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BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically., Hepatology, 75 (4), ISSN:0270-9139, ISSN:1527-3350

Orchestrated recruitment of neutrophils to inflamed tissue is essential during initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it is still unclear how these factors influence the migration of neutrophils to and at the site of inflammation either during their transmigration through the blood-endothelial cell barrier, or their motility in the interstitial space. Here, we reveal that activation of the Hypoxia Inducible Factor-2 (HIF2α) due to deficiency of HIF-prolyl hydroxylase domain protein-2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNAseq analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the here identified novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation as it promotes neutrophil movement through highly confined tissue landscapes.

Significance During infection, extracellular “labile” heme, released from damaged red blood or parenchymal cells, acts as prototypical alarmin stimulating myeloid cells. A characteristic hallmark of myeloid cell activation is the development of trained immunity, specified as long-lasting adaptations based on transcriptional and epigenetic modifications. In vivo, this is maintained by the rerouting of hematopoiesis. We found that heme is a previously unrecognized trained immunity inducer promoting resistance to bacterial infection in mice. This goes along with extensive long-lasting epigenetic memory in hematopoietic stem cells provoking drastic changes in the transcription factor–binding landscape of myeloid progenitor cells. Given the critical role of heme during infections, we propose that trained immunity is a more general component of innate immunity than previously suggested., Trained immunity defines long-lasting adaptations of innate immunity based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors [M. Divangahi et al., Nat. Immunol. 22, 2–6 (2021)]. Innate immune cells, however, do not exclusively differentiate between foreign and self but also react to host-derived molecules referred to as alarmins. Extracellular “labile” heme, released during infections, is a bona fide alarmin promoting myeloid cell activation [M. P. Soares, M. T. Bozza, Curr. Opin. Immunol. 38, 94–100 (2016)]. Here, we report that labile heme is a previously unrecognized inducer of trained immunity that confers long-term regulation of lineage specification of hematopoietic stem cells and progenitor cells. In contrast to previous reports on trained immunity, essentially mediated by pathogen-associated molecular patterns, heme training depends on spleen tyrosine kinase signal transduction pathway acting upstream of c-Jun N-terminal kinases. Heme training promotes resistance to sepsis, is associated with the expansion of self-renewing hematopoetic stem cells primed toward myelopoiesis and to the occurrence of a specific myeloid cell population. This is potentially evoked by sustained activity of Nfix, Runx1, and Nfe2l2 and dissociation of the transcriptional repressor Bach2. Previously reported trained immunity inducers are, however, infrequently present in the host, whereas heme abundantly occurs during noninfectious and infectious disease. This difference might explain the vanishing protection exerted by heme training in sepsis over time with sustained long-term myeloid adaptations. Hence, we propose that trained immunity is an integral component of innate immunity with distinct functional differences on infectious disease outcome depending on its induction by pathogenic or endogenous molecules.

Several lines of clinical and experimental evidence suggest that immune cell plasticity is a central player in tumorigenesis, tumor progression, and metastasis formation. Neutrophils are able to promote or inhibit tumor growth. Through their interaction with tumor cells or their crosstalk with other immune cell subsets in the tumor microenvironment, they modulate tumor cell survival. Here, we summarize current knowledge with regards to the mechanisms that underlie neutrophil–mediated effects on tumor establishment and metastasis development. We also discuss the tumor-mediated effects on granulopoiesis and neutrophil precursors in the bone marrow and the involvement of neutrophils in anti-tumor therapeutic modalities.

