mortality (p = .01) with increasing WIfI stage as well as across the individual component scores was noted. This analysis further supports the application of the WIfI classification system early during patient care to stratify risk and identify the need for early intervention and a multispecialty team approach to potentially improve outcomes in the severe multicomorbid patient.

Functional Characterization of Neutrophils Allows Source Control Evaluation in a Murine Sepsis Model.

INTRODUCTION: Current surgical guidelines for the treatment of intra-abdominal sepsis recommend interventional source control as the key element of therapy, alongside resuscitation and antibiotic administration. Past trials attempted to predict the success of interventional source control to assess whether further interventional therapy is needed. However, no predictive score could be developed. MATERIALS AND METHODS: We utilized an established murine abdominal sepsis model, the cecal ligation and puncture (CLP), and performed a successful surgical source control intervention after full development of sepsis, the CLP-excision (CLP/E). We then sought to evaluate the success of the source control by characterizing circulating neutrophil phenotype and functionality 24 h postintervention. RESULTS: We showed a significant relative increase of neutrophils and a significant absolute and relative increase of activated neutrophils in septic mice. Source control with CLP/E restored these numbers back to baseline. Moreover, main neutrophil functions, the acidification of cell compartments, such as lysosomes, and the production of Tumor Necrosis Factor-alpha (TNF-α), were impaired in septic mice but restored after CLP/E intervention. CONCLUSIONS: Neutrophil characterization by phenotyping and evaluating their functionality indicates successful source control in septic mice and can serve as a prognostic tool. These findings provide a rationale for the phenotypic and functional characterization of neutrophils in human patients with infection. Further studies will be needed to determine whether a predictive score for the assessment of successful surgical source control can be established.

IFNγ and TNFα mediate CCL22/MDC production in alveolar macrophages after hemorrhage and resuscitation.

Acute lung injury is a major complication of hemorrhagic shock and the required resuscitation with large volumes of crystalloid fluids and blood products. We previously identified a role of macrophage-derived chemokine (CCL22/MDC) pulmonary inflammation following hemorrhage and resuscitation. However, further details regarding the induction of CCL22/MDC and its precise role in pulmonary inflammation after trauma remain unknown. In the current study we used in vitro experiments with a murine alveolar macrophage cell line, as well as an in vivo mouse model of hemorrhage and resuscitation, to identify key regulators in CCL22/MDC production. We show that trauma induces expression of IFNγ, which leads to production of CCL22/MDC through a signaling mechanism involving p38 MAPK, NF-κB, JAK, and STAT-1. IFNγ also activates TNFα production by alveolar macrophages, potentiating CCL22/MDC production via an autocrine mechanism. Neutralization of IFNγ or TNFα with specific antibodies reduced histological signs of pulmonary injury after hemorrhage and reduced inflammatory cell infiltration into the lungs.

Fibrotic liver has prompt recovery after ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (I/R) is a major complication of liver resection, trauma, and liver transplantation; however, liver repair after I/R in diseased liver has not been studied. The present study sought to determine the manner in which the fibrotic liver repairs itself after I/R. Liver fibrosis was established in mice by CCl4 administration for 6 wk, and then liver I/R was performed to investigate liver injury and subsequent liver repair in fibrotic and control livers. After I/R, fibrotic liver had more injury compared with nonfibrotic, control liver; however, fibrotic liver showed rapid resolution of liver necrosis and reconstruction of liver parenchyma. Marked accumulation of hepatic stellate cells and macrophages were observed specifically in the fibrotic septa in early reparative phase. Fibrotic liver had higher numbers of hepatic stellate cells, macrophages, and hepatic progenitor cells during liver recovery after I/R than did control liver, but hepatocyte proliferation was unchanged. Fibrotic liver also had significantly greater number of phagocytic macrophages than control liver. Clodronate liposome injection into fibrotic mice after I/R caused decreased macrophage accumulation and delay of liver recovery. Conversely, CSF1-Fc injection into normal mice after I/R resulted in increased macrophage accumulation and concomitant decrease in necrotic tissue during liver recovery. In conclusion, fibrotic liver clears necrotic areas and restores normal parenchyma faster than normal liver after I/R. This beneficial response appears to be directly related to the increased numbers of nonparenchymal cells, particularly phagocytic macrophages, in the fibrotic liver.NEW & NOTEWORTHY This study is the first to reveal how diseased liver recovers after ischemia-reperfusion (I/R) injury. Although it was not completely unexpected that fibrotic liver had increased hepatic injury after I/R, a novel finding was that fibrotic liver had accelerated recovery and repair compared with normal liver. Enhanced repair after I/R in fibrotic liver was associated with increased expansion of phagocytic macrophages, hepatic stellate cells, and progenitor cells.

