Sterile Peritonitis Research Articles

1438MicroRNA editing is integral for interleukin-6 trans-signalling and leukocyte trafficking to ischemic tissues

Abstract Background/Aim Adenosine to inosine RNA editing is an essential post-transcriptional RNA modification catalysed by adenosine deaminase acting on RNA-1 and -2 (ADAR1; ADAR2). Endothelial cells (ECs) attract and guide leukocytes to sites of ischemic tissue injury. Here we studied the role of RNA editing in ischemic disease. Methods Primary human and murine vascular endothelial cell cultures were used to assess the EC responses to interleukin-6 (IL-6) or ischemia. For the animal studies, the effect of ADAR2 in acute and chronic ischemic disease was evaluated in cremaster muscle microcirculation by intravital microscopy, in peritoneal cavity after sterile peritonitis and in gastrocnemius muscle after hind-limb ischemia by 8-colour flow cytometry and immunohistochemistry (IHC) studies of Adar2−/−/tg as well as of i(nducible)EC-ADAR2 knockout (KO) mice. For the mechanistic studies, deep RNA sequencing, qRT-PCR, western blot, confocal microscopy, target-specific microRNA (miRNA) editing studies, RNA-immunoprecipitation, miRNA/plasmid silencing/overexpression and luciferase reporter assays were used among others. For human studies, ischemic tissues derived from patients with acute or chronic ischemic heart disease were processed. Results ADAR2, but not ADAR1, expression is induced by >2-fold in hypoxic ECs and in ischemic vascular ECs in mice and humans. Unbiased gene ontology analysis of the EC transcriptome indicated that ADAR2 controls inflammatory responses and predominantly the expression of interleukin-6-signal transducer (IL6ST), the co-receptor of IL-6. Subsequently, ADAR2 controls IL-6 trans-signalling in ECs as documented by the STAT3 phosphorylation and expression of the downstream leukocyte adhesion molecules, E-selectin and VCAM-1. IL-6-inflamed cremaster muscles showed that rolling and adhesion of leukocyte subsets to vascular wall were severely impaired in Adar2−/−/tg mice. Leukocyte transmigration was also diminished by >2-fold in Adar2−/−/tg and in iEC-ADAR2 KO mice in response to IL-6 or ischemia. Similar results were obtained for leukocyte rolling, adhesion and infiltration after acute (4h) and chronic (3d; 21d) ischemia from iEC-ADAR2 KO mice and human ischemic muscle tissues. Next we studied how ADAR2 controls IL6ST expression. ADAR2-deficient vascular EC miRNAome revealed the upregulation of a conserved group of miRNAs targeting the IL6ST mRNA including miR-199a-5p and miR-335-3p. At a single-nucleotide level, ADAR2-induced RNA editing of the stem loops of the primary miR-199a1/2 and miR-335 directly disrupted Drosha recruitment to both and thus inhibited their maturation process. Accordingly, rescue experiments using miRNA-inhibitors restored IL6ST levels after ADAR2 deficiency. Conclusion Taking together, inhibition of the microRNA maturation process by ADAR2-mediated RNA editing is integral for IL-6 trans-signalling in vascular endothelium and subsequent leukocyte trafficking to ischemic tissues in mice and humans.

34: Hematopoietic Stem/Progenitor Cell Dependent Participation of Innate Lymphoid Cells in Low-Intensity Sterile Inflammation

Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. They act as early orchestrators of immunity and as such they make up interesting therapeutic targets for several diseases and possibly a means to peripheral tolerance. Taking advantage of an animal model of sterile peritonitis, induced by intraperitoneal injection of zymosan, a fungal derived TLR-2 ligand we studied the progenitor–successor relationship between the hematopoietic stem/progenitor cells (HSPC) and ILCs at the site of sterile inflammation. Our results demonstrate that the strength of the inflammatory signal affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections triggered the appearance of mature ILCs in the peritoneal cavity. Only in low-dose injected mice, the recovered ILCs seemed to be dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose, the stronger inflammatory environment seemed to be able to induce the emergence of ILCs independently of BM-derived HSPCs. To further elucidate the importance of the inflammatory environment per se for the attraction of mature ILCs, an anti- inflammatory drug was used. Treatment of mice with alpha-1 anti-trypsin (AAT) 15 min after injection of either low- or high- dose of zymosan, led to the blockade of the migration of HSPCs into the peritoneal cavity resulting in a loss of even mature ILCs in mice injected with the low-dose of zymosan. Contrary, in mice injected with high-dose, progenitor cells of type 2 of ILCs could be still recovered, but not mature ILC2 cells. We suggest that the relationship between HSPCs and ILCs seem to be affected by the strength of the inflammatory stimuli and that the use of anti-inflammatory molecules like A1 antitrypsin may attenuate the inflammation and to some extent prevent the migration of HSCPs to the site of inflammation and the emergence of mature ILCs.

Platelet PI3K Modulates Innate Leukocyte Extravasation during Acid-Induced Acute Lung Inflammation.

Blood platelets are increasingly recognized as modulators of leukocyte effector functions in various pathologies including acute lung injury (ALI). ALI is a life-threatening disease, caused by damage to the alveolar epi- and endothelium. Excessive accumulation of leukocytes leads to severe lung inflammation, resulting in impaired lung function and hypoxemia. Since leukocyte migration is modulated by activated platelets and phosphatidylinositol 3-kinase (PI3K) signaling is involved in platelet function, we aimed to elucidate the effect of PI3K on platelet-mediated immune responses. We generated a mouse model with a platelet-specific deletion of p85α, the most important regulatory subunit of the class IA PI3K, and evaluated platelet function and platelet-leukocyte interactions. Moreover, we analyzed the impact of platelet-specific p85α gene deficiency during sterile peritonitis and acid-induced ALI. In vitro analyses of platelets revealed that lack of p85α led to decreased downstream signaling and diminished expression of surface activation markers, for example, CD62P and CD63, as well as reduced platelet aggregation. Moreover, platelet PI3K essentially mediated direct interactions of platelets with monocytes and neutrophils. In mice, platelet-specific p85α deficiency prevented leukocyte infiltration into the peritoneum and the bronchoalveolar compartment during sterile peritonitis and ALI, respectively. Additionally, the release of the inflammatory cytokine interleukin-12/23 was diminished in platelet p85α-deficient mice during ALI. In contrast to PI3K, neither overexpression nor depletion of platelet phosphatase and tensin homolog, the endogenous antagonist of PI3K, significantly modulated platelet function. Our data indicate a crucial role of platelet PI3K signaling for leukocyte extravasation upon inflammatory stimuli in various diseases models.

Differential attenuation of β2 integrin–dependent and –independent neutrophil migration by Ly6G ligation

AbstractAntibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on β2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally. By contrast, Ly6G ligation had no impact on integrin-independent neutrophil migration into inflamed lung. In peritoneum, the role of β2 integrins varied with stimulus, proving dispensable for neutrophil entry in Escherichia coli peritonitis but contributory in interleukin 1 (IL-1)–mediated sterile peritonitis. Correspondingly, Ly6G ligation attenuated only IL-1 peritonitis, disrupting the molecular association between integrins and Ly6G and inducing cell-intrinsic blockade restricted to integrin-dependent migration. Consistent with this observation, Ly6G ligation impaired integrin-mediated postadhesion strengthening for neutrophils arresting on activated cremaster endothelium in vivo. Together, these findings identify selective inhibition of integrin-mediated neutrophil emigration through Ly6G ligation, highlighting the marked site and stimulus specificity of β2 integrin dependence in neutrophil migration.