Efficient inflammation resolution is important not only for the termination of the inflammatory response but also for the restoration of tissue integrity. An integral process to resolution of inflammation is the phagocytosis of dying cells by macrophages, known as efferocytosis. This function is mediated by a complex and well-orchestrated network of interactions amongst specialized phagocytic receptors, bridging molecules, as well as "find-me" and "eat-me" signals. Efferocytosis serves not only as a waste disposal mechanism (clearance of the apoptotic cells) but also promotes a pro-resolving phenotype in efferocytic macrophages and thereby termination of inflammation. Alterations in cellular metabolism are critical for shaping the phenotype and function of efferocytic macrophages, thus, representing an important determinant of macrophage plasticity. Impaired efferocytosis can result in inflammation-associated pathologies or autoimmunity. The present mini review summarizes current knowledge regarding the mechanisms regulating macrophage efferocytosis during clearance of inflammation.

Summary Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with β-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of β-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from β-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of β-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis., Graphical Abstract, Highlights • Trained innate immunity (TII) promotes anti-tumor activity • TII is linked to transcriptomic and epigenetic rewiring of granulopoiesis • Trained granulopoiesis promotes an anti-tumor phenotype in neutrophils • Trained granulopoiesis might bear potential for cancer immunotherapy, Pre-treatment of mice with β-glucan, an agonist of trained immunity responses, leads to epigenetic changes in granulopoiesis and neutrophil function to drive anti-tumor immune responses.

Integrins constitute a large group of adhesion receptors that are formed as heterodimers of α and β subunits. Their presence and activation status on the surface of leukocytes modulate a broad spectrum of processes in inflammation and immunity. This mini review critically outlines research advances with regard to the function of leukocyte integrins in regulating and integrating the onset and resolution of acute inflammation. Specifically, we summarize and discuss relevant, current literature that supports the multifunctional role of integrins and their partners. The latter include molecules that physically associate with integrins or regulate their activity in the context of the following: 1) leukocyte recruitment to an inflamed tissue, 2) recognition and phagocytosis of apoptotic neutrophils (efferocytosis), and 3) egress of efferocytic macrophages from the inflamed site to lymphoid tissues. The understanding of the fine-tuning mechanisms of the aforementioned processes by integrins and their functional partners may enable the design of therapeutic tools to counteract destructive inflammation and promote more efficient resolution of inflammation.

Trained immunity defines long-lasting adaptive responses of innate immunity, mediated by transcriptional and epigenetic modifications of myeloid cells. Here, we report that labile heme, an alarmin released extracellularily upon tissue damage and hemolysis, induces trained immunity in human and murine primary cells and in mice. Heme-trainning in monocytes is associated with epigenetic and transcriptional profiles that overlap to some extent with those seen in β-glucan-training, the prototype agonist of trained immunity. In sharp contrast to β-glucan training however, heme-training relied on activation of the Syk/JNK-pathway. Heme training in mice was associated with long-lasting expansion of myeloid-biased progenitor cells as well as with altered chromatin accessibility in hematopoietic stem and progenitor cells. Finally, heme-induced training was protective against bacterial sepsis in mice.In conclusion, we reveal that heme, a bona fide alarmin, induces innate immune memory regulating host response to infection.

Summary Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery., Graphical Abstract, Highlights • Trained immunity (TI) modulates hematopoietic progenitors in bone marrow • TI is associated with adaptations in cell metabolism in progenitors • TI increases expansion of hematopoietic progenitors and myelopoiesis • TI promotes beneficial responses to systemic inflammation and chemotherapy, Modulation of hematopoietic stem and progenitor cells during trained immunity allows a sustained response of myeloid cells to a secondary challenge despite their short lifespan in circulation.

Wound healing is a complex homeostatic response to injury that engages numerous cellular activities, processes, and cell-to-cell interactions. The complement system, an intricate network of proteins with important roles in immune surveillance and homeostasis, has been implicated in many physiological processes; however, its role in wound healing remains largely unexplored. In this study, we employ a murine model of excisional cutaneous wound healing and show that C3−/− mice exhibit accelerated early stages of wound healing. Reconstitution of C3−/− mice with serum from C3+/+ mice or purified human C3 abrogated the accelerated wound-healing phenotype. Wound histology of C3−/− mice revealed a reduction in inflammatory infiltrate compared with C3+/+ mice. C3 deficiency also resulted in increased accumulation of mast cells and advanced angiogenesis. We further show that mice deficient in the downstream complement effector C5 exhibit a similar wound-healing phenotype, which is recapitulated in C5aR1−/− mice, but not C3aR−/− or C5aR2−/− mice. Taken together, these data suggest that C5a signaling through C5aR may in part play a pivotal role in recruitment and activation of inflammatory cells to the wound environment, which in turn could delay the early stages of cutaneous wound healing. These findings also suggest a previously underappreciated role for complement in wound healing, and may have therapeutic implications for conditions of delayed wound healing.

