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Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. Here, we report that male mice with myeloid-specificCnr1deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. MaleCnr1deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in bone marrow derived macrophages and blunted by estradiol. Kinase activity profiling revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally,CNR1expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, hinting to a possible implication of CB1-dependent regulation in human pathophysiology. In conclusion, impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The importance of macrophage CB1 signaling seems to be more pronounced in male mice.Abstract FigureGraphical summary(created withBioRender.com)

Identifying novel pathways regulating the adaptive immune response in chronic inflammatory diseases such as atherosclerosis is of particular interest in view of developing new therapeutic drugs. Here we report that the lipid receptor GPR55 is highly expressed by splenic B cells and inversely correlates with atheroma plaque size in mice. In human carotid endarterectomy specimen, GPR55 transcript levels were significantly lower in unstable compared to stable carotid plaques. To study the impact of GPR55 deficiency in atherosclerosis, we crossedGpr55knockout mice with apolipoprotein E (ApoE) knockout mice and subjected the mice to Western diet for 4 to 16 weeks. Compared toApoE-/-controls,ApoE-/-Gpr55-/-mice developed larger plaques with increased necrotic core size, associated with elevated circulating and aortic leukocyte counts. Flow cytometry, immunofluorescence and RNA-sequencing analysis of splenic B cells in these mice revealed a hyperactivated B cell phenotype with disturbed plasma cell maturation and immunoglobulin (Ig)G antibody overproduction. The specific contribution of B cell GPR55 in atherosclerosis was further studied in mixedGpr55-/-/µMTbone marrow chimeras on low density receptor deficiency (Ldlr-/-) background, revealing that B-cell specific depletion ofGpr55was sufficient to promote plaque development. Conversely, adoptive transfer of wildtype B cells intoApoE-/-Gpr55-/-mice blunted the proatherogenic phenotype.In vitrostimulation of splenocytes with the endogenous GPR55 ligand LPI promoted plasma cell proliferation and enhanced B cell activation marker expression, which was inhibited by the GPR55 antagonist CID16020046. Collectively, these discoveries provide new evidence for GPR55 as key modulator of the adaptive immune response in atherosclerosis. Targeting GPR55 could be useful to limit inflammation and plaque progression in patients suffering from atherosclerosis.

Background: Sepsis is a serious, heterogeneous clinical entity produced by a severe and systemic host inflammatory response to infection. Methotrexate (MTX) is a folate-antagonist that induces the generation of adenosine and also inhibits JAK/STAT pathway, MTX it is widely used as an anti-inflammatory drug to control the immune system. Objective: The aim of this study was to assess the beneficial effects of a single and low dose of MTX in the systemic response and acute lung injury (ALI) induced by sepsis. As in the clinics, we treated our animals with antibiotics and fluids and performed the source control to mimic the current clinic treatment. Methods and main results: Sepsis was induced in rats by a cecal ligation puncture (CLP) procedure. Six hours after induction of sepsis, we proceeded to the source control, fluids and antibiotics were administered at 6 h and 24 h after CLP. MTX (2.5 mg/Kg) was administered 6 h after the first surgery in one CLP experimental group and to one Sham group. A protective effect of MTX was observed through a significant reduction of pro-inflammatory cytokines and a decrease infiltration of inflammatory cells in the lung. In addition, we found a regulation in adenosine receptor A2aR and the metalloproteinases by MTX. Conclusion: A single, low dose of MTX attenuates sepsis lung-associated damage by decreasing pro-inflammatory response, infiltration of pro-inflammatory cells and avoiding defective tissue lung remodeling.

In this study we aimed to determine the effect of the exosomes derived from Mesenchymal stromal cells (MSC) on cell proliferation, and to study how pre-conditioning the MSCs to a septic environment, changes their exosomes’ content and consequently, their regenerative capacity. MSC were isolated from male rats’ femora and tibiae and cultured in non pre-stimulated and pre-stimulated (with LPS) conditions. The secreted exosomes were obtained via standard ultracentrifugation protocol. Cell proliferation experiments were conducted by treating an epithelial cell line with exosomes from non pre-stimulated MSC (Control exosomes) and pre-stimulated MSC (LPS exosomes) through an in vitro scratch assay. Exosomal protein profile was analyzed by a liquid chromatography-mass spectrometry analysis. The treatment of epithelial cells with LPS exosomes increases a 10% its capacity to proliferate and to heal the wound in comparison with the cells treated with Control exosomes (Fig.1a). The analysis of the protein content of both types of exosomes confirmed that pre-conditioning MSCs to a septic environment modifies the protein cargo of its exosomes (Fig. 1b), resulting in the appearance proteins related to cell cycle regulation and cell proliferation. In conclusion, pre-stimulating MSC with LPS enhances their paracrine activity and improves the reparative capacity of their secreted exosomes.