An unusual presentation of a known condition: Q fever manifesting as an acute abdomen

Q fever is an endemic zoonotic infection in Australia cause by Coxiella burnetii. It has been recognised in other parts of the world, especially among livestock rearing occupations, stock yard and abattoir workers. Majority (65%) of patients infected with C.burnetti are asymptomatic while symptoms similar to those of respiratory and hepatitis are the most common making diagnosis difficult in the early stages. We report a case of a young man who was exposed to and infected with Q fever as an occupational hazard. He presented in an unusual way with the predominant initial symptoms of abdominal pain, fever, hepatitis and sterile peritonitis necessitating an emergency surgical procedure to explore a suspected surgical abdomen. Respiratory involvement ensued only several days later. The diagnosis of Q fever was confirmed with positive convalescent serology phase II IgM and IgG antibodies to Coxiella burnetii. A marked clinical response to doxycycline pending serological confirmation was supportive of this highly suspected diagnosis in an at-risk patient.

LXR Suppresses Inflammatory Gene Expression and Neutrophil Migration through cis-Repression and Cholesterol Efflux

SUMMARYThe activation of liver X receptor (LXR) promotes cholesterol efflux and repression of inflammatory genes with anti-atherogenic consequences. The mechanisms underlying the repressive activity of LXR are controversial and have been attributed to cholesterol efflux or to transrepression of activator protein-1 (AP-1) activity. Here, we find that cholesterol efflux contributes to LXR repression, while the direct repressive functions of LXR also play a key role but are independent of AP-1. We use assay for transposase-accessible chromatin using sequencing (ATAC-seq) to show that LXR reduces chromatin accessibility in cis at inflammatory gene enhancers containing LXR binding sites. Targets of this repressive activity are associated with leukocyte adhesion and neutrophil migration, and LXR agonist treatment suppresses neutrophil recruitment in a mouse model of sterile peritonitis. These studies suggest a model of repression in which liganded LXR binds in cis to canonical nuclear receptor binding sites and represses pro-atherogenic leukocyte functions in tandem with the induction of LXR targets mediating cholesterol efflux.

Plasmin-Mediated Fibrinolysis Enables Macrophage Migration Via Liberation from Fibrin-αMβ2 Interactions

Mounting evidence ties both fibrin(ogen) and plasmin(ogen) to inflammatory diseases. Indeed, both fibrin(ogen) and plasmin(ogen) have been linked to critical macrophage functions in multiple disease processes. Migration of macrophages to sites of sterile inflammation is, at least partially, dependent on plasmin(ogen). Mice lacking plasminogen, when challenged with sterile thioglycollate-induced peritonitis, have both diminished overall leukocyte migration and decreased macrophage migration. Additionally, macrophage migration defects have been identified in both mice lacking plasminogen and plasminogen receptors. Plasmin has many targets that may play a role in supporting macrophage migration. In addition to proteolysis of fibrin(ogen), plasmin activates matrix metalloprotease (MMP) 2 and MMP9 and cleaves collagen and laminin. Indeed, mice that lack MMP9 have a migration defect similar to mice that lack plasminogen, suggesting that MMP9 is a biologically relevant proteolytic target in this context. To further examine the targets of plasmin that regulate macrophage migration, we challenged animals that have individual and combined genetic deficiencies in fibrinogen and plasminogen with thioglycollate-induced peritonitis. We have found that mice that lack fibrinogen alone have a significantly increased migration of macrophages to the peritoneal cavity. Mice that lack plasminogen alone demonstrated the expected diminution in macrophage migration to the peritoneal cavity. However, mice that were deficient in both plasminogen and fibrinogen demonstrated macrophage migration that was indistinguishable from wildtype. These data suggest that fibrin(ogen) impedes macrophage migration to the peritoneal cavity. To further confirm this mechanism, we examined macrophage migration in a transwell assay in vitro. Here, a macrophage cell line (RAW 264.7 or BMDM) migration was examined in the absence and presence of fibrin matrices. Macrophages, in the presence of plasminogen, did demonstrate a modest, but statistically significant, increase in migration across the transwell membrane in the absence of fibrinogen. When a fibrin matrix was generated on the transwell membrane, macrophages were essentially unable to cross in the absence of plasminogen. These data further support the concept that macrophages require plasmin(ogen) to cross fibrin matrices. We further sought to determine if the fibrin-αMβ2 interaction was implicated in the macrophage migration phenotype. To do this, we first examined macrophage migration in vivo in mice expressing a form of fibrinogen that cannot interact with αMβ2, Fibγ390-396A mice. Similar to mice lacking fibrinogen, an increase in peritoneal macrophages was observed at 72 hours following a challenge with 4% thioglycollate. To confirm that this was related to the fibrin-αMβ2 interaction, and not due to abnormal factor XIII crosslinking, factor XIII deficient animals were also challenged with thioglycollate induced peritonitis. Mice lacking factor XIII exhibited no difference from wildtype in this model of peritonitis. We further confirmed in the in vitro transwell migration assay that macrophages were able to cross a fibrin barrier, derived from Fibγ390-396A mice, in the absence of plasminogen. Taken together, these data suggest that plasmin allows macrophage migration via liberation from the fibrin-αMβ2 interaction. DisclosuresMullins:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees.