Therapeutic Inhaled Sphingosine for Treating Lung Infection in a Mouse Model of Critical Illness.

BACKGROUND/AIMS: Sphingosine, a sphingoid long chain base, is a natural lipid with antimicrobial properties. Recent animal studies have shown that preventive sphingosine inhalation can rescue susceptible mice, such as cystic fibrosis-, burn injured- or aged mice from bacterial pulmonary infection. While preventing lung infections in susceptible patients has obvious clinical merit, treatment strategies for an established infection are also direly needed, particularly in the times of rising antibiotic resistance. Here, we tested the potential of sphingosine in treating an established pulmonary infection. METHODS: We used a cecal ligation and puncture (CLP) model in male CF-1 mice and a Pseudomonas aeruginosa strain that was isolated from a septic patient (P. aeruginosa 762). We determined susceptibility to intranasal infection and ascertained when the pulmonary infection was established by continuous core body temperature monitoring. We quantified sphingosine levels in the tracheal epithelium by immunohistochemistry and studied the effects on sphingosine on bacterial membrane permeabilization and intracellular acidification using fluorescent probes. RESULTS: We firstdetermined that septic mice are highly susceptible to P. aeruginosa infection 2 days after indu-cing sepsis. Additionally, at this time, sphingosine levels in the tracheal epithelium are significantly reduced as compared to levels in healthy mice. Secondly, upon intranasal Pseudomonasinoculation, we ascertained that pulmonary infection was established as early as 2.5 h after inoculation as evidenced by a significant drop in core body temperature. Using these times of infection susceptibility and detection (2 days post CLP, 2.5h after inoculation) we treated with inhaled sphingosine and observed pulmonary bacterial loads reduced to levels found in infected healthy mice after inoculation and decreased infection-associated mortality. Further, our data demonstrate that sphingosine induces outer membrane permeabilization, disrupting the membrane potential and leading to intracellular acidification of the bacteria. CONCLUSION: Sphingosine shows efficacy in treating P. aeruginosa lung infections not only prophylactically, but also therapeutically.

Intraperitoneal Neutrophil IL-10 production is promoted by interferon γ in a murine model of sepsis model in the acute phase of sepsis.

The disease burden of sepsis continues to increase, with intraabdominal contamination being a significant source of infection. Sepsis is a syndrome involving both an increase in systemic inflammation as well as a regulatory component. We have previously demonstrated that neutrophils are significant IL-10 producers in the abdomen during sepsis. Here, we sought to further characterize these neutrophils and elucidate potential underlying mechanisms resulting in IL-10 generation. Using transcriptional reporter mice, we observed that IL-10 producing neutrophils were activated, non-apoptotic, and expressed C-X-C chemokine receptor type 4-expressing. Further, we observed that active Signal Transducer and Activator of Transcription 1 expression was significantly increased in IL-10 producing versus non-IL-10 producing neutrophils. During sepsis, IFN-γ blockade lead to a decrease of neutrophil IL-10 production, while peritoneal CD4 T cells were found to be the most numerous acute producers of IFN-γ. Altogether, this report demonstrates that during sepsis, mature neutrophils can potentially dampen local inflammation by IL-10 production and this can be orchestrated by CD4 T cells through an IFN-γ dependent manner.

Consumptive coagulopathy is associated with organ dysfunction during PICS.