Background Atherosclerosis is accompanied by an imbalance between resolving and pro-inflammatory lipid mediators. Targeting lipid signaling pathways might offer a new anti-inflammatory therapy for improving the clinical outcome in cardiovascular disease patients. We considered lysophosphatidylinositol (LPI) and its receptor G protein-coupled receptor (GPR)55 as a potential modulator of atherosclerosis. Its role in regulating atherosclerosis and B cell function is unknown. Hypothesis We assessed the hypothesis that GPR55 signaling causally affects atherosclerosis and whether it has a specific role in regulating B cell function in this disease. Methods Atherosclerotic plaques were compared between apolipoprotein E deficient (ApoE−/−) and ApoE−/−Gpr55−/− mice after 4 to 16 weeks Western Diet (WD; 0.15% cholesterol; n=12–15 per group). To specifically test the role of B cell GPR55 in atherosclerosis, we generated mixed chimeras by lethally irradiating low density lipoprotein receptor deficient (Ldlr−/−) mice and reconstituting with a mixture of μMT and wildtype (control) or μMT and Gpr55−/− bone marrow cells. Circulating B cells were sorted and bulk RNA sequencing analysis was performed. We performed lipid and immunostainings of murine aortic root plaques, qPCR and ELISA of tissue lysates, as well as multiplex analysis of plasma immunoglobulins. Leukocyte plasma and tissue counts were determined by flow cytometry. Results GPR55 expression in mouse and human atherosclerotic plaques was detected by immunostaining. Furthermore, we confirmed murine Gpr55 mRNA expression on sorted circulating B220+B cells via qPCR, which was higher compared to CD3+ T cells, while CD11+ myeloid cells as well as NK cells had only low Gpr55 mRNA levels. ApoE−/−Gpr55−/− mice had significantly larger plaques after 4&16 weeks WD compared to ApoE−/− controls, with more pronounced body weight increases and higher cholesterol levels at the 16 weeks WD time point. In addition, global Gpr55 deficiency resulted in enhanced aortic pro-inflammatory cytokine mRNA expression (IL-1β, IL-6, TNFα) and a massively upregulated IgG1 plasma levels and increased percentages of splenic germinal center and plasma cells. B-cell RNA-seq analysis showed 460 differential expressed regulated genes in the ApoE−/−Gpr55−/− compared to ApoE−/−. The main pathways affected were calcium ion transport, immunoglobulin production, negative regulation of phosphorylation, and cellular component morphogenesis, suggesting a dsysregulation of B cell function. B cell specific Gpr55 deficiency blunted the metabolic effects on body weight and cholesterol, but still translated in larger atherosclerotic plaques and elevated plasma IgG levels compared to the respective controls. Conclusion Both global and B cell-restricted Gpr55 deficiency promotes atherosclerosis and is associated with a more pro-inflammatory phenotype. Our findings suggest a novel role for GPR55 in regulating B cell development and function. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Background: Recent evidence suggests the beneficial effects of cell therapies and nebulized anticoagulants for acute respiratory distress syndrome (ARDS). Aim: Determine the therapeutic benefit of a combined cell therapy (alveolar type II cells (ATII), mesenchymal stem cells (MSC), supernatant (SN) ATII, SN MSC) with antithrombin in vitro in a coculture of ATII and alveolar macrophages injured with lipopolysaccharide (LPS). Methods: Cocultured ATII and alveolar macrophages isolated from rat lungs were injured with LPS (10 ng/ml). Two hours after the injury a combined cell therapy (ATII, MSC, SN ATII, SN MSC) with antithrombin (0.1 ng/ml) was administered. Proinflammatory mediators and coagulant factors were analyzed via qRT-PCR at 18 h. Data are expressed as mean±SEM. Statistical analysis was performed using One-Way-ANOVA and Bonferroni post-hoc test (statistical significance p≤0.05). Results: Proinflammatory mediators and coagulant factors were significantly increased after LPS administration. After administering the different treatments compared the expression of IL1β, IL6, TF PAI-1 and Plasminogen was not as high as in the LPS group. Conclusions: The results indicate that combined cell therapy with antithrombin are able to attenuate inflammation and coagulation in vitro in a coculture of ATII and macrophages. Both treatments together are able to face different processes involved in lung injury.

Objective— Palmitoylethanolamide is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N -acyl phosphatidylethanolamine phospholipase D. Its biological actions are primarily mediated by PPAR-α (peroxisome proliferator-activated receptors α) and the orphan receptor GPR55. Palmitoylethanolamide exerts potent anti-inflammatory actions but its physiological role and promise as a therapeutic agent in chronic arterial inflammation, such as atherosclerosis remain unexplored. Approach and Results— First, the polarization of mouse primary macrophages towards a proinflammatory phenotype was found to reduce N -acyl phosphatidylethanolamine phospholipase D expression and palmitoylethanolamide bioavailability. N -acyl phosphatidylethanolamine phospholipase D expression was progressively downregulated in the aorta of apolipoprotein E deficient (ApoE −/− ) mice during atherogenesis. N -acyl phosphatidylethanolamine phospholipase D mRNA levels were also downregulated in unstable human plaques and they positively associated with smooth muscle cell markers and negatively with macrophage markers. Second, ApoE − /− mice were fed a high-fat diet for 4 or 16 weeks and treated with either vehicle or palmitoylethanolamide (3 mg/kg per day, 4 weeks) to study the effects of palmitoylethanolamide on early established and pre-established atherosclerosis. Palmitoylethanolamide treatment reduced plaque size in early atherosclerosis, whereas in pre-established atherosclerosis, palmitoylethanolamide promoted signs of plaque stability as evidenced by reduced macrophage accumulation and necrotic core size, increased collagen deposition and downregulation of M1-type macrophage markers. Mechanistically, we found that palmitoylethanolamide, by activating GPR55, increases the expression of the phagocytosis receptor MerTK (proto-oncogene tyrosine-protein kinase MER) and enhances macrophage efferocytosis, indicative of proresolving properties. Conclusions— The present study demonstrates that palmitoylethanolamide protects against atherosclerosis by promoting an anti-inflammatory and proresolving phenotype of lesional macrophages, representing a new therapeutic approach to resolve arterial inflammation.

Objective— Cardiovascular diseases and depression are the leading causes of disability in Western countries. Clinical data on potential cardiovascular effects of serotonin reuptake inhibitors (SSRIs), the most commonly used antidepressant drugs, are controversial. In addition to blocking serotonin reuptake transporter in the brain, SSRIs deplete the major peripheral serotonin (5-hydroxytryptamine [5-HT]) storage by inhibiting serotonin reuptake transporter–mediated uptake in platelets. In this study, we aimed to investigate the effect of chronic SSRI intake on the development of atherosclerosis. Approach and Results— Treatment of apolipoprotein E–deficient mice with the SSRI fluoxetine for 2, 4, or 16 weeks increased atherosclerotic lesion formation, with most pronounced effect during early plaque development. Intravital microscopy of carotid arteries revealed enhanced myeloid cell adhesion on fluoxetine treatment. Mechanistically, we found that fluoxetine augmented vascular permeability and increased chemokine-induced integrin-binding activity of circulating leukocytes. In vitro stimulation of murine blood demonstrated that fluoxetine, but not 5-HT, could directly promote β1 and β2 integrin activation provided C-C motif chemokine ligand 5 was also present. Similar effects were observed with the SSRI escitalopram. Enhanced C-C motif chemokine ligand 5–induced integrin activation by fluoxetine was also confirmed in a human neutrophil-like cell line. In contrast to the proatherogenic properties of fluoxetine, pharmacological inhibition of the peripheral 5-HT synthesizing enzyme tryptophan hydroxylase 1 did not promote atherosclerosis, suggesting that the proatherogenic effect of fluoxetine occurs independent of peripheral 5-HT depletion. Conclusions— SSRI intake may promote atherosclerosis and therefore potentially increase the risk for acute cardiovascular events by a mechanism that is independent of 5-HT depletion.