MiR-125b regulates chemotaxis and survival of bone marrow derived granulocytes in vitro and in vivo.

The evolutionary conserved miR-125b is highly expressed in hematopoietic stem cells (HSC) enhancing self-renewal and survival. Accordingly, over-expression of miR-125b in HSC may induce myeloproliferative neoplasms and leukemia with long latency. During hematopoietic cell maturation miR-125b expression decreases, and the function of miR-125b in mature granulocytes is not yet known. We here use transplantation of miR-125b over-expressing HSC into syngeneic hosts to generate and analyse miR-125b over-expressing granulocytes. Under steady state conditions, miR-125b over-expression inhibits granulocytic chemotaxis and LPS- but not PMA- and TNFα- induced cell death. Inflammatory signals modulate the effects of miR-125b over-expression as demonstrated in a sterile peritonitis and a polymicrobial sepsis model. In particular, survival of mice with miR-125b over-expressing granulocytes is significantly reduced as compared to controls in the polymicrobial sepsis model. These data demonstrate inflammation dependent effects of miR-125b in granulocytes and may point to therapeutic intervention strategies in the future.

Post natal rupture of meconium pseudocyst in a neonate with delayed diagnosis

Meconium peritonitis is a rare fetal disease with an increased rate of morbidity and mortality in the neonatal period. Meconium pseudocyst is a complication of meconium peritonitis which is a sterile chemical peritonitis due to intrauterine bowel perforation. When the perforation in the intestine does not heal and communication with the cyst persist postnatal that can lead to cyst expansion, infection of the cyst or rupture of pseudocyst. This is a case report of a neonate with rupture of meconium pseudocyst causing perforation peritonitis. There was a delay in diagnosis in our case, but was successfully managed with staged enterostomy.

Prominent Levels of the Profibrotic Chemokine CCL18 during Peritonitis: In Vitro Downregulation by Vitamin D Receptor Agonists.

Peritoneal dialysis (PD) is used as a renal replacement therapy, which can be limited by peritoneal membrane ultrafiltration failure (UFF) secondary to fibrotic processes. Peritonitis, a frequent complication of PD, is a major risk factor for peritoneal membrane fibrosis and UFF. Low peritoneal levels of the chemokine CCL18 are associated with preservation of peritoneal membrane function in PD. Given that CCL18 is involved in fibrotic processes and recurrent peritonitis, it is a risk factor for peritoneal membrane failure; thus, we evaluated CCL18 concentrations in peritoneal effluents from patients undergoing peritonitis episodes. Pharmacological interventions aimed at diminishing the production of CCL18 were also explored. Fivefold higher CCL18 peritoneal concentrations were found during acute bacterial peritonitis, in parallel with the increased infiltration of macrophages. Unexpectedly, CCL18 was also highly (50-fold) increased during sterile eosinophilic peritonitis, and peritoneal eosinophils were found to express CCL18. In vitro treatment of peritoneal macrophages with the vitamin D receptor agonist paricalcitol was able to reduce the secretion and the expression of CCL18 in isolated peritoneal macrophages. In conclusion, our study suggests that the chemokine CCL18 can be a mediator of peritoneal membrane failure associated with peritonitis episodes as well as providing a new potential therapeutic target.