Patients who survive the acute phase of sepsis can progress to persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Although sepsis is characterized by early hypercoagulability and delayed hypocoagulability, coagulopathy during chronic critical illness is not fully understood. The objective of this study was to determine whether sepsis-induced PICS is associated with coagulation abnormalities. Using our previously described murine PICS model, outbred mice underwent cecal ligation and puncture, and coagulability was characterized after 8 days. We found that during PICS the spleen became markedly enlarged with increased splenocytes and splenic megakaryocytes without a concomitant increase in circulating platelets. Microscopy revealed a nearly sevenfold increase in pulmonary microvascular thrombi in PICS mice, along with significantly decreased pulmonary tidal volumes and inspiratory times and with significantly increased respiratory rates. Thromboelastometry showed that PICS mice had significantly delayed clot initiation time but increased clot firmness. Finally, PICS mice displayed delayed thrombin production and decreased overall thrombin concentrations. All together, these data demonstrate a general dysregulation of coagulation resulting in microthrombus formation and compromised lung function. On the basis of these findings, we propose that consumptive coagulopathy constitutes another cardinal feature of PICS and may contribute to the ongoing tissue damage and multiple organ failure that can occur in chronic critical illness.

Cell-specific regulatory effects of CXCR2 on cholestatic liver injury.

The CXC chemokine receptor 2 (CXCR2) is critical for neutrophil recruitment and hepatocellular viability but has not been studied in the context of cholestatic liver injury following bile duct ligation (BDL). The present study sought to elucidate the cell-specific roles of CXCR2 on acute liver injury after BDL. Wild-type and CXCR2-/- mice were subjected BDL. CXCR2 chimeric mice were created to assess the cell-specific role of CXCR2 on liver injury after BDL. SB225002, a selective CXCR2 antagonist, was administrated intraperitoneally after BDL to investigate the potential of pharmacological inhibition. CXCR2-/- mice had significantly less liver injury than wild-type mice at 3 and 14 days after BDL. There was no difference in biliary fibrosis among groups. The chemokines CXCL1 and CXCL2 were induced around areas of necrosis and biliary structures, respectively, both areas where neutrophils accumulated after BDL. CXCR2-/- mice showed significantly less neutrophil accumulation in those injured areas. CXCR2Liver+/Myeloid+ and CXCR2Liver-/Myeloid- mice recapitulated the wild-type and CXCR2-knockout phenotypes, respectively. CXCR2Liver+/Myeloid+ mice suffered higher liver injury than CXCR2Liver+/Myeloid- and CXCR2Liver-/Myeloid+; however, only those chimeras with knockout of myeloid CXCR2 (CXCR2Liver+/Myeloid- and CXCR2Liver-/Myeloid-) showed reduction of neutrophil accumulation around areas of necrosis. Daily administration of SB225002 starting after 3 days of BDL reduced established liver injury at 6 days. In conclusion, neutrophil CXCR2 guides the cell to the site of injury, while CXCR2 on liver cells affects liver damage independent of neutrophil accumulation. CXCR2 appears to be a viable therapeutic target for cholestatic liver injury.NEW & NOTEWORTHY This study is the first to reveal cell-specific roles of the chemokine receptor CXCR2 in cholestatic liver injury caused by bile duct ligation. CXCR2 on neutrophils facilitates neutrophil recruitment to the liver, while CXCR2 on liver cells contributes to liver damage independent of neutrophils. CXCR2 may represent a viable therapeutic target for cholestatic liver injury.

Amitriptyline Reduces Inflammation and Mortality in a Murine Model of Sepsis.

BACKGROUND/AIMS: During sepsis, an unchecked pro-inflammatory response can be detrimental to the host. We investigated the potential protective effect of amitriptyline (AT). METHODS: We used two murine models of sepsis: Cecal ligation and puncture and endotoxemia following LPS challenge. Aural temperatures were taken and cytokines quantified by cytometric bead assay. Lung injury was determined histologically and by protein determination in bronchoalveolar lavage fluid. Cell accumulation in the peritoneum was analyzed by flow cytometry, as well as cytokine production and p38-phosphorylation. Neutrophil chemotaxis was evaluated using an in vitro transwell assay. RESULTS: Our findings demonstrate that AT-treated septic mice have improved survival and are protected from pulmonary edema. Treatment with AT significantly decreased serum levels of KC and monocyte chemoattractant protein-1, as well as the accumulation of neutrophils and monocytes in the peritoneum of septic mice. Peritoneal IL-10 levels in septic mice were increased upon AT treatment. Direct treatment of septic mice with IL-10 recapitulated the effects of AT. Endotoxemic mice also exhibited enhanced IL-10 production upon AT-administration and peritoneal macrophages were identified as the ATinfluenced producers of IL-10. Treatment of these cells with AT in vitro resulted in increased p38-phosphorylation and IL-10 generation, whereas ceramide and p38 inhibition had the opposite effect. CONCLUSION: Altogether, AT treatment improved survival, increased IL-10 levels, and mitigated a pro-inflammatory response during sepsis. We conclude that AT is a promising therapeutic to temper inflammation during septic shock.

Antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide.

Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.

Staphylococcus aureus Alpha-Toxin Disrupts Endothelial-Cell Tight Junctions via Acid Sphingomyelinase and Ceramide.

Staphylococcus aureus (S. aureus) infections are among the most common and severe infections, garnering notoriety in an era of increasing resistance to antibiotics. It is therefore important to define molecular mechanisms by which this pathogen attacks host cells. Here, we demonstrate that alpha-toxin, one of the major toxins of S. aureus, induces activation of acid sphingomyelinase and concomitant release of ceramide in endothelial cells treated with the toxin. Activation of acid sphingomyelinase by alpha-toxin is mediated via ADAM10. Infection experiments employing alpha-toxin-deficient S. aureus and the corresponding wild-type strain reveal that activation of acid sphingomyelinase in endothelial cells requires alpha-toxin expression by the pathogen. Activation of acid sphingomyelinase is linked to degradation of tight junctions in endothelial cells in vitro, which is blocked by pharmacological inhibition of acid sphingomyelinase. Most importantly, alpha-toxin induces severe degradation of tight junctions in the lung and causes lung edema in vivo, which is prevented by genetic deficiency of acid sphingomyelinase. These data indicate a novel and important role of the acid sphingomyelinase/ceramide system for the endothelial response to toxins and provide a molecular link between alpha-toxin and the degradation of tight junctions. The data also suggest that inhibition of acid sphingomyelinase may provide a novel treatment option to prevent lung edema caused by S. aureus alpha-toxin.

Pulmonary infection of cystic fibrosis mice with Staphylococcus aureus requires expression of α-toxin.

Pulmonary infections of cystic fibrosis (CF) patients with Staphylococcus aureus (S. aureus) occur very early in the disease. The molecular details that cause infection-susceptibility of CF patients to and mediate infection with S. aureus are poorly characterized. Therefore, we aimed to identify the role of α-toxin, a major S. aureus toxin, for pulmonary infection of CF mice. Infection with S. aureus JE2 resulted in severe pneumonia in CF mice, while wildtype mice were almost unaffected. Deficiency of α-toxin in JE2-Δhla reduced the pathogenicity of S. aureus in CF mice. However, CF mice were still more susceptible to the mutant S. aureus strain than wildtype mice. The S. aureus JE2 induced a marked increase of ceramide and a downregulation of sphingosine and acid ceramidase expression in bronchi of CF mice. Deletion of α-toxin reduced these changes after infection of CF mice. Similar changes were observed in wildtype mice, but at much lower levels. Our data indicate that expression of α-toxin is a major factor causing S. aureus infections in CF mice. Wildtype S. aureus induces a marked increase of ceramide and a reduction of sphingosine and acid ceramidase expression in bronchial epithelial cells of wildtype and CF mice, changes that determine infection susceptibility.

Acid Sphingomyelinase Inhibition in Stored Erythrocytes Reduces Transfusion-Associated Lung Inflammation.