Hematopoietic stem cells (HSCs) remain mostly quiescent under steady-state conditions but switch to a proliferative state following hematopoietic stress, e.g., bone marrow (BM) injury, transplantation, or systemic infection and inflammation. The homeostatic balance between quiescence, self-renewal, and differentiation of HSCs is strongly dependent on their interactions with cells that constitute a specialized microanatomical environment in the BM known as the HSC niche. Here, we identified the secreted extracellular matrix protein Del-1 as a component and regulator of the HSC niche. Specifically, we found that Del-1 was expressed by several cellular components of the HSC niche, including arteriolar endothelial cells, CXCL12-abundant reticular (CAR) cells, and cells of the osteoblastic lineage. Del-1 promoted critical functions of the HSC niche, as it regulated long-term HSC (LT-HSC) proliferation and differentiation toward the myeloid lineage. Del-1 deficiency in mice resulted in reduced LT-HSC proliferation and infringed preferentially upon myelopoiesis under both steady-state and stressful conditions, such as hematopoietic cell transplantation and G-CSF- or inflammation-induced stress myelopoiesis. Del-1-induced HSC proliferation and myeloid lineage commitment were mediated by β3 integrin on hematopoietic progenitors. This hitherto unknown Del-1 function in the HSC niche represents a juxtacrine homeostatic adaptation of the hematopoietic system in stress myelopoiesis.

Background: The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the -effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3. Methods: Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150 minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n= 5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls. Results: Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11 +/- 0.34% vs 3.12 +/- 0.48% control activation;P

Summary Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of in vitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5 μm), for 120 or 240 min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5 μm). The results indicate that incubation of spermatozoa with 2.5 μm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p

SummaryPlatelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood–islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.Supplementary Material to this article is available online at www.thrombosis-online.com.

The involvement of IL-4 in liver regeneration has not yet been recognized. In this article, we show that IL-4, produced by NKT cells that accumulate in regenerating livers after partial hepatectomy, contributes to this process by regulating the activation of complement after liver resection in mice. The mechanism of this regulation was associated with the maintenance of an appropriate level of IgM in mouse blood, because IgM deposited in liver parenchyma most likely initiated complement activation during liver regeneration. By controlling complement activation, IL-4 regulated the induction of IL-6, thereby influencing a key pathway involved in regenerating liver cell proliferation and survival. Furthermore, the secretion of IL-4 was controlled by complement through the recruitment of NKT cells to regenerating livers. Our study thus reveals the existence of a regulatory feedback mechanism involving complement and IL-4 that controls liver regeneration.

Familial Mediterranean fever (FMF) is an autoinflammatory disease, characterized by MEFV gene mutations and self-limited recurrent episodes of fever and localized serositis. Complement system is a key regulator of the inflammatory process. The aim of this study was to investigate the genetic alterations and mRNA expression pattern of C5aR and C5L2 genes in neutrophils from attack-free FMF patients. No mutations were observed in the two receptors' genes, while the genetic alteration observed in the C5aR1 gene was identified as N279 K polymorphic variant. Furthermore, lower mRNA expression of C5L2 gene was observed in neutrophils from FMF patients compared to control subjects. The binding capacity of rhC5a and the ability to produce reactive oxygen species was similar in neutrophils from healthy subjects and FMF patients and independent of the presence of N279 K polymorphism or mRNA expression of C5L2.