Objective— Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI. Approach and Results— Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6C high monocyte surface expression levels of chemokine receptors (CCR) were ≈2-fold higher at the beginning of the active phase, Zeitgeber Time (ZT) 13 compared with ZT5. This was because of enhanced receptor surface expression at ZT13, whereas no significant changes in total cellular protein levels were found. Most blood Ly6C high monocytes were CCR2 high , whereas only a minority was CCR1 high and CCR5 high . We also found diurnal changes of classical monocyte blood counts in humans, being higher in the evening, while exhibiting enhanced CCR2 surface expression in the morning. In support of monocyte oscillations between blood and tissue, murine cardiac Ly6C high monocyte counts were highest at ZT13, accompanied by an upregulation of cardiac CC chemokine ligand 2 mRNA. Mice subjected to MI at ZT13 had an even higher upregulation of CCR2 surface expression on circulating monocytes compared with noninfarcted mice and more elevated cardiac CC chemokine ligand 2 protein expression and more pronounced Ly6C high monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6C high monocyte infiltration after ZT13 MI but not ZT5 MI. Conclusions— CCR2 surface expression on Ly6C high monocytes changes in a time-of-day–dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.

Background The animal experimental counterpart of human acute respiratory distress syndrome (ARDS) is acute lung injury (ALI). Most models of ALI involve reproducing the clinical risk factors associated with human ARDS, such as sepsis or acid aspiration; however, none of these models fully replicates human ARDS. Aim To compare different experimental animal models of ALI, based on direct or indirect mechanisms of lung injury, to characterize a model which more closely could reproduce the acute phase of human ARDS. Materials and methods Adult male Sprague-Dawley rats were subjected to intratracheal instillations of (1) HCl to mimic aspiration of gastric contents; (2) lipopolysaccharide (LPS) to mimic bacterial infection; (3) HCl followed by LPS to mimic aspiration of gastric contents with bacterial superinfection; or (4) cecal ligation and puncture (CLP) to induce peritonitis and mimic sepsis. Rats were sacrificed 24 h after instillations or 24 h after CLP. Results At 24 h, rats instilled with LPS or HCl-LPS had increased lung permeability, alveolar neutrophilic recruitment and inflammatory markers (GRO/KC, TNF-α, MCP-1, IL-1β, IL-6). Rats receiving only HCl or subjected to CLP had no evidence of lung injury. Conclusions Rat models of ALI induced directly by LPS or HCl-LPS more closely reproduced the acute phase of human ARDS than the CLP model of indirectly induced ALI.

The use of cell therapies has recently increased for the treatment of pulmonary diseases. Mesenchymal stem/stromal cells (MSCs) and alveolar type II cells (ATII) are the main cell-based therapies used for the treatment of acute respiratory distress syndrome (ARDS). Many pre-clinical studies have shown that both therapies generate positive outcomes; however, the differences in the efficiency of MSCs or ATII for reducing lung damage remains to be studied. We compared the potential of both cell therapies, administering them using the same route and dose and equal time points in a sustained acute lung injury (ALI) model. We found that the MSCs and ATII cells have similar therapeutic effects when we tested them in a hydrochloric acid and lipopolysaccharide (HCl-LPS) two-hit ALI model. Both therapies were able to reduce proinflammatory cytokines, decrease neutrophil infiltration, reduce permeability, and moderate hemorrhage and interstitial edema. Although MSCs and ATII cells have been described as targeting different cellular and molecular mechanisms, our data indicates that both cell therapies are successful for the treatment of ALI, with similar beneficial results. Understanding direct cell crosstalk and the factors released from each cell will open the door to more accurate drugs being able to target specific pathways and offer new curative options for ARDS., This work was supported by the Ministerio de Economía y Competitividad– Instituto de Salud Carlos III (PI12/02548 to A.A., and PI12/02451 and PI15/00482 to R.H.), by the Spanish Society of Critical Care (SEMICYUC); we also got funding from Fondo Europeo de Desarrollo Regional (FEDER), Marta Camprubí got a PhD grant by Catalan Society of Pneumology (SOCAP); the CIBER de Enfermedades Respiratorias and a grant from Fundació Parc Taulí obtained by Ferranda Puig supported also this study.

Background During acute respiratory distress syndrome, proinflammatory mediators inhibit natural anticoagulant factors, which alter the normal balance between coagulation and fibrinolysis leading to a procoagulant state. We hypothesize that pulmonary administration of anticoagulants might be beneficial to treat acute respiratory distress syndrome for their anticoagulant and antiinflammatory effects and reduce the risk of systemic bleeding. Objectives Our aim is to study the effects of nebulized antithrombin (AT) and combined AT and heparin in an animal model of acute lung injury. Methods Acute lung injury was induced in rats by the intratracheal administration of hydrochloric acid and lipopolysaccharide. AT alone (500 IU/kg body weight) or combined with heparin (1000 IU/kg body weight) were nebulized after the injury. Control groups received saline instead. Blood, lung tissue, bronchoalveolar lavage, and alveolar macrophages (AM) isolated from bronchoalveolar lavage were collected after 48 hours and analyzed. Results Nebulized anticoagulant treatments reduced protein concentration in the lungs and decreased injury-mediated coagulation factors (tissue factor, plasminogen activator inhibitor-1, plasminogen, and fibrinogen degradation product) and inflammation (tumor necrosis factor α and interleukin 1β) in the alveolar space without affecting systemic coagulation and no bleeding. AT alone reduced fibrin deposition and edema in the lungs. Heparin did not potentiate AT coagulant effect but promoted the reduction of macrophages infiltration into the alveolar compartment. Anticoagulants reduced nuclear factor-kB downstream effectors in AM. Conclusions Nebulized AT and heparin attenuate lung injury through decreasing coagulation and inflammation without altering systemic coagulation and no bleeding. However, combined AT and heparin did not produce a synergistic effect.