Mono Sodium Urate Crystal-induced Peritonitis for in vivoAssessment of Inflammasome Activation.

Due to its particulate material, mono-sodium urate (MSU) crystals are potent activators of the NOD-like receptor NLRP3. Upon activation, NLRP3 induces the formation of inflammasome complexes, which lead to the production and release of mature IL-1β. Bioactive IL-1β is a potent activator of innate immune responses and promotes recruitment of inflammatory cells, including neutrophils from the blood into damaged/inflamed tissues. This protocol describes a method to study in vivo inflammasome activation via intraperitoneal injection of MSU crystals. MSU-injection results in a drastic increase of intraperitoneal IL-1β levels, promoting neutrophil infiltration. Early-stage neutrophil numbers correlate with the amount of released IL-1β and can be used as a read-out for the extent of in vivo inflammasome activation. In addition, this protocol might also be used as a sterile peritonitis model, to investigate mechanisms of neutrophil recruitment to the peritoneum, or as a means to obtain large numbers of in vivo activated neutrophils.

Resolution of inflammation and sepsis survival are improved by dietary \u03a9-3 fatty acids

Critical conditions such as sepsis following infection or traumatic injury disturb the complex state of homeostasis that may lead to uncontrolled inflammation resulting in organ failure, shock and death. They are associated with endogenous mediators that control the onset of acute inflammatory response, but the central problem remains the complete resolution of inflammation. Omega-3 enriched lipid emulsions (Ω-3+ LEs) were used in experimental studies and clinical trials to establish homeostasis, yet with little understanding about their role on the resolution of inflammation and tissue regeneration. Here, we demonstrate that Ω-3 lipid emulsions (LEs) orchestrate inflammation-resolution/regeneration mechanism during sterile peritonitis and murine polymicrobial sepsis. Ω-3+ LEs recessed neutrophil infiltration, reduced pro-inflammatory mediators, reduced the classical monocyte and enhanced the non-classical monocytes/macrophages recruitment and finally increased the efferocytosis in sepsis. The actions of Ω-3+ LE were 5-lipoxygenase (5-LOX) and 12/15-lipoxygenase (12/15-LOX) dependent. Ω-3+ LEs shortened the resolution interval by 56%, stimulated the endogenous biosynthesis of resolution mediators lipoxin A4, protectin DX and maresin 1 and contributed to tissue regeneration. Ω-3+ LEs protected against hypothermia and weight loss and enhanced survival in murine polymicrobial sepsis. We highlighted a role of Ω-3+ LEs in regulating key mechanisms within the resolution terrain during murine sepsis. This might form the basis for a rational design of sepsis specific clinical nutrition.

Progressive Fibrosis: A Progesterone- and KLF11-Mediated Sexually Dimorphic Female Response.

Progressive scarring is ubiquitous postoperatively and in an array of chronic systemic diseases. Recent studies indicate that such scarring has a high female propensity; females are also almost exclusively affected by endometriosis, a common sex steroid-dependent fibrotic disease. Endometriosis-related fibrosis is regulated epigenetically through transcription factor Krüppel-like factor 11 (KLF11). In response to surgical induction of endometriosis, Klf11-/- female mice develop significant fibrosis in contrast to wild-type mice. We therefore hypothesized that female fibrotic predilection was mediated by differential sex steroid regulation of KLF11/collagen 1a1 signaling and investigated the fibrotic response in wild-type and Klf11-/- male and female animals using a sterile peritonitis model. Fibrosis selectively developed in Klf11-/- females. Fibrosis in these animals was almost completely abrogated by ovariectomy. Ovariectomized animals were selectively supplemented with estradiol, medroxyprogesterone acetate (MPA), or dihydrotestosterone; fibrosis was only observed in mice exposed to MPA. Fibrosis therefore selectively developed in Klf11-/- female mice in response to physiological or pharmacological progesterone. The fibrotic response in these animals was also mitigated in response to antiprogestin therapy. Profibrotic gene expression was activated in a primary human peritoneal cell line in response to KLF11 short hairpin RNA and MPA but not estradiol. KLF11/collagen 1a1 signaling previously shown to be linked to fibrosis was thus selectively dysregulated in MPA-treated cells. Our in vivo and in vitro findings in an animal model and human cells, respectively, suggest that progressive fibrotic scarring is a sexually dimorphic response irrespective of etiology; moreover, it is responsive to novel, individualized therapeutic intervention.