Familial Mediterranean fever (FMF) inflammatory attacks are often triggered by metabolic or physical stress. mTOR signaling and autophagy modulate cellular responses to metabolic danger signals. In this study, we investigated the implication of mTOR inhibition and autophagy in FMF pathophysiology. mTOR inhibition induced MEFV gene expression in polymorphonuclear cells (PMNs) from healthy individuals, whereas it had no effect on PMNs from attack-free FMF patients. A significant reduction in pyrin levels in PMNs from FMF patients after mTOR inhibition was also observed. Pyrin levels in control PMNs remained unaffected. Moreover, the basal autophagic status in PMNs from FMF patients was reduced, as indicated by the lower LC3B-II/I ratio and ATG mRNA expression levels. However, mTOR inhibition had similar effects on the induction of autophagy in the two groups. The differential pyrin expression after metabolic stress induction and the impaired basal autophagy suggest a potential role in the triggering of FMF attacks.

The screening for JAK2 V617F mutation in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis offers crucial information for the final diagnosis of these disorders. Recently, several JAK2 exons 12 and 14 mutations have been detected in V617F-negative patients with idiopathic erythrocytosis. The need for a rapid and accurate assay for the mutation screening in both exons 12 and 14 prompted us for the application of a method for the analysis of the entire coding region between exons 11 and 15. We applied the non-isotopic RNase cleavage assay and the accuracy of the method was verified in a series of V617F-positive, V617F-negative patients and healthy individuals, with no false results. This method can be applied in any laboratory without the requirement of specific sophisticated equipment.

Introduction Leptin is an adipocyte-derived cytokine primarily involved in the regulation of body weight and energy balance. In vivo studies suggest that leptin promotes platelet aggregation and thrombosis. Neutrophils are involved in the crosstalk between inflammation and thrombosis in clinical disorders. Leptin is also involved in the regulation of inflammation. Aim We examined the in vitro effects of leptin on the expression of tissue factor (TF), the primary initiator of coagulation, in healthy neutrophils. Materials and methods/results The effects on TF expression were assayed functionally using a modified prothrombin time (mPT), as well as at mRNA and protein levels. The same experiments were performed in parallel with PBMC. Leptin induced functional TF and increased TF mRNA and protein expression in both cell types, as determined by mPT, real-time RT-PCR, western blot, flow cytometry, immunocytochemistry. Inhibition studies revealed that the effect of leptin on TF expression is mediated, at least in part, by JAK2 and PI3K. Our findings, after neutralising TNFα in supernatants of leptin-treated cells, also suggest the involvement of TNFα in the leptin-induced TF expression in leukocytes. Conclusions This study indicates a novel link between inflammation, obesity and thrombosis by showing that leptin is able to trigger the extrinsic coagulation cascade. This work suggests a possible mechanism of the thrombotic effects of hyperleptinemic-associated clinical disorders.

Reactive oxygen species (ROS) production above critical levels affects the genetic and functional integrity of spermatozoa by causing oxidative stress. Spermatozoa are susceptible to oxidative stress in terms of motility and fertilization capacity. Crocin (crocetin di-gentiobiose ester), a main constituent of Crocus Sativus L. (saffron), is known for its antioxidant activity by scavenging ROS, especially superoxide anion. The aim of the present study is to evaluate the effect of crocin on the quality characteristics of spermatozoa and fertilization rate. Frozen-thawed and washed spermatozoa from four different bulls were incubated with three different concentrations of crocin (0.5, 1, and 2 mM), for 120 and 240 minutes, in the presence of a negative control, and were evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. The most potent concentration of crocin (1 mM) was also added in the fertilization medium to test its impact on fertilization outcome. The results indicate that the incubation of spermatozoa with 1 mM of crocin resulted in a statistically significant lower production of ROS, lower lipid peroxidation and in better maintenance of motility, viability, and acrosomal integrity, with a very small number of fragmented cells, compared to the control and the other treated groups (P

