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

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

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

Targeting CD44 Expressed on Neutrophils Inhibits Lung Damage in Abdominal Sepsis.

Neutrophil infiltration is an insidious feature in septic lung injury, although the specific adhesive mechanisms regulating pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. The aim of this present study was to define the role of CD44 in sepsis-induced neutrophil infiltration and lung damage. Mice were treated with a monoclonal antibody against CD44 before cecal ligation and puncture (CLP) induction. Edema formation, bronchoalveolar accumulation of neutrophils, myeloperoxidase activity, and macrophage inflammatory protein-2 (MIP-2) levels in the lung were determined after CLP. Expression of Mac-1 and CD44 on neutrophils was quantified by using flow cytometry. In separate experiments, fluorescent-labeled neutrophils co-incubated with an anti-CD44 antibody were adoptively transferred to CLP mice. CLP triggered clear-cut lung damage characterized by edema formation, neutrophil infiltration, and increased levels of MIP-2 in the lung. Notably, immunoneutralization of CD44 reduced CLP-induced pulmonary accumulation of neutrophils. In addition, functional inhibition of CD44 decreased CLP-induced lung damage and edema. However, formation of MIP-2 in the lung and neutrophil expression of Mac-1 were intact in septic mice pretreated with the anti-CD44 antibody. Adoptive transfer experiments revealed that neutrophil rather than lung CD44 mediates neutrophil accumulation in septic lung injury. Moreover, administration of hyaluronidase had no effect on CLP-induced neutrophil recruitment and tissue damage in the lung. Our data demonstrate that CD44 contributes to pulmonary infiltration of neutrophils and lung damage associated with abdominal sepsis. Thus, these novel findings suggest that CD44 may serve as a target to protect against lung injury in polymicrobial sepsis

c-Abl kinase regulates neutrophil extracellular trap formation, inflammation, and tissue damage in severe acute pancreatitis

Neutrophil extracellular traps (NETs) are involved in acute pancreatitis (AP) but mechanisms controlling NET expulsion in AP are incompletely understood. Herein, we examined the role of c-Abelson (c-Abl) kinase in NET formation and tissue damage in severe AP. AP was induced by taurocholate infusion into pancreatic duct or intraperitoneal administration of l-arginine in mice. Pancreatic, lung, and blood samples were collected and levels of phosphorylated c-Abl kinase, citrullinated histone 3, DNA-histone complexes, myeloperoxidase, amylase, cytokines, and CXC chemokines were quantified. Citrullinated histone 3, reactive oxygen species (ROS), and NET formation were determined in bone marrow neutrophils. Taurocholate challenge increased phosphorylation of c-Abl kinase and levels of citrullinated histone 3 in the pancreas as well as DNA-histone complexes in the plasma. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase but also decreased levels of citrullinated histone 3 in the pancreas and DNA-histone complexes in the plasma of animals with AP. Moreover, GZD824 decreased plasma levels of amylase, IL-6, and MMP-9 as well as edema, acinar cell necrosis, hemorrhage, CXC chemokine formation, and neutrophil infiltration in the inflamed pancreas. A beneficial effect of c-Abl kinase inhibition was confirmed in l-arginine-induced pancreatitis. In vitro, inhibition of c-Abl kinase reduced TNF-α-induced formation of ROS, histone 3 citrullination, and NETs in isolated bone marrow neutrophils. Our findings demonstrate that c-Abl kinase regulates NET formation in the inflamed pancreas. In addition, inhibition of c-Abl kinase reduced pancreatic tissue inflammation, and damage in AP. Thus, targeting c-Abl kinase might be a useful way to protect the pancreas in severe AP

Accuracy of MRI in early rectal cancer: national cohort study

Radiological staging of rectal cancer dictates subsequent patienttreatment. In early-stage disease, local excision is associatedwith reduced morbidity, mortality, and costs, and maintainsbowel continuity compared with surgery, where the whole orpart of the rectum is resected1–3. Nearly 90 per cent of patientswith T1 rectal cancer have N0 disease and are therefore potentially curable with local resection, yet the majority undergo major resection4–6. MRI is the primary staging investigation used to predictlocal stage in rectal cancer7, mainly owing to its ability to allocatepatients in need of neoadjuvant treatment8–10. There is potentially inaccuracy in MRI staging for nodal involvement and differentiation of T1 from T2 tumours6,7,11. Consequently, cT1 and cT2 areoften combined and comprice tumours considered for local resection. Apart from a recent study6 reporting 54 per cent accuracy forMRI cT1–2 category, combined cT1–2 status has not beeninvestigated.The aim of this large nationwide retrospective cohort study wasto investigate the staging accuracy of MRI, from a clinical perspective, in early rectal cancer when combining cT1 and cT2 categories

Platelet secretion of CXCL4 is Rac1-dependent and regulates neutrophil infiltration and tissue damage in septic lung damage.

Platelets are potent regulators of neutrophil accumulation in septic lung damage. We hypothesized that platelet-derived CXCL4 might support pulmonary neutrophilia in a murine model of abdominal sepsis