The haptoglobin beta subunit sequesters HMGB1 toxicity in sterile and infectious inflammation.

Extra-corpuscular haemoglobin is an endogenous factor enhancing inflammatory tissue damage, a process counteracted by the haemoglobin-binding plasma protein haptoglobin composed of alpha and beta subunits connected by disulfide bridges. Recent studies established that haptoglobin also binds and sequesters another pro-inflammatory mediator, HMGB1, via triggering CD163 receptor-mediated anti-inflammatory responses involving heme oxygenase-1 expression and IL-10 release. The molecular mechanism underlying haptoglobin-HMGB1 interaction remains poorly elucidated. Haptoglobin β subunits were tested for HMGB1-binding properties, as well as efficacy in animal models of sterile liver injury (induced by intraperitoneal acetaminophen administration) or infectious peritonitis (induced by cecal ligation and puncture, CLP, surgery) using wild-type (C57BL/6) or haptoglobin gene-deficient mice. Structural-functional analysis demonstrated that the haptoglobin β subunit recapitulates the HMGB1-binding properties of full-length haptoglobin. Similar to HMGB1-haptoglobin complexes, the HMGB1-haptoglobin β complexes also elicited anti-inflammatory effects via CD163-mediated IL-10 release and heme oxygenase-1 expression. Treatment with haptoglobin β protein conferred significant protection in mouse models of polymicrobial sepsis as well as acetaminophen-induced liver injury, two HMGB1-dependent inflammatory conditions. Haptoglobin β protein offers a novel therapeutic approach to fight against various inflammatory diseases caused by excessive HMGB1 release.

Intestinal goblet cell carcinoid presenting with recurrent sterile peritonitis in a patient on peritoneal dialysis: a case report

BackgroundGoblet cell carcinoid is a rare variant of appendiceal carcinoid with mixed endocrine and exocrine features. The most common symptom and signs are abdominal pain, acute appendicitis and palpable mass. Additionally, abdominal pain is common in patient on peritoneal dialysis, which may confound the diagnosis in such patient.Case presentationWe report a 71- years- old woman on peritoneal dialysis that experienced several episodes of abdominal cramping pain and sterile peritonitis. She had one episode of severe pain and underwent an appendectomy for suspicion of appendicitis. Goblet cell carcinoid was diagnosed. She had no further abdominal pain after she received appendectomy.ConclusionsMalignant dialysate was rarely reported in patient with peritoneal dialysis. However, goblet cell carcinoid can initially present with acute appendicitis, chronic intermittent abdominal pain and mimicking peritonitis. In systemically reviewing the literature, this is the first case report of sterile peritonitis with peritoneal dialysis caused by goblet cell carcinoid.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activationof CD47. The AMD-associated CFH(H402) variantmarkedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Use of Toll-like receptor assays for the detection of bacterial contaminations in icodextrin batches released for peritoneal dialysis.