Time to clearance of pathogens is probably critical to outcome in septic shock. Current guidelines recommend intervention for source control within 12 hours after diagnosis. We aimed to determine the epidemiology of source control in the management of sepsis and to analyze the impact of timing to source control on mortality.Prospective observational analysis of the Antibiotic Intervention in Severe Sepsis study, a Spanish national multicenter educational intervention to improve antibiotherapy in sepsis.Ninety-nine medical-surgical ICUs in Spain.We enrolled 3,663 patients with severe sepsis or septic shock during three 4-month periods between 2011 and 2013.Source control and hospital mortality.A total of 1,173 patients (32%) underwent source control, predominantly for abdominal, urinary, and soft-tissue infections. Compared with patients who did not require source control, patients who underwent source control were older, with a greater prevalence of shock, major organ dysfunction, bacteremia, inflammatory markers, and lactic acidemia. In addition, compliance with the resuscitation bundle was worse in those undergoing source control. In patients who underwent source control, crude ICU mortality was lower (21.2% vs 25.1%; p = 0.010); after adjustment for confounding factors, hospital mortality was also lower (odds ratio, 0.809 [95% CI, 0.658-0.994]; p = 0.044). In this observational database analysis, source control after 12 hours was not associated with higher mortality (27.6% vs 26.8%; p = 0.789).Despite greater severity and worse compliance with resuscitation bundles, mortality was lower in septic patients who underwent source control than in those who did not. The time to source control could not be linked to survival in this observational database.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. Methods We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse side effects that appeared within 12 months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12 months of follow-up were evaluated. Results No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12 months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. Conclusions Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF.

Endocannabinoids are a group of arachidonic acid-derived lipid mediators binding to cannabinoid receptors CB1 and CB2. An overactivity of the endocannabinoid system plays a pathophysiological role in the development of visceral obesity and insulin resistance. Moreover, elevated circulating endocannabinoid levels are also prevalent in atherosclerosis. The pathophysiological increase of endocannabinoid levels is due to an altered expression of endocannabinoid synthesizing and degrading enzymes induced by inflammatory mediators such as cytokines or lipids. Emerging experimental evidence suggests that enhanced endocannabinoid signalling affects atherosclerosis via multiple effects, including a modulation of vascular inflammation, leukocyte recruitment, macrophage cholesterol metabolism and consequently atherosclerotic plaque stability. In addition, recent findings in various metabolic disease models highlight the relevance of peripheral CB1 cannabinoid receptors in adipose tissue, liver and pancreas, which crucially regulate lipid and glucose metabolism as well as macrophage properties in these organs. This suggests that targeting the endocannabinoid system in the vasculature and peripheral organs might have a therapeutic potential for atherosclerosis by inhibiting vascular inflammation and improving metabolic risk factors. This review will provide a brief update on the effects of endocannabinoid signalling in atherosclerosis and related metabolic complications.

Background:The acute respiratory distress syndrome (ARDS) is characterized by protein-rich oedema in the alveolar spaces, a feature in which Fas-mediated apoptosis of the alveolar epithelium has been involved.Objective:To determine whether Fas activation increases protein permeability by mechanisms involving disruption of the paracellular tight junction (TJ) proteins in the pulmonary alveoli.Methods: Protein permeability and the expression of TJ proteins were assessed in vivo in wild-type and Fas-deficient lpr mice 16 hours after the intratracheal instillation of recombinant human soluble Fas ligand (rh-sFasL), and at different time points in vitro in human pulmonary alveolar epithelial cells (HPAEpiC) exposed to rh-sFasLResults:Activation of the Fas pathway increased protein permeability in mouse lungs and altered the expression of the TJ proteins occludin and zonula occludens-1 in the alveolar–capillary membrane in vivo and in human alveolar epithelial cell monolayers in vitro. Blockade of caspase-3, but not inhibition of tyrosine kinase dependent pathways, prevented the alterations in TJ protein expression and permeability induced by the Fas/FasL system in human alveolar cell monolayers in vitro. We also observed that both the Fas-induced increase of protein permeability and disruption of TJ proteins occurred before cell death could be detected in the cell monolayers in vitro.Conclusion:Targeting caspase pathways could prevent the disruption of TJs and reduce the formation of lung oedema in the early stages of ARDS.

Acute lung injury(ALI) is a respiratory failure caused by a response of the lung against local or systemic injury. In the early stages C-C chemokine ligand-2(CCL2) is upregulated and classically activated chemokine receptor 2(CCR2+)monocytes/macrophages are recruited to the lung. Monocytes/macrophages infiltration into the alveoli enhances tissue inflammation. Aim: To inhibit the recruitment of CCR2+ monocytes/macrophages into the lung by the administration of CCR2 antagonist or an anti-CCL2 antibody. Methods: Lung injury was induced in Sprague-Dawley rats by intratracheal instillation (it) of HCl (0.1mol/L), followed 2h later by it of LPS of Escherichia coli O55:B5(30μg/g body weight). Control rats were treated with saline. 9h later the corresponding animals were it with CCR2 antagonist(1000ug/ml) or an anti-CCL2 antibody(10ug/ml). Animals were sacrificed at day 3. Leucocytes subsets were measured by flow cytometry in blood, bronchoalveolar lavage (BAL), spleen and bone marrow. One-Way-ANOVA and Bonferroni post-hoc test was applied. P Results: Both treatments,CCR2 antagonist and an anti-CCL2 antibody, reduced lung weight and decreased CD11b+cells (neutrophils and monocytes) in the BAL; proving less inflammatory cell infiltration. No changes in monocytes numbers were observed in bone marrow or spleen. Inflammatory markers expression (IL-1β,IL-6 and TNF-α) was significantly reduced in lung tissue in treated animals. Conclusion: Locally CCR2 or CCL2 blockade reduced infiltrated monocyte/macrophages into the lung, without affecting the systemic production. The reduction of CCR2+monocytes/macrophages was associated with a decrease of inflammation in the lung