OBJECTIVE: We aimed to identify the role of the enzyme acid sphingomyelinase in the aging of stored units of packed red blood cells (pRBCs) and subsequent lung inflammation after transfusion. SUMMARY BACKGROUND DATA: Large volume pRBC transfusions are associated with multiple adverse clinical sequelae, including lung inflammation. Microparticles are formed in stored pRBCs over time and have been shown to contribute to lung inflammation after transfusion. METHODS: Human and murine pRBCs were stored with or without amitriptyline, a functional inhibitor of acid sphingomyelinase, or obtained from acid sphingomyelinase-deficient mice, and lung inflammation was studied in mice receiving transfusions of pRBCs and microparticles isolated from these units. RESULTS: Acid sphingomyelinase activity in pRBCs was associated with the formation of ceramide and the release of microparticles. Treatment of pRBCs with amitriptyline inhibited acid sphingomyelinase activity, ceramide accumulation, and microparticle production during pRBC storage. Transfusion of aged pRBCs or microparticles isolated from aged blood into mice caused lung inflammation. This was attenuated after transfusion of pRBCs treated with amitriptyline or from acid sphingomyelinase-deficient mice. CONCLUSIONS: Acid sphingomyelinase inhibition in stored pRBCs offers a novel mechanism for improving the quality of stored blood.

Staphylococcus aureus α-Toxin Induces Inflammatory Cytokines via Lysosomal Acid Sphingomyelinase and Ceramides.

BACKGROUND/AIMS: Staphylococcus aureus (S. aureus) infections are a major clinical problem and range from mild skin and soft-tissue infections to severe and even lethal infections such as pneumonia, endocarditis, sepsis, osteomyelitis, and toxic shock syndrome. Toxins that are released from S. aureus mediate many of these effects. Here, we aimed to identify molecular mechanisms how α-toxin, a major S. aureus toxin, induces inflammation. METHODS: Macrophages were isolated from the bone marrow of wildtype and acid sphingomyelinase-deficient mice, stimulated with S. aureus α-toxin and activation of the acid sphingomyelinase was quantified. The subcellular formation of ceramides was determined by confocal microscopy. Release of cathepsins from lysosomes, activation of inflammasome proteins and formation of Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor-α (TNF-α) were analyzed by western blotting, confocal microscopy and ELISA. RESULTS: We demonstrate that S. aureus α-toxin activates the acid sphingomyelinase in ex vivo macrophages and triggers a release of ceramides. Ceramides induced by S. aureus α-toxin localize to lysosomes and mediate a release of cathepsin B and D from lysosomes into the cytoplasm. Cytosolic cathepsin B forms a complex with Nlrc4. Treatment of macrophages with α-toxin induces the formation of IL-1ß and TNF-α. These events are reduced or abrogated, respectively, in cells lacking the acid sphingomyelinase and upon treatment of macrophages with amitriptyline, a functional inhibitor of acid sphingomyelinase. Pharmacological inhibition of cathepsin B prevented activation of the inflammasome measured as release of IL-1ß, while the formation of TNF-α was independent of cathepsin B. CONCLUSION: We demonstrate a novel mechanism how bacterial toxins activate the inflammasome and mediate the formation and release of cytokines: S. aureus α-toxin triggers an activation of the acid sphingomyelinase and a release of ceramides resulting in the release of lysosomal cathepsin B and formation of pro-inflammatory cytokines.

Neutrophils Kill Reactive Oxygen Species-Resistant Pseudomonas aeruginosa by Sphingosine.

BACKGROUND/AIMS: Cystic fibrosis (CF) is dominated by chronic inflammation and infection of the lung resulting in lung destruction and early death of patients. The lungs of CF patients are characterized by a massive accumulation of neutrophils. It requires definition why these massive numbers of neutrophils fail to eliminate typical CF pathogens like Staphylococcus aureus and Pseudomonas aeruginosa (P. aeruginosa) in CF lungs. METHODS: We determined ceramide, sphingosine and reactive oxygen species (ROS) in neutrophils from wildtype and CF mice and determined the effect of sphingosine and ROS alone or in combination on killing of different P. aeruginosa strains. RESULTS: We demonstrate that wildtype neutrophils are able to kill non-mucoid and mucoid clinical P. aeruginosa strains, while neutrophils from CF mice are insufficient to kill these P. aeruginosa strains, although both types of neutrophils infected with P. aeruginosa produce comparable levels of superoxide. All three analyzed P. aeruginosa strains are resistant to reactive oxygen species. The inability of CF neutrophils to kill P. aeruginosa is caused by a marked decrease of surface sphingosine levels in CF neutrophils. Wildtype neutrophils contain much higher concentrations of surface sphingosine than CF neutrophils. Further, wildtype neutrophils, but not CF neutrophils, release sphingosine, most likely as microparticles, upon infection. Sphingosine kills P. aeruginosa in vitro at low micromolar concentrations. Reconstitution of sphingosine in CF neutrophils restores their ability to kill these pathogens, demonstrating the significance of sphingosine for bacterial killing. CONCLUSION: The data provide evidence for a new paradigm explaining how neutrophils kill ROS-resistant P. aeruginosa, i.e. by sphingosine that kills P. aeruginosa at low concentrations. This mechanism is defective in CF neutrophils.