Icodextrin is a starch derivative used for preparing solutions of peritoneal dialysis. Unfortunately, peptidoglycans (PGN) and lipopolysaccharides (LPS) have been reported to contaminate certain icodextrin batches and to contribute to the development of sterile peritonitis. The decision of selecting or rejecting icodextrin batches is however difficult, because of limitations in the detection of these bacterial contaminants. Besides monocyte activation tests of cytokine release, a number of bio-assays using stably TLR-transfected cell lines have been developed. Here, we compared the efficacy of TLR2- and TLR4-transfected cells to detect bacterial contamination with the responses of monocytes exposed to the same icodextrin samples. In contrast to monocyte models of cytokine release, we found that TLR2- and TLR4-transfected cell lines are highly sensitive to detect little PGN and LPS contaminations in the presence of icodextrin. With the intent to increase PGN reactivity, mutanolysin was used to generate soluble fragments in icodextrin samples. We found that such an enzymatic treatment led to an enhanced response of TLR2-transfected cells, even though parental icodextrin samples were poorly reactive. Altogether, these findings indicate that the use of TLR2- and TLR4-transfected cell lines is a valuable approach for helping to the decision of selecting icodextrin batches for peritoneal dialysis.

Plasmin(ogen) Mediates Macrophage Migration in a Fibrin(ogen) Dependent Mechanism

Mounting evidence ties both fibrin(ogen) and plasmin(ogen) to inflammatory diseases. Indeed, both fibrin(ogen) and plasmin(ogen) have been linked to critical macrophage functions in multiple disease processes. Migration of macrophages to sites of sterile inflammation is, at least partially, dependent on plasmin(ogen). Mice lacking plasminogen, when challenged with sterile thioglycollate-induced peritonitis, have both diminished overall leukocyte migration and decreased macrophage migration. Additionally, macrophage migration defects have been identified in both mice lacking plasminogen and plasminogen receptors. Plasmin has many targets that may play a role in supporting macrophage migration. In addition to proteolysis of fibrin(ogen), plasmin activates matrix metalloprotease (MMP) 2 and MMP9 and cleaves collagen and laminin. Indeed, mice that lack MMP9 have a migration defect similar to mice that lack plasminogen, suggesting that MMP9 is a biologically relevant proteolytic target in this context. To further examine the targets of plasmin that regulate macrophage migration, we challenged animals that have individual and combined genetic deficiencies in fibrinogen and plasminogen with thioglycollate-induced peritonitis. We have found that mice that lack fibrinogen alone have a significantly increased migration of macrophages to the peritoneal cavity. Mice with lack of plasminogen alone demonstrated the expected diminution in macrophage migration to the peritoneal cavity. However, mice that were deficient in both plasminogen and fibrinogen demonstrated macrophage migration that was indistinguishable from wildtype. These data suggest that fibrin(ogen) impedes macrophage migration to the peritoneal cavity. To further confirm this mechanism, we examined macrophage migration in a transwell assay in vitro, in response to macrophage chemoattractant protein-1 (MCP-1). Here, a macrophage cell line (RAW 264.7) migration was examined in the absence and presence of fibrin matrices. Macrophages, in the absence of plasminogen, did demonstrate a modest, but statistically significant, increase in migration across the transwell membrane in the absence of fibrinogen. When a fibrin matrix was generated on the transwell membrane, macrophages were essentially unable to cross in the absence of plasminogen. These data further support the concept that macrophages require plasmin(ogen) to cross fibrin matrices. To further explore the plasmin(ogen)-fibrin(ogen) interaction in macrophage migration, we assessed the migration of macrophages to fibrin degradation products (FDPs). First, we examined macrophage transwell migration in response to MCP-1 in the presence of FDPs to assess if FDPs impede macrophage migration. Instead of impeding macrophage migration, FDPs significantly increased macrophage migration across the transwell membrane. Indeed FDPs initiated macrophage migration even in the absence of MCP-1. To confirm that this was FDP induced migration, and not direct plasmin signaling on macrophages, we examined macrophage migration to FDPs in the presence of an irreversible plasmin inhibitor. We again found that plasmin degradation of fibrin was needed for migration, however, further plasmin activity was not required. Taken together, these data suggest that macrophages require plasmin(ogen) to navigate fibrin matrices and that the by-product of this degradation (FDPs) is a signal for additional macrophage migration to sites of fibrin deposition. DisclosuresMullins:Baxalta (now part of Shire): Honoraria; US WorldMeds: Membership on an entity's Board of Directors or advisory committees.