[Background] Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by excess production of inflammatory factors. Alveolar type II (ATII) cells help repair damaged lung tissue, rapidly proliferating and differentiating into alveolar type I cells after epithelial cell injury. In ALI, the lack of viable ATII favors progression to more severe lung injury. ATII cells regulate the immune response by synthesizing surfactant and other anti-inflammatory proteins and lipids. Cross-talk between ATII and other cells such as macrophages may also be part of the ATII function. The aim of this study was to test the anti-inflammatory and reparative effects of ATII cells in an experimental model of ALI. [Methods ]In this study ATII cells (2.5 × 106 cells/animal) were intratracheally instilled in rats with HCl and lipopolysaccharide (LPS)-induced ALI and in healthy animals to check for side effects. The specific effect of ATII cells was compared with fibroblast transplantation. [Results] ATII cell transplantation promoted recovery of lung function, decrease mortality and lung inflammation of the animals with ALI. The primary mechanisms for benefit were paracrine effects of prostaglandin E2 (PGE2) and surfactant protein A (SPA) released from ATII cells that modulate alveolar macrophages to an anti-inflammatory phenotype. To our knowledge, these data are the first to provide evidence that ATII cells secrete PGE2 and SPA, reducing pro-inflammatory macrophage activation and ALI. [Conclusion] ATII cells and their secreted molecules have shown an ability to resolve ALI, thereby highlighting a potential novel therapeutic target.

Background: The endocannabinoid system plays a pathophysiological role in metabolic and cardiovascular disorders. G protein-coupled receptor (GPR) 55 is a novel cannabinoid receptor expressed by various lymphocyte subsets, in particular γδT cells, innate lymphoid cells and B1 cells. Its role in regulating immune functions and atherosclerosis is unknown. Methods: We studied early and advanced atherosclerotic plaques and inflammatory parameters in apolipoprotein E deficient (ApoE-/-) and ApoE-/-GPR55-/- mice after 4 or 16 weeks Western Diet (WD), respectively (n=12-15 per group). GPR55 mRNA expression was assessed in human carotid artery plaques (n=29) and healthy control vessels (left internal thoracic arteries; n=28). Results: ApoE-/-GPR55-/- mice had significantly 1.9 to 2.3-fold increased plaque sizes after 4 and 16 weeks WD compared to ApoE-/- controls with higher macrophage content at early stage, but less macrophage and more collagen content at advanced stage. This was accompanied by enhanced aortic pro-inflammatory cytokine mRNA expression (IL-6, TNFα, IL-1β, iNOS) as well as massively upregulated IgG1 plasma levels. Moreover, GPR55 deficiency was associated with increased circulating, splenic and aortic lymphocyte counts after 4 and 16 weeks WD. The most striking (2 to 4-fold) increase was found in γδT cells, with less pronounced increases in the total CD3+ T cell population and CD4+ and CD8+ subsets. This was paralleled by systemic increases in blood monocyte and neutrophil counts, likely due to enhanced myelopoiesis as suggested by enhanced bone marrow myeloid cell counts. At advanced atherosclerosis, ApoE-/-GPR55-/- had significantly higher NK cell numbers in blood and lymphoid organs, suggesting a possible regulation of cytotoxic lymphocyte responses. In view of a potential relevance for human pathophysiology, GPR55 mRNA levels were significantly higher in human plaque samples compared to non-atherosclerotic control vessels. Conclusion: GPR55 deficiency promotes atherosclerosis associated with a more pro-inflammatory phenotype. We may speculate that GPR55 negatively regulates the proatherogenic activity of lymphocyte subsets such as γδT cells, which deserves further investigation.

Aims Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. Methods and results Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. Conclusion These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Acute respiratory distress syndrome (ARDS) presents a complex pathophysiology characterized by pulmonary activated coagulation and reduced fibrinolysis. Despite advances in supportive care of this syndrome, morbidity and mortality remains high, leading to the need of novel therapies to combat this disease. Focus these therapies in the inhibition of ARDS development pathophysiology is essential. Beneficial effects of anticoagulants in ARDS have been proved in preclinical and clinical trials, thanks to its anticoagulant and anti-inflammatory properties. Moreover, local administration by nebulization in the alveolar compartment increases local efficacy and does not produce systemic bleeding. In this review the coagulation and fibrinolytic pathway and its pharmacological targets to treat ARDS are summarized.

Alveolar macrophages (AM) play a key role in acute lung injury (ALI). Its depletion study would help us to evaluate the role of these cells in ALI and whether AM can be potential targets for the treatment of this syndrome. Aim: Evaluate the effect of AM in ALI model, through the depletion of these cells by the administration of clodronate liposomes. Sprague-Dawley rats (~300g) underwent intratracheal administration of HCl and Lipopolysaccharide (LPS)(30μg/g body weight) 2h later. Liposomes of PBS or clodronate (8µg/g body weight) were instilled 9h and 33h after HCl administration. Animals were sacrificed 72h after injury. AM depletion was determined by cell count and flow cytometry analysis. Body weight, lung weight and proteins in bronchoalveolar lavage (BAL) were assessed. Pro and anti-inflammatory mediators, markers of recruitment and apoptosis were analyzed in lung tissue and AM isolated from BAL. Data expressed as media±SEM, relative to GAPDH and fold over saline group (n=8 for all the study groups). One-Way-ANOVA and Newman Keuls post-hoc test were performed (p≤0.05). Clodronate liposomes reduced the percentage of AM to a 50% and did not produce any changes in body weight, lung weight, proteins levels or neutrophils infiltration. In the ALI model the expression of all the evaluated markers were increased. The depletion of AM in ALI model reduced IL1β and increased IL6 in lung tissue. Furthermore, the recruitment of de novo macrophages (CCL2) was reduced, and no changes were observed in neutrophils recruitment (CXCL1). Altogether indicate that AM have a major role in the ALI development and resolution. AM depletion in ALI model does not ameliorate lung injury, indicating that the presence of AM is essential to treat ALI.

