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Objectives We investigate the possibility to exploit high-field MRI to acquire 3D images of Purkinje network which plays a crucial role in cardiac function. Since Purkinje fibers (PF) have a distinct cellular structure and are surrounded by connective tissue, we investigated conventional contrast mechanisms along with the magnetization transfer (MT) imaging technique to improve image contrast between ventricular structures of differing macromolecular content. Methods Three fixed porcine ventricular samples were used with free-running PFs on the endocardium. T1, T2*, T2, and M0 were evaluated on 2D slices for each sample at 9.4 T. MT parameters were optimized using hard pulses with different amplitudes, offset frequencies and durations. The cardiac structure was assessed through 2D and 3D T1w images with isotropic resolutions of 150 µm. Histology, immunofluorescence, and qPCR were performed to analyze collagen contents of cardiac tissue and PF. Results An MT preparation module of 350 ms duration inserted into the sequence with a B1 = 10 µT and frequency offset = 3000 Hz showed the best contrast, approximately 0.4 between PFs and myocardium. Magnetization transfer ratio (MTR) appeared higher in the cardiac tissue (MTR = 44.7 ± 3.5%) than in the PFs (MTR = 25.2 ± 6.3%). Discussion MT significantly improves contrast between PFs and ventricular myocardium and appears promising for imaging the 3D architecture of the Purkinje network.

Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4−/− but not in trpv4−/− ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4+/+ but not in trpv4−/− myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

Background Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder involving first and second branchial arches derivatives, mainly characterised by asymmetric ear anomalies, hemifacial microsomia, ocular defects and vertebral malformations. Although numerous chromosomal abnormalities have been associated with OAVS, no causative gene has been identified so far. Objectives We aimed to identify the first causative gene for OAVS. Methods As sporadic cases are mostly described in Goldenhar syndrome, we have performed whole exome sequencing (WES) on selected affected individuals and their unaffected parents, looking for de novo mutations. Candidate gene was tested through transient knockdown experiment in zebrafish using a morpholino-based approach. A functional test was developed in cell culture in order to assess deleterious consequences of mutations. Results By WES, we identified a heterozygous nonsense mutation in one patient in the myelin transcription factor 1 ( MYT1 ) gene. Further, we detected one heterozygous missense mutation in another patient among a cohort of 169 patients with OAVS. This gene encodes the MYT1 . Functional studies by transient knockdown of myt1a , homologue of MYT1 in zebrafish, led to specific craniofacial cartilage alterations. Treatment with all-trans retinoic acid (RA), a known teratogenic agent causing OAVS, led to an upregulation of cellular endogenous MYT1 expression. Additionally, cellular wild-type MYT1 overexpression induced a downregulation of RA receptor β ( RARB) , whereas mutated MYT1 did not. Conclusion We report MYT1 as the first gene implicated in OAVS, within the RA signalling pathway.

Background: A large Dispersion of Repolarization (DoR) is associated with an increased arrhythmogenic risk. This can be measured clinically by calculating the Activation Recovery Interval (ARI) to estimate Action Potential Duration (APD). However, the ability of ARI to accurately predict APD dispersion in patients with repolarization abnormality has not been determined. Objective: Compare ARI calculated from patients with optical mapping of human hearts to establish the validity of ARI as a surrogate for APD. Methods: Optical mapping (OM) was performed on the left ventricles of 4 explanted human hearts. APD and repolarization times were measured endo- and epicardially on the anterior of the LV. Electroanatomic mapping was performed with CARTO over the entire endo- and epicardial surfaces of 3 patients. Activation and repolarization were calculated, dispersion of ARI was measured. Results: APD and ARI were consistent between mapping methods over most of the sub-regions studied. Epicardium ARI dispersion was consistently higher than that of the endocardium in both OM and CARTO datasets. Conclusion: APD distribution, and consequently DoR, agree between mapping methods. Measuring DoR by ARI accurately assesses the underlying repolarization abnormalities in patients.

