Platelet-dependent pulmonary recruitment of neutrophils in abdominal sepsis

Sepsis and subsequent multiple organ failure remain the major cause of mortality in intensive care units. Leukocyte-mediated tissue damage is a key feature in septic lung injury. Accumulating data suggest that platelets play a role in inflammation and tissue injury. However, the role of platelets in sepsis-induced leukocyte recruitment and lung edema formation in abdominal sepsis is not demonstrated yet. We hypothesized that platelets may play a significant role in pulmonary neutrophil recruitment and tissue damage in abdominal sepsis. For this purpose, we used the mice cecal ligation and puncture (CLP) model of abdominal sepsis. CLP causes significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines and increased edema formation in the lung. CLP also provoked Mac-1 expression on circulating neutrophils. Interestingly, depletion of platelets reduced CLP-induced lung damage, neutrophil recruitment in the bronchoalveolar space and edema formation as well as up-regulation of Mac-1 on neutrophils. However, blocking of platelet-neutrophil aggregates formation did not attenuate CLP-induced lung damage and neutrophil activation suggesting that platelets regulate sepsis-induced lung damage via up-regulation of Mac-1 in a contact independent manner. We also found that plasma levels of soluble CD40L was significantly increased in septic mice. Use of CD40L-deficient mice confirmed that platelet-derived CD40L is a pivotal mediator of neutrophil activation and recruitment in abdominal sepsis and this platelet mediated neutrophil activation was indirect and mediated via formation of MIP-2 and CXCR2 signaling. In addition, we observed a significant increase of soluble CD40L levels in septic patients. Interestingly, we found that inhibition of matrix MMPs reduced Mac-1 up-regulation on neutrophils and CXC chemokine formation in the septic lung injury. We also found that MMP-9 levels are significantly increased in septic mice but not MMP-2. In vitro studies revealed that activated platelets up-regulate surface expression of MMP-9 and that inhibition of MMP-9 decreased platelet shedding of CD40L. Use of MMP-9-deficient mice suggested that MMP-9 regulates platelet CD40L shedding in abdominal sepsis. Moreover, pulmonary infiltration of neutrophils as well as edema formation and lung injury were markedly decreased in septic animals lacking MMP-9. Plasma levels of MMP-9 were significantly increased in patients with septic shock compared to healthy controls. Taken together, platelets regulate neutrophil activation in abdominal sepsis via MMP-9-dependent shedding of platelet-derived CD40L. Thus, MMP-9 and CD40L may constitute novel and effective therapeutic targets in abdominal sepsis

Streptococcal M1 protein triggers farnesyltransferase-dependent formation of CXC chemokines in alveolar macrophages and neutrophil infiltration in the lung.

M1 serotype of Streptococcus pyogenes plays an important role in streptococcal toxic shock syndrome. Simvastatin, a HMG-CoA reductase inhibitor, has been shown to inhibit streptococcal M1 protein-induced acute lung damage although downstream mechanisms remain elusive. Protein isoprenylation, such as farnesylation and geranylgeranylation, has been suggested to regulate anti-inflammatory effects exerted by statins. Herein, we examined the effect of a farnesyltransferase inhibitor (FTI-277) on M1 protein-triggered lung inflammation. Male C57BL/6 mice were treated with FTI-277 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Flow cytometry was used to determine Mac-1 expression on neutrophils. Gene expression of CXC chemokines was determined in alveolar macrophages by using quantitative RT-PCR. We found that administration of FTI-277 markedly decreased M1 protein-induced accumulation of neutrophils, edema formation and tissue damage in the lung. Notably, inhibition of farnesyltransferase abolished M1 protein-evoked production of CXC chemokines in the lung and gene expression of CXC chemokines in alveolar macrophages. Moreover, FTI-277 completely inhibited chemokine-induced neutrophil migration in vitro. However, farnesyltransferase inhibition had no effect on M1 protein-induced expression of Mac-1 on neutrophils. Our findings suggest that farnesyltransferase is a potent regulator of CXC chemokine formation in alveolar macrophages and that inhibition of farnesyltransferase not only reduces neutrophil recruitment but also attenuates acute lung injury provoked by streptococcal M1 protein. We conclude that farnesyltransferase activity is a potential target in order to attenuate acute lung damage in streptococcal infections

The role of extracellular vesicle fusion with target cells in triggering systemic inflammation

Extracellular vesicles (EVs) play a crucial role in intercellular communication by transferring bioactive molecules from donor to recipient cells. As a result, EV fusion leads to the modulation of cellular functions and has an impact on both physiological and pathological processes in the recipient cell. This study explores the impact of EV fusion on cellular responses to inflammatory signaling. Our findings reveal that fusion renders non-responsive cells susceptible to inflammatory signaling, as evidenced by increased NF-κB activation and the release of inflammatory mediators. Syntaxin-binding protein 1 is essential for the merge and activation of intracellular signaling. Subsequent analysis show that EVs transfer their functionally active receptors to target cells, making them prone to an otherwise unresponsive state. EVs in complex with their agonist, require no further stimulation of the target cells to trigger mobilization of NF-κB. While receptor antagonists were unable to inhibit NF-κB activation, blocking of the fusion between EVs and their target cells with heparin mitigated inflammation in mice challenged with EVs.</p