Local macrophage clean-up Infection, especially by helminths or bacteria, can cause tissue damage (see the Perspective by Bouchery and Harris). Minutti et al. studied mouse models of helminth infection and fibrosis. They expressed surfactant protein A (a member of the complement component C1q family) in the lung, which enhanced interleukin-4 (IL-4)-mediated proliferation and activation of alveolar macrophages. This activation accelerated helminth clearance and reduced lung injury. In the peritoneum, C1q boosted macrophage activation for liver repair after bacterial infection. By a different approach, Bosurgi et al. discovered that after wounding caused by migrating helminths in the lung or during inflammation in the gut of mice, IL-4 and IL-13 act only in the presence of apoptotic cells to promote tissue repair by local macrophages. Science , this issue p. 1076 , p. 1072 ; see also p. 1014

Background Alveolar Type II cell transplantation has been proposed as a cell therapy for the treatment of idiopathic pulmonary fibrosis. Its long-term benefits include repair of lung fibrosis, but its success partly depends on the restoration of lung homeostasis. Our aim was to evaluate surfactant protein restoration after alveolar Type II cell transplantation in an experimental model of bleomycin-induced lung fibrosis in rats. Methods Lung fibrosis was induced by intratracheal instillation of bleomycin. Alveolar Type II cells were obtained from healthy animals and transplanted 14 days after bleomycin was administered. Furthermore, one group transplanted with alveolar macrophages and another group treated with surfactant were established to evaluate the specificity of the alveolar Type II cell transplantation. The animals were euthanized at 21 days after bleomycin instillation. Lung fibrosis was confirmed by a histologic study and an evaluation of the hydroxyproline content. Changes in surfactant proteins were evaluated by mRNA expression, Western blot and immunofluorescence studies. Results The group with alveolar Type II cell transplantation was the only one to show a reduction in the degree of lung fibrosis and a complete recovery to normal levels of surfactant proteins. Conclusion One of the mechanisms involved in the beneficial effect of alveolar Type II cell transplantation is restoration of lung surfactant protein levels, which is required for proper respiratory function. © 2014 International Society for Heart and Lung Transplantation., This work was supported by Grant FIS PS09/02362 (Spanish Ministry of Health), by MTV3122410, by SEPAR (Sociedad Española de Neumología y Cirugía Torácica) and by FUCAP. Raquel Guillamat-Prats was supported by a Grant from CIBERES (Centro de investigaciones Biomédicas en Red de Enfermedades Respiratorias)

Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI.Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6CCCR2 surface expression on Ly6C

International audience; BACKGROUND: In the early stages of acute respiratory distress syndrome (ARDS), pro-inflammatory mediators inhibit natural anticoagulant factors and initiate an increase in procoagulant activity. Previous studies proved the beneficial effects of heparin in pulmonary coagulopathy, which derive from its anticoagulant and anti-inflammatory activities, although it is uncertain whether heparin works. Understanding the specific effect of unfractioned heparin on cell lung populations would be of interest to increase our knowledge about heparin pathways and to treat ARDS. METHODS: In the current study, the effect of heparin was assessed in primary human alveolar macrophages (hAM), alveolar type II cells (hATII), and fibroblasts (hF) that had been injured with LPS. RESULTS: Heparin did not produce any changes in the Smad/TGFß pathway, in any of the cell types evaluated. Heparin reduced the expression of pro-inflammatory markers (TNF-α and IL-6) in hAM and deactivated the NF-kß pathway in hATII, diminishing the expression of IRAK1 and MyD88 and their effectors, IL-6, MCP-1 and IL-8. CONCLUSIONS: The current study demonstrated that heparin significantly ameliorated the cells lung injury induced by LPS through the inhibition of pro-inflammatory cytokine expression in macrophages and the NF-kß pathway in alveolar cells. Our results suggested that a local pulmonary administration of heparin through nebulization may be able to reduce inflammation in the lung; however, further studies are needed to confirm this hypothesis.

Objective Alveolar macrophages play a key role in the development and resolution of acute respiratory distress syndrome (ARDS), modulating the inflammatory response and the coagulation cascade in lungs. Anti-coagulants may be helpful in the treatment of ARDS. This study investigated the effects of nebulized heparin on the role of alveolar macrophages in limiting lung coagulation and inflammatory response in an animal model of acute lung injury (ALI). Methods Rats were randomized to four experimental groups. In three groups, ALI was induced by intratracheal instillation of lipopolysaccharide (LPS) and heparin was nebulized at constant oxygen flow: the LPS/Hep group received nebulized heparin 4 and 8 hours after injury; the Hep/LPS/Hep group received nebulized heparin 30 minutes before and 4 and 8 hours after LPS-induced injury; the LPS/Sal group received nebulized saline 4 and 8 hours after injury. The control group received only saline. Animals were exsanguinated 24 hours after LPS instillation. Lung tissue, bronchoalveolar lavage fluid (BALF) and alveolar macrophages isolated from BALF were analysed. Results LPS increased protein concentration, oedema and neutrophils in BALF as well as procoagulant and proinflammatory mediators in lung tissue and alveolar macrophages. In lung tissue, nebulized heparin attenuated ALI through decreasing procoagulant (tissue factor, thrombin–anti-thrombin complexes, fibrin degradation products) and proinflammatory (interleukin 6, tumour necrosis factor alpha) pathways. In alveolar macrophages, nebulized heparin reduced expression of procoagulant genes and the effectors of transforming growth factor beta (Smad 2, Smad 3) and nuclear factor kappa B (p-selectin, CCL-2). Pre-treatment resulted in more pronounced attenuation. Conclusion Nebulized heparin reduced pulmonary coagulopathy and inflammation without producing systemic bleeding, partly by modulating alveolar macrophages.

The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation.Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice.Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability.Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse cholesterol transport.

Acute Lung Injury(ALI) is a clinical manifestation of respiratory failure caused by a response of the lung against an injury. The damage to the alveolar barrier is a critical event and it could be repaired by alveolar type IIcells(ATII) or by mesenchymal stem cells(MSC)trasplantation. Objective: Compare the involved pathways in the ALI resolution after ATII and MSC cells administration. Sprague-Dawley rats(∼300g) were sedated and lung injury was induced by intratracheal instillation of HCl(0.1mol/L),followed 2h later by instillation of LPS of E.coli(30μg/g body weight).Control rats were treated with saline.9h after the second instillation ATII cells or MSC (2.5x10 6 cells)were transplanted intratracheally.Data are expressed as mean±SEM. Statistical analysis was performed with OneWay-ANOVA and NewmanKeuls post-hoc.Statistical significance p The instillation of HCl+LPS caused an increase in lung weight,TNFα, IL1s and IL6. Transplantation of both cell populations reduced significantly all of them. ATII and MSC cell therapy presented marked differences.ATII cells had a huge antinflammatory activity, reducing to level controls IL4 and IL13. MSC transplantation did not produce any change in IL4 or IL13 compared to the HCl+LPSgroup. On the other hand,MSC transplantation induces a reduction in GM-CSF indicating a less number of monocytes recruited to the lung. ATII cells transplantation produces a better recovering of the lung injury evaluated by histological analysis. Conclusion: Both populations of cells are effective in lung injury recovery. The study of the specific pathways involved in the recovery of lung injury allows us to improve a better therapy for ALI/ARDS.