Introduction Transient Receptor Potential Vanilloid 4 (TRPV4) channels, belonging to the TRP channels superfamily, are Ca2+-permeable channels activated by hypo-osmotic stress. In the heart, TRPV4 channels are expressed at the protein level in mouse and rat ventricular myocytes and fibroblasts. They are involved in valve development during embryogenesis, in ischemia-reperfusion injuries and in reactive fibrosis. Whether TRPV4 channel can participate in ventricular action potential (AP) is still unknown. Objective The aim of this study is to pharmacologically unmask the contribution of TRPV4 channels in left ventricular AP. Methods Left Ventricular myocytes were isolated from TRPV4 + / + and TRPV4 − / − mice and AP were recorded using the patch-clamp technique. Pharmacological activator (GSK1016790A) and inhibitors (HC-067047 and GSK2193874) were perfused during the recordings to evaluate the effect on AP parameters. Results TRPV4 activator GSK1016790A induces a transient and dose dependent increase in AP duration at 90% of repolarization (APD90) in TRPV4 + / + but not in TRPV4 − / − myocytes (+ 19.4 ± 3.8%; n = 11 vs − 0.6 ± 1.8%; n = 7 at 500 nM respectively). The activator mediated-APD increase is abolished by TRPV4 channels inhibitors and CamKII inhibitor KN93 at 1 μM. Conversely, TRPV4 channel inhibitor (GSK2193874) at 100 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 7.8 ± 2.6%; n = 11 vs 1.0 ± 1.3%; n = 7 respectively). Similarly, HC-067047 at 300 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 10.9 ± 5.0%; n = 6 vs − 1.2 ± 2.4%;n = 4 respectively). At the concentrations mentioned above, activator and inhibitors have no effect on the other AP parameters. Conclusion This study shows that TRPV4 channels exert a basal contribution in ventricular AP and could affect repolarization. It is thus a potential target for pharmacological approaches against ventricular arrhythmias.

Introduction The RVOT is involved in arrhythmias in several diseases such as the Brugada syndrome (BrS). In a subpopulation of patients with BrS and early repolarization (ER) syndrome, a K-ATP gain-of-function (GOF) mutation has previously been described and was suggested to contribute to arrhythmogenicity in this context. Objective To determine the role of regional K-ATP channels expression on RV repolarization heterogeneity and in arrhythmia occurrence and modulation. Methods Pig right ventricles (RV) were perfused by both right and left coronary arteries, paced at 1 or 1.5 Hz and the electrical activity optically-mapped (di-4-ANEPPS, 20 μM). The preparation was perfused with pinacidil (PINA, 20 μM), and diazoxide (DIAZ, 100 μM), and subjected to 20 min no-flow (NF) ischemia episodes followed by 20 min reperfusion (RF). Arrhythmia susceptibility was assessed using a 20 Hz burst protocol while spontaneous arrhythmias were also recorded. Protein expression was assessed by western blot. Results PINA-induced action potential duration (APD) shortening was larger in RVOT than in the free wall (FW). DIAZ induced a slight but significant reduction in APD across the RV. SUR2A expression was greater in the RVOT epicardium vs. FW but Kir6.1/2 and SUR1 expression was similar in both regions. PINA induced spontaneous ventricular fibrillation (VF) in 80% of the preparations with higher dominant frequency (DF) and regularity index in the RVOT than the FW. DF of burst-induced VF were higher in PINA but not in DIAZ compared to CTRL VF. Similarly to PINA, NF-induced APD shortening was greater in the RVOT than in the FW. No spontaneous arrhythmia occurred during NF but slow ventricular tachycardias arose in 36% of the preparations during RF. Conclusion K-ATP activation leads to an increase in RV repolarization dispersion which is likely to contribute to arrhythmogenicity in BrS or ER patients with a K-ATP channel GOF mutation and may underlie BrS phenocopy induced by RV ischemia.

Introduction We recently found the presence of a pro-arrhythmic electrophysiological substrate with increased collagen content and slow conduction in the healthy right ventricular outflow tract (RVOT). An epicardial layer of fat is commonly found in the RVOT but whether fat penetrates in the myocardium and regulates RVOT structure and electrophysiology remains unknown. Objective To assess the presence and localization of fat infiltrations (FI) in the healthy RVOT. Methods The presence of FI was assessed histologically in Masson's trichrome and Oil-Red-O stained sections from young healthy pigs (n = 4) and human donor hearts with no cardiac disease history (n = 5). High resolution MRI (9.4 T Bruker Biospin) was used to localize fat in formaldehyde-fixed human RVOTs. The expression of selected targets was assessed by qPCR and Western Blot in the subepicardial layer (EPI) of the RV free wall (RVFW) and RVOT of healthy pigs. Results Sporadic FIs could be observed in the pig RVOT but were absent in the RVFW. In human hearts, FIs were present in the RVFW, LV free wall but were larger in the RVOT. High resolution MRI showed the presence of 3 layers in the human RVOT wall with a thick epicardial fat layer, a transition layer composed of myocardial fibers and fat (up to 50% of wall thickness) and a muscular endocardial layer. Intramyocardial fat content increased with age. The mRNA expression of perilipin (PLIN1) and the adipogenic hormone adiponectin (ADIPOQ) was significantly higher in the RVOT EPI compared to the RVFW (P Conclusion FIs are in direct contact with the adjacent myocardium in the healthy porcine and human RVOT. Activin-A is likely to contribute to the higher collagen content found in the healthy RVOT as observed in the atria. Thus, these infiltrations are likely to participate to RVOT slower conduction and arrhythmogenesis.