Neutrophil derived microparticles increase mortality and the counter-inflammatory response in a murine model of sepsis.

Although advances in medical care have significantly improved sepsis survival, sepsis remains the leading cause of death in the ICU. This is likely due to a lack of complete understanding of the pathophysiologic mechanisms that lead to dysfunctional immunity. Neutrophil derived microparticles (NDMPs) have been shown to be the predominant microparticle present at infectious and inflamed foci in human models, however their effect on the immune response to inflammation and infection is sepsis has not been fully elucidated. As NDMPs may be a potential diagnostic and therapeutic target, we sought to determine the impact NDMPs on the immune response to a murine polymicrobial sepsis. We found that peritoneal neutrophil numbers, bacterial loads, and NDMPs were increased in our abdominal sepsis model. When NDMPs were injected into septic mice, we observed increased bacterial load, decreased neutrophil recruitment, increased expression of IL-10 and worsened mortality. Furthermore, the NDMPs express phosphatidylserine and are ingested by F4/80 macrophages via a Tim-4 and MFG-E8 dependent mechanism. Finally, upon treatment, NDMPs decrease macrophage activation, increase IL-10 release and decrease macrophage numbers. Altogether, these data suggest that NDMPs enhance immune dysfunction in sepsis by blunting the function of neutrophils and macrophages, two key cell populations involved in the early immune response to infection. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Burn injury influences the T cell homeostasis in a butyrate-acid sphingomyelinase dependent manner.

Following burn injury, a key factor for patients susceptible to opportunistic infections is immune suppression. Butyrate levels are important in maintaining a functional immune system and these levels can be altered after injury. The acid sphingomyelinase (Asm) lipid signaling system has been implicated in a T cell actions with some evidence of being influenced by butyrate. Here, we hypothesized that burn-injury changes in butyrate levels would mediate Asm activity and, consequently, T cell homeostasis. We demonstrate that burn injury temporally decreases butyrate levels. We further determined that T cell Asm activity is increased by butyrate and decreased after burn injury. We additionally observed decreased T cell numbers in Asm-deficient, burn-injured, and microbiota-depleted mice. Finally, we demonstrate that butyrate reduced T cell death in an Asm-dependent manner. These data suggest that restoration of butyrate after burn injury may ameliorate the T cell lost observed in burn-injured patients by Asm regulation.

Nanoparticles prepared from porcine cells support the healing of cutaneous inflammation in mice and wound re-epithelialization in human skin.

Previous reports have demonstrated that cell-derived nanoparticles (CDNPs) composed of bovine or porcine protein complexes exerted therapeutic effects against viral infections and cancer in mice and humans. Based on these observations, we asked whether CDNPs would improve inflammatory skin disorders. To address this, we utilized two distinct mouse models of cutaneous inflammation: the autoimmune skin-blistering disease epidermolysis bullosa acquisita (EBA) as an example of an autoantibody-induced cutaneous inflammation, and Leishmania major (L. major) infection as an example of a pathogen-induced cutaneous inflammation. In both models, we observed that CDNPs increased mRNA expression of the Th2 cytokine IL-4. Clinically, CDNPs decreased inflammation due to EBA and increased L. major-specific IgG1 levels without major effects on infected skin lesions. In addition, CDNPs supported the growth of keratinocytes in human skin cultures. In vitro studies revealed that CDNPs were taken up predominantly by macrophages, leading to a shift towards the expression of anti-inflammatory cytokine genes. Altogether, our data demonstrate that treatment with porcine CDNPs may be a new therapeutic option for the control of autoimmune-mediated inflammatory skin disorders.