Acute Respiratory Distress Syndrome (ARDS) is the major cause of respiratory insufficiency in patients and is associated with high mortality and morbidity. Most patients with sepsis suffer ARDS, characterized by generalized inflammation with edema and inflammatory cells infiltration. Methotrexate (MTX) is an anti-inflammatory drug currently used in autoimmune diseases. This drug reduces lymphocyte proliferation and cytokines produced by neutrophils and macrophages. Our main hypothesis is that MTX controls the inflammatory response and reduces the damage in the lung. To carry out this project we used a murine model of sepsis induced by cecal ligation and puncture. After 6 hours we did the source control removing the cecum and administrating antibiotics, fluid and analgesic. In addition, MTX (2.5 mg/Kg) was administered i.p. to a group of rats. 48 h later, the animals were sacrificed and samples of lung tissue, bronchoalveolar lavage fluid and blood were collected. The corresponding control groups were performed. Our results show that MTX reduces weight of the lung, infiltration of neutrophil and lymphocyte and bronchoalveolar protein concentration. This drug also reduces the circulating lymphocytes in blood. In addition, MTX significantly reduces the expression of proinflamatory markers in lung tissue. MTX can control the proinflammatory cascade activated in the early stages of lung inflammation, reducing lung injury in a murine model.

Acute Respiratory Distress Syndrome(ARDS) is an acute failure that develops in all ages patients with diverse clinical disorders and high mortality. ARDS pathogenesis involves inflammatory and procoagulant mechanisms. There is an increased inflammatory pulmonary edema with a great number of neutrophils and platelets,and endothelial and epithelial injury. The equilibrium between coagulation and fibrinolysis is disrupted and replaced by a procoagulant state. Antihombin III(ATIII) neutralizes several enzymes in the coagulation cascade,including thrombin,one of the most important procoagulant proteins found in ARDS patients. The aim of the study was to test the antinflammatory effect of ATIII in an invitro model of acute injury. We isolated human alveolar typeII cells from surgical resections. Acute damage was induced with Lipopolysaccharide of E. coli(100ng/ml) and 2h later we administered ATIII(5IU/ml) to one cell group. After 24h the expression of iNOS, TNFα, IL1s and IL12p40 was evaluated by qRT-PCR. Data are expressed as mean±SEM; one-way ANOVA and Bonferroni post-hoc test was performed(n=6). Statistical differences are considered with p≤0.05. The expression of iNOS,TNFα,IL1s and IL12p40 were increased in our invitro acute injury model. ATIII was able to reverse significantly the increase of all the proinflammatory markers. In conclusion ATIII reduces proinflammatory activation of Alveolar typeII cells and could help to recover ARDS.

Acute Lung Injury(ALI) inflammation is initiated by alveolar macrophages(AM) which release proinflammatory mediators highly regulated for other pulmonary cells. Coagulation pathways are involved in the injury progress. The beneficial effect of anticoagulants is due to their anti-inflammatory effect and their anticoagulant activity. Objective: Evaluate the specific effect of heparin in different pulmonary cells after ALI induction. Human alveolar type II cells(ATII), AM and fibroblasts were isolated from pulmonary biopsies. Heparin (0,1IU/ml) was administered to the cells after the induction of an ALI with a pro-inflammatory stimulus with Lipopolysaccharide from E. coli 055:B5(LPS). The effect of heparin was assessed by the analysis of pro and anti-inflammatory markers and their pathways via qRT-PCR. Data are expressed as mean±SEM. Statistical analysis was performed using One-Way-ANOVA and Newman-Keuls post-hoc test. Statistical significance p≤0.05 is considered. Results: Heparin was able to modify the inflammatory response of all cell populations via NFkB pathway, decreasing it significantly in the case of ATII. Heparin did not produce any changes in the SMAD/TGFβ pathway. Conclusions: Heparin has an immunomodulatory effect in alveolar cells and reduces inflammation in ATII and macrophages. To go in deep in heparin interaction with pulmonary cells promote the development of specific treatments for ALI.

The majority of the animal models of acute lung injury (ALI) are focused on the acute phase. This limits the studies of the mechanisms involved in later phases and the effects of long-term treatments. Thus the goal of this study was to develop an experimental ALI model of aspiration pneumonia, in which diffuse alveolar damage continues for 72 h. Rats were intratracheally instilled with one dose of HCl (0.1 mol/l) followed by another instillation of one dose of LPS (0, 10, 20, 30, or 40 μg/g body weight) 2 h later, which models aspiration of gastric contents that progresses to secondary lung injury from bacteria or bacterial products. The rats were euthanized at 24, 48, and 72 h after the last instillation. The results showed that HCl and LPS at all doses caused activation of inflammatory responses, increased protein permeability and apoptosis, and induced mild hypoxemia in rat lungs at 24 h postinstillation. However, this lung damage was present at 72 h only in rats receiving HCl and LPS at the doses of 30 and 40 μg/g body wt. Mortality (~50%) occurred in the first 48 h and only in the rats treated with HCl and LPS at the highest dose (40 μg/g body wt). In conclusion, intratracheal instillation of HCl followed by LPS at the dose of 30 μg/g body wt results in severe diffuse alveolar damage that continues at least 72 h. This rat model of aspiration pneumonia-induced ALI will be useful for testing long-term effects of new therapeutic strategies in ALI.

The EuroSciCon's 2015 Innate Immunity Summit, London, UK, 17–19 November 2015 A first line of defense against viral infection is prompted by the innate immune system. Viruses activate both extracellular and intracellular events that lead to a war between the virus and the host. In addition to vaccines which induce adaptive T- and B-cell response in readiness for infection, other therapies that potentiate the host immune response are in development, such as those that induce an increase in restriction factor activity or diminish inflammation through Toll-like receptors’ antagonists. Other modulators of immune response, such as thymosin α-1, contribute to the inhibition of HIV-1 and human T lymphotropic virus 1 infection. Understanding the mechanisms by which the innate immune response combats pathogen invasion will enable the generation of novel therapeutic strategies to cure viral infection.

Acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are a clinical manifestation of respiratory failure caused by a response of the lung to local or systemic injury [1]. Damage of alveolar barrier is a critical event in the early stage of ALI/ARDS. Currently there is no effective treatment for this disease. Alveolar type II cells (ATII) are implicated in the alveoli reparation [2] and the transplant of these cells could be a promising ALI treatment.

Introduction: Acute Lung Injury (ALI) is characterized by a promptly release of proinflammatory mediators started by macrophages that propagate the coagulant response. Anticoagulants could be effective for their anti-inflammatory effect additionally their anticoagulant activity. Objective: To evaluate the effect of heparin in human alveolar cells and macrophages after induce an ALI. Methods: Human alveolar primary cells and alveolar macrophages from pulmonary biopsies were seeded. Heparin (100 UI/ml) was administered to the cells after the induction of an ALI with a pro-inflammatory stimulus (Cytomix: mix of TNFα, IL1β and IFNγ; 50 ng/ml). The effect of heparin was assessed by the analysis of proinflammatory markers (IL12p40 and iNOS), cell proliferation and permeability (measuring transmembrane resistance). Data are expressed as mean±SEM (units are relative to the expression of control group). Statistical analysis was performed using One-Way-ANOVA and post-hoc (Newman Keuls) test. Statistical significance p≤0.05 is considered. Results: Heparin was able to modify the inflammatory response of both cell populations, decreasing it significantly in the case of macrophages ( iNOS: Control:1±0.09, Injured group:41.68±4.86, Heparin group:0.54±0.06. IL12p40: Control:1±0.11, Injured group:46.74±4.32, Heparin group:0.15±0.009). The permeability of the monolayer and cell proliferation of alveolar cells did not show changes. Conclusions: Heparin has an immunomodulatory effect in alveolar cells and reduces inflammation in macrophages. This mechanism produced by heparin could have a beneficial effect in ALI.

Introduction: Acute lung injury (ALI) is a clinical manifestation of respiratory failure caused by a response of the lung against local or systemic injury. The damage to the alveolar barrier is a critical event in the early stage of ALI that could be repaired by alveolar type II cells (ATII). In previous studies with transplantation of ATII in ALI we obtained promising results now we want to study them in depth. Objective: Determine the role of ATII cells in the pro and anti-inflammatory pathways in ALI. Methods: Sprague-Dawley rats (225-250g) were anesthetized with isoflurane. The lesion was induced by intratracheal instillation of HCl (0.1 mol/L), followed by instillation of LPS of Escherichia coli O55:B5 (30μg/g body weight) 2h later. Control rats were treated with saline. 9h after the second instillation, a group were transplanted intratracheally with ATII cells (2.5x10 6 cells). Data are expressed as mean±SEM. Statistical analysis was performed with One-Way-ANOVA and Newman Keuls post-hoc. Statistical significance p Results: The instillation of HCl+LPS caused an increase in lung weight, which was significantly reduced by transplantation (C:1.3±0.2g; HCL+LPS:1.8±0.2; TRP:0.15g±1.5),(p

Lung surfactant protein A (SP-A) plays an important function in modulating inflammation in the lung. However, the exact role of SP-A and the mechanism by which SP-A affects IFN-γ-induced activation of alveolar macrophages (aMfs) remains unknown. To address these questions, we studied the effect of human SP-A on rat and human aMfs stimulated with IFN-γ, LPS, and combinations thereof and measured the induction of proinflammatory mediators as well as SP-A's ability to bind to IFN-γ or IFN-γR1. We found that SP-A inhibited (IFN-γ + LPS)-induced TNF-a, iNOS, and CXCL10 production by rat aMfs. When rat macrophages were stimulated with LPS and IFN-γ separately, SP-A inhibited both LPS-induced signaling and IFN-γ-elicited STAT1 phosphorylation. SP-A also decreased TNF-a and CXCL10 secretion by ex vivo-cultured human aMfs and M-CSF-derived macrophages stimulated by either LPS or IFN-γ or both. Hence, SP-A inhibited upregulation of IFN-γ-inducible genes (CXCL10, RARRES3, and ETV7) as well as STAT1 phosphorylation in human M-CSF-derived macrophages. In addition, we found that SP-A bound to human IFN-γ (KD = 11 6 0.5 nM) in a Ca2+-dependent manner and prevented IFN-γ interaction with IFN-γR1 on human aMfs. We conclude that SP-A inhibition of (IFN-γ + LPS) stimulation is due to SP-A attenuation of both inflammatory agents and that the binding of SP-A to IFN-γ abrogates IFN-γ effects on human macrophages, suppressing their classical activation and subsequent inflammatory response.

Over the last years, proteomics has provided us a lot of information about the spectrum of all the proteins that are expressed by an organism in pathological and non-pathological processes. The advantage of studying the proteome over other omics (genomics, epigenetics, transcriptomics and metabolomics) is that proteins reflect the final effectors in all the complex network of replication, transcription and translation. Sepsis is a systemic inflammatory response caused by infection and that could produce multiple organ dysfunctions. The study of the secreted proteins would improve the knowledge of molecular mechanisms and pathways implicated in the septic process and consequently, data will allow us to find new therapeutic targets. The objective of this review is to summarize the proteomics updates of preclinical and clinical studies of sepsis in fields like pathophysiology, treatment, diagnosis or prognosis, providing new perspectives and directions of sepsis. Proteomics is a useful technique for the understanding of the pathophysiology of sepsis infection, the identification of new molecules for an early diagnosis and the prognosis, and the follow-up of treatment progress. The validation of new biomarkers needs a large cohort of patients and the use of other additional methods. Nevertheless, together with other techniques, proteomics has added important elements to the understanding of sepsis and other diseases. Despite current limitations, proteomic techniques improvement with bioinformatics tools might help results interpretation. Besides, the bettering in sensitivity and sensibility may facilitate further sepsis studies with these techniques.