Introduction The right ventricular outflow tract (RVOT) has a distinct embryonic development leading to specific electrophysiological properties in the healthy adult heart. These include shorter action potential duration (APD), which is likely to contribute to RVOT high susceptibility to arrhythmias in idiopathic and pathological conditions. How these differences are altered in ischemia remains unknown. Objective We sought to determine whether RVOT repolarization undergoes a specific adaptation to ischemia. Methods Pig right ventricles (RV) were perfused by both right and left anterior descending coronary arteries, paced at 1.5 Hz and the electrical activity optically-mapped on epicardial (EPI) and endocardial (ENDO) surfaces (di-4-ANEPPS, 20 μM). The preparation was perfused with pinacidil (PINA, 20 μM), diazoxide (DIAZ, 100 μM) and glibenclamide (GLIB, 10 μM), and subjected to 20 min no-flow ischemia episodes followed by 20 min reperfusion. Protein expression was assessed by western blot and localization by immunohistochemistry. Results Ischemia significantly shortened APD across the whole wedge, with a larger effect in RVOT than RVFW EPI (−50.94% ± 9.58% vs. −36.18% ± 8.38% P Conclusion RVOT repolarization seems to be more sensitive to ischemia than the RVFW. Katp channels are involved in this difference despite similar Kir6.1 and Kir6.2 expression, suggesting a role for sulphonylurea receptors. This increase in repolarization heterogeneity is likely to facilitate re-entrant arrhythmias in this context.

Introduction The right ventricular outflow tract (RVOT) is the main origin of idiopathic ventricular tachycardia (VT). Although these arrhythmias appear to be cAMP-dependent, the actual mechanisms leading to VT remain uncertain. Here we studied β-adrenergic regulation of action potential duration (APD) in the healthy pig right ventricle (RV). Objective To determine regional β-adrenergic regulation of APD and arrhythmias in the RVOT. Methods The RV of 4 pig hearts were isolated and perfused via both the coronary arteries. Epicardial electrical activity was optically mapped (di-4-ANEPPS 20 μM) and APD measured at 80% repolarization (APD80) in the RVOT and posterior RV free wall region (RVFW). Regional mRNA and protein expression of β-adrenergic receptors was determined respectively by RT-PCR and Western-Blot. Results The expression of β1-adrenergic receptor (β1-AR) was lower in the RVOT at the mRNA level (P Conlcusion β-adrenergic stimulation differentially regulates APD80 in the RVOT and RVFW. Although β1-AR expression was similar in the 2 regions, ISO preferentially shortened APD and triggered arrhythmia in the RVOT. These results suggest the presence of a high sensitivity of RVOT electrophysiology to β-adrenergic stimulation.

Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder involving first and second branchial arches derivatives, mainly characterised by asymmetric ear anomalies, hemifacial microsomia, ocular defects and vertebral malformations. Although numerous chromosomal abnormalities have been associated with OAVS, no causative gene has been identified so far.We aimed to identify the first causative gene for OAVS.As sporadic cases are mostly described in Goldenhar syndrome, we have performed whole exome sequencing (WES) on selected affected individuals and their unaffected parents, looking for de novo mutations. Candidate gene was tested through transient knockdown experiment in zebrafish using a morpholino-based approach. A functional test was developed in cell culture in order to assess deleterious consequences of mutations.By WES, we identified a heterozygous nonsense mutation in one patient in the myelin transcription factor 1 (We report

Ventricular arrhythmias are frequent in patients with repaired tetralogy of Fallot but their underlying mechanisms remain unclear. In this study, ventricular electrical and structural remodelling was assessed in an animal model that mimics postoperative tetralogy of Fallot.Piglets underwent a tetralogy of Fallot repair-like surgery (rTOF N=6) or were sham-operated (Sham N=5). Following cardiac function assessment in vivo by MRI 3–4 months after surgery, pigs were euthanized and their hearts rapidly excised. Electrophysiological properties of right (RV) and left ventricles (LV) were obtained by optical mapping. Fibrosis was assessed histologically.RV dysfunction was evident while LV function remained unaltered in rTOF pigs. LV action potential duration (APD) was significantly longer on the epicardium (Sham 280±50ms; rTOF 390±76ms) and endocardium (Sham 301±20ms; rTOF 403±34ms) of rTOF animals (P

Introduction Les cellules phagocytaires des lignees monocytaire et neutrophile sont essentielles a la defense de l’organisme au cours d’une infection a Candida spp. Cependant in vitro, C. albicans , C. glabrata et C. lusitaniae sont capables de resister a la phagocytose. Notre hypothese est que les lipides, et en particulier les acides gras, pourraient constituer une ressource energetique privilegiee pour la levure afin de resister a la phagocytose. Chez les levures Ascomycetes, le catabolisme des acides gras est presume etre exclusivement localise dans les peroxysomes et repose