Infection or sterile inflammation triggers site-specific attraction of leukocytes. Leukocyte recruitment is a process comprising several steps orchestrated by adhesion molecules, chemokines, cytokines and endogenous regulatory molecules. Distinct adhesive interactions between endothelial cells and leukocytes and signalling mechanisms contribute to the temporal and spatial fine-tuning of the leukocyte adhesion cascade. Central players in the leukocyte adhesion cascade include the leukocyte adhesion receptors of the β2-integrin family, such as the αLβ2 and αMβ2 integrins, or of the β1-integrin family, such as the α4β1- integrin. Given the central involvement of leukocyte recruitment in different inflammatory and autoimmune diseases, the leukocyte adhesion cascade in general, and leukocyte integrins in particular, represent key therapeutic targets. In this context, the present review focuses on the role of leukocyte integrins in the leukocyte adhesion cascade. Experimental evidence that has implicated leukocyte integrins as targets in animal models of inflammatory disorders, such as experimental autoimmune encephalomyelitis, psoriasis, inflammatory bone loss and inflammatory bowel disease as well as preclinical and clinical therapeutic applications of antibodies that target leukocyte integrins in various inflammatory disorders are presented. Finally, we review recent findings on endogenous inhibitors that modify leukocyte integrin function, which could emerge as promising therapeutic targets.

Xenotransplantation ( xeno-Tx) is considered as an alternative solution to overcome the shortage of human donor organs. However, the success of xeno-Tx is hindered by immune reactions against xenogeneic cells (e. g. of porcine origin). More specifically, activation of innate immune mechanisms such as complement and triggering of the coagulation cascade occur shortly after xeno-Tx, and adhesion of human leukocytes to porcine endothelium is another early critical step mediating the immune attack. To investigate the therapeutic potential of complement inhibition in the context of xenogeneic interactions, we have employed a whole-blood model in the present study. Incubation of human blood with porcine endothelial cells (PAECs) led to activation of complement and coagulation as well as to increased leukocyte adhesion. The observed responses can be attributed to the pig-to-human xenogeneicity, since the presence of human endothelium induced a minor cellular and plasmatic inflammatory response. Importantly, complement inhibition using a potent complement C3 inhibitor, compstatin analogue Cp40, abrogated the adhesion of leukocytes and, more specifically, the attachment of neutrophils to porcine endothelium. Moreover, Cp40 inhibited the activation of PAECs and leukocytes, since the levels of the adhesion molecules E-selectin, ICAM-1, ICAM-2, and VCAM-1 on PAECs and the surface expression of integrin CD11b on neutrophils were significantly decreased. Along the same line, inhibition of CD11b resulted in decreased leukocyte adhesion. Taken together, our findings provide a better understanding of the mechanisms regulating the acute innate immune complications in the context of xeno-Tx and could pave the way for complement-targeting therapeutic interventions.

Background Terpenoids and isoprenoids are an important class of natural products, which includes currently used drugs, high value bioactive and industrial compounds, and fuel candidates. Due to their industrial application, there is increasing interest in the development of S. cerevisiae strains capable of producing high levels of terpenoids. Results Aiming to identify new gene targets which can be manipulated to increase sesquiterpene production, a set of HMG2 positive genetic interactors were assessed as single and digenic heterozygous deletions in the presence or absence of stable HMG2(K6R) overexpression. Upon single allele deletion, most genes examined led to increased sesquiterpene production in yeast cells. Tandem heterozygous deletion of a set of three genes, the ubiquitin ligases ubc7 and ssm4/doa10, and the ER resident protein pho86, led to an 11-fold increase in caryophyllene yields (125 mg/L in shake flasks) compared to cells lacking these modifications. The effect of the heterozygous deletions appears to be due to Hmg1p and Hmg2p stabilization. Conclusion Heterozygous deletions cause significant reductions in protein levels but do not lead to growth impediments frequently seen in haploid strains. By exploiting desirable haploinsufficiencies in yeast, we identified a new set of genes that can be disrupted in tandem and cause significant stabilization of Hmgp and a substantial increase in sesquiterpene production. The approach presented here allows new genetic perturbations to be compiled on yeast cell factory strains without negatively impacting cell growth and viability.