The role of extracellular vesicle fusion with target cells in triggering systemic inflammation

Extracellular vesicles (EVs) play a crucial role in intercellular communication by transferring bioactive molecules from donor to recipient cells. As a result, EV fusion leads to the modulation of cellular functions and has an impact on both physiological and pathological processes in the recipient cell. This study explores the impact of EV fusion on cellular responses to inflammatory signaling. Our findings reveal that fusion renders non-responsive cells susceptible to inflammatory signaling, as evidenced by increased NF-κB activation and the release of inflammatory mediators. Syntaxin-binding protein 1 is essential for the merge and activation of intracellular signaling. Subsequent analysis show that EVs transfer their functionally active receptors to target cells, making them prone to an otherwise unresponsive state. EVs in complex with their agonist, require no further stimulation of the target cells to trigger mobilization of NF-κB. While receptor antagonists were unable to inhibit NF-κB activation, blocking of the fusion between EVs and their target cells with heparin mitigated inflammation in mice challenged with EVs.</p

A molecular map of murine lymph node blood vascular endothelium at single cell resolution

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis

Platelet-dependent pulmonary recruitment of neutrophils in abdominal sepsis [Elektronisk resurs]

Popular Abstract in Swedish Sepsis, blodförgiftning, är ett potentiellt allvarligt tillstånd där bakterier eller deras toxiner aktiverar immunsystemet I blodbanan. Svår sepsis är associerad med organdysfunktion och hög mortalitet (30-60%). Cirka 200 per 100 000 invånare i Sverige drabbas årligen av svår sepsis. Akut lungskada är en central komponent hos patienter med sepsis och experimentella studier har visat att aktivering och ackumulering av vita blodkroppar är ett hastighetsberoende steg i sepsis-associerad lungskada. Trombocyter är kända för sin viktiga roll vid blödning och sårläkning men nyare data indikerar också att trombocyter är också viktiga vid inflammatoriska reaktioner. Den här avhandlingen fokuserar på den potentiella betydelsen av trombocyter vid sepsis. I det första arbetet observerades att om man tog bort trombocyterna från möss minskade aktiveringen och rekrytering av vita blodkroppar, neutropfila granulocyter, till lunga med minskad vävnadsskada som följd. Inhibering av PSGL-1 fullständigt blockerade aggregat bildningen mellan trombocyter och neutrofiler vid sepsis. Det vill säga att den här trombocyt-beroende aktivering av neutrofiler visade sig vara oberoende av fysisk kontakt mellan trombocyterna och neutrofilerna. Istället kunde det konstateras att någon eller några faktorer som utsöndras i löslig form från trombocyter i sin tur aktiverade cirkulerande neutrofiler vid sepsis. I arbete nummer två observerade vi att löslig form av CD40L ökade kraftigt i blodet vid sepsis och att den här ökningen försvann helt om man tog bort trombocyterna före induktion av sepsis. I det här arbetet identifierades CD40L vara den molekyl som utsöndras från trombocyter och som aktiverar neutrofiler i blodbanan. Blockering av CD40L minskade inte bara aktivering av neutrofiler utan reducerade också sepsis inducerad lungskada. CD40L-medierad aktivering av neutrofiler visade sig vara indirekt via bildningen av MIP-2 som är en potent stimulator av neutrofiler. I det tredje arbetet visade det sig att lösligt CD40L också ökade i blodet på patienter med sepsis jämfört med friska kontroller. Löslig CD40L ökade inte bara vid septisk chock utan också vid chock orsakad av andra faktorer än bakterier. De här resultaten indikerar att fynden i de två första djurexperimentella arbetena kan vara relevanta också hos patienter med sepsis. I arbete fyra undersöktes mekanismer som kan förklara hur CD40L frisätts från trombocyter vid sepsis. Blockering av en grupp av enzym, metalloproteinaser (MMP), visade sig hindra frisättning av CD40L från trombocyter och därmed aktiveringen av neutrofiler samt minskade lungskadan vid sepsis. Efter att ha konstaterat att något MMP kan vara involverat, mättes bildningen av relevanta kandidater, MMP-2 och MMP-9, i blodet. Det visade sig att MMP-9 men inte MMP-2 ökade i blodet vid sepsis. Med hjälp av möss som saknar MMP-9 kunde det fastställas att MMP-9 reglerade CD40L frisättningen från trombocyter vid sepsis. I direkta försök på isolerade trombocyter kunde det konstateras att MMP-9 ökar på ytan av aktiverade trombocyter och spelar en direkt avgörande roll för frisättning av CD40L. Det visade sig också att patienter hade förhöjda nivåer av MMP-9 i blodet jämfört med friska kontroller vilket skulle kunna betyda att MMP-9 också spelar en funktionell roll vid sepsis. Sammanfattningsvis kan det konstateras att trombocyter spelar en viktig roll vid sepsis genom att aktivera cirkulerande neutrofiler via frisättning av CD40L. Dessutom visar den här avhandlingen att MMP-9 upregleras på aktiverade trombocyter och frisätter CD40L ligand. Mot bakgrund av att dessa molekyler också ökar vid hos patienter med sepsis skulle CD40L och MMP-9 kunna utgöra nya och mer specifika måltavlor för behandling av patienter med svår sepsis.Sepsis and subsequent multiple organ failure remain the major cause of mortality in intensive care units. Leukocyte-mediated tissue damage is a key feature in septic lung injury. Accumulating data suggest that platelets play a role in inflammation and tissue injury. However, the role of platelets in sepsis-induced leukocyte recruitment and lung edema formation in abdominal sepsis is not demonstrated yet. We hypothesized that platelets may play a significant role in pulmonary neutrophil recruitment and tissue damage in abdominal sepsis. For this purpose, we used the mice cecal ligation and puncture (CLP) model of abdominal sepsis. CLP causes significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines and increased edema formation in the lung. CLP also provoked Mac-1 expression on circulating neutrophils. Interestingly, depletion of platelets reduced CLP-induced lung damage, neutrophil recruitment in the bronchoalveolar space and edema formation as well as up-regulation of Mac-1 on neutrophils. However, blocking of platelet-neutrophil aggregates formation did not attenuate CLP-induced lung damage and neutrophil activation suggesting that platelets regulate sepsis-induced lung damage via up-regulation of Mac-1 in a contact independent manner. We also found that plasma levels of soluble CD40L was significantly increased in septic mice. Use of CD40L-deficient mice confirmed that platelet-derived CD40L is a pivotal mediator of neutrophil activation and recruitment in abdominal sepsis and this platelet mediated neutrophil activation was indirect and mediated via formation of MIP-2 and CXCR2 signaling. In addition, we observed a significant increase of soluble CD40L levels in septic patients. Interestingly, we found that inhibition of matrix MMPs reduced Mac-1 up-regulation on neutrophils and CXC chemokine formation in the septic lung injury. We also found that MMP-9 levels are significantly increased in septic mice but not MMP-2. In vitro studies revealed that activated platelets up-regulate surface expression of MMP-9 and that inhibition of MMP-9 decreased platelet shedding of CD40L. Use of MMP-9-deficient mice suggested that MMP-9 regulates platelet CD40L shedding in abdominal sepsis. Moreover, pulmonary infiltration of neutrophils as well as edema formation and lung injury were markedly decreased in septic animals lacking MMP-9. Plasma levels of MMP-9 were significantly increased in patients with septic shock compared to healthy controls. Taken together, platelets regulate neutrophil activation in abdominal sepsis via MMP-9-dependent shedding of platelet-derived CD40L. Thus, MMP-9 and CD40L may constitute novel and effective therapeutic targets in abdominal sepsis

Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

Matrix metalloproteinase-9 (MMP-9) regulates platelet shedding of CD40L in abdominal sepsis. However, the signaling mechanisms controlling sepsis-induced shedding of CD40L from activated platelets remain elusive. Rac1 has been reported to regulate diverse functions in platelets; we hypothesized herein that Rac1 might regulate platelet shedding of CD40L in sepsis. The specific Rac1 inhibitor NSC23766 (N6-[2-[[4-(diethylamino)-1-methylbutyl] amino]-6-methyl-4-pyrimidinyl]-2 methyl-4, 6-quinolinediamine trihydrochloride) was administered to mice undergoing cecal ligation and puncture (CLP). Levels of CD40L and MMP-9 in plasma, platelets, and neutrophils were determined by use of ELISA, western blot, and confocal microscopy. Platelet depletion abolished the CLP-induced increase in plasma levels of CD40L. Rac1 activity was significantly increased in platelets from septic animals. Administration of NSC23766 abolished the CLP-induced enhancement of soluble CD40L levels in the plasma. Moreover, Rac1 inhibition completely inhibited proteinase-activated receptor-4-induced surface mobilization and secretion of CD40L in isolated platelets. CLP significantly increased plasma levels of MMP-9 and Rac1 activity in neutrophils. Treatment with NSC23766 markedly attenuated MMP-9 levels in the plasma from septic mice. In addition, Rac1 inhibition abolished chemokine-induced secretion of MMP-9 from isolated neutrophils. Finally, platelet shedding of CD40L was significantly reduced in response to stimulation with supernatants from activated MMP-9-deficient neutrophils compared with supernatants from wild-type neutrophils, indicating a direct role of neutrophil-derived MMP-9 in regulating platelet shedding of CD40L. Our novel data suggest that sepsis-induced platelet shedding of CD40L is dependent on Rac1 signaling. Rac1 controls surface mobilization of CD40L on activated platelets and MMP-9 secretion from neutrophils. Thus, our findings indicate that targeting Rac1 signaling might be a useful way to control pathologic elevations of CD40L in the systemic circulation in abdominal sepsis.Laboratory Investigation advance online publication, 21 July 2014; doi:10.1038/labinvest.2014.92