Extracellular ATP Induces Vascular Inflammation and Atherosclerosis via Purinergic Receptor Y2 in Mice.

A solid body of evidence supports a role of extracellular ATP and its P2 receptors in innate and adaptive immunity. It promotes inflammation as a danger signal in various chronic inflammatory diseases. Thus, we hypothesize contribution of extracellular ATP and its receptor P2Y2 in vascular inflammation and atherosclerosis. Extracellular ATP induced leukocyte rolling, adhesion, and migration in vivo as assessed by intravital microscopy and in sterile peritonitis. To test the role of extracellular ATP in atherosclerosis, ATP or saline as control was injected intraperitoneally 3× a week in low-density lipoprotein receptor(-/-) mice consuming high cholesterol diet. Atherosclerosis significantly increased after 16 weeks in ATP-treated mice (n=13; control group, 0.26 mm2; ATP group, 0.33 mm2; P=0.01). To gain into the role of ATP-receptor P2Y2 in ATP-induced leukocyte recruitment, ATP was administered systemically in P2Y2-deficient or P2Y2-competent mice. In P2Y2-deficient mice, the ATP-induced leukocyte adhesion was significantly reduced as assessed by intravital microscopy. P2Y2 expression in atherosclerosis was measured by real-time polymerase chain reaction and immunohistochemistry and demonstrates an increased expression mainly caused by influx of P2Y2-expressing macrophages. To investigate the functional role of P2Y2 in atherogenesis, P2Y2-deficient low-density lipoprotein receptor(-/-) mice consumed high cholesterol diet. After 16 weeks, P2Y2-deficient mice showed significantly reduced atherosclerotic lesions with decreased macrophages compared with P2Y2-competent mice (n=11; aortic arch: control group, 0.25 mm(2); P2Y2-deficient, 0.14 mm2; P=0.04). Mechanistically, atherosclerotic lesions from P2Y2-deficient mice expressed less vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 RNA. We show that extracellular ATP induces vascular inflammation and atherosclerosis via activation of P2Y2.

MerTK cleavage limits proresolving mediator biosynthesis and exacerbates tissue inflammation

The acute inflammatory response requires a coordinated resolution program to prevent excessive inflammation, repair collateral damage, and restore tissue homeostasis, and failure of this response contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated in part by long-chain fatty acid-derived lipid mediators called specialized proresolving mediators (SPMs). However, how SPMs are regulated during the inflammatory response, and how this process goes awry in inflammatory diseases, are poorly understood. We now show that signaling through the Mer proto-oncogene tyrosine kinase (MerTK) receptor in cultured macrophages and in sterile inflammation in vivo promotes SPM biosynthesis by a mechanism involving an increase in the cytoplasmic:nuclear ratio of a key SPM biosynthetic enzyme, 5-lipoxygenase. This action of MerTK is linked to the resolution of sterile peritonitis and, after ischemia-reperfusion (I/R) injury, to increased circulating SPMs and decreased remote organ inflammation. MerTK is susceptible to ADAM metallopeptidase domain 17 (ADAM17)-mediated cell-surface cleavage under inflammatory conditions, but the functional significance is not known. We show here that SPM biosynthesis is increased and inflammation resolution is improved in a new mouse model in which endogenous MerTK was replaced with a genetically engineered variant that is cleavage-resistant (Mertk(CR)). Mertk(CR) mice also have increased circulating levels of SPMs and less lung injury after I/R. Thus, MerTK cleavage during inflammation limits SPM biosynthesis and the resolution response. These findings contribute to our understanding of how SPM synthesis is regulated during the inflammatory response and suggest new therapeutic avenues to boost resolution in settings where defective resolution promotes disease progression.