Background: Sepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation. Methodology/Principal Findings: In this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling. Conclusions/Significance: This study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis. © 2012 Kambas et al.

Objective. The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)– thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc. Methods. Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ETA) were investigated. Collagen production and migratory activity of HCMFs were further assessed. Results. HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ETA expression. ETA overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs. Conclusion. Tissue factor–thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET A expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle.

Background Gout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β. Methodology/Principal Findings Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra. Conclusions/Significance These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.

article Autophagy is a homeostatic process promoting cell survival in periods of stress. The induction of the autophagic machinery has also been implicated in both innate and adaptive immunity. Leishmania donovani, which is the causative pathogen of visceral leishmaniasis, is an intracellular parasite that invades and multiplies in bone marrow macrophages. We describe the induction of host cell autophagic machinery during acute natural bone marrow infection by L. donovani complex, detected by LC3B immunoblot. The successful treatment with liposomal amphotericin B resulted in the resolution of this phenomenon. Even though the role of autophagy in parasite biology has been previously studied, our findings show for the first time the in vivο host cell LC3B conversion as a marker of the induction of the autophagic machinery during infection with Leishmania parasite in real time conditions.

Acute respiratory distress syndrome (ARDS) is characterized by the presence of fibrin-rich inflammatory exudates in the intra-alveolar spaces and the extensive migration of neutrophils into alveoli of the lungs. Tissue factor (TF)-dependent procoagulant properties of bronchoalveaolar lavage fluid (BALF) obtained from ARDS patients favor fibrin deposition, and are likely the result of cross-talk between inflammatory mediators and hemostatic mechanisms. However, the regulation of these interactions remains elusive. Prompted by previous findings suggesting that neutrophils, under certain inflammatory conditions, can express functional TF, we investigated the contribution of intra-alveolar neutrophils to the procoagulant properties of BALF from patients with ARDS. Our results confirm that the procoagulant properties of BALF from ARDS patients are the result of TF induction, and further indicate that BALF neutrophils are a main source of TF in intra-alveolar fluid. We also found that BALF neutrophils in these patients express significantly higher levels of TF than peripheral blood neutrophils. These results suggest that the alveolar microenvironment contributes to TF induction in ARDS. Additional experiments indicated that the ability of BALF to induce TF expression in neutrophils from healthy donors can be abolished by inhibiting C5a or TNF-α signaling, suggesting a primary role for these inflammatory mediators in the up-regulation of TF in alveolar neutrophils in ARDS. This cross-talk between inflammatory mediators and the induction of TF expression in intra-alveolar neutrophils may be a potential target for novel therapeutic strategies to limit ARDS-associated disturbances of coagulation.

Innate immune cells can develop long-term memory after stimulation by microbial products during infections or vaccinations. Here, we report that metabolic signals can induce trained immunity. Pharmacological and genetic experiments reveal that activation of the cholesterol synthesis pathway, but not the synthesis of cholesterol itself, is essential for training of myeloid cells. Rather, the metabolite mevalonate is the mediator of training via activation of IGF1-R and mTOR and subsequent histone modifications in inflammatory pathways. Statins, which block mevalonate generation, prevent trained immunity induction. Furthermore, monocytes of patients with hyper immunoglobulin D syndrome (HIDS), who are mevalonate kinase deficient and accumulate mevalonate, have a constitutive trained immunity phenotype at both immunological and epigenetic levels, which could explain the attacks of sterile inflammation that these patients experience. Unraveling the role of mevalonate in trained immunity contributes to our understanding of the pathophysiology of HIDS and identifies novel therapeutic targets for clinical conditions with excessive activation of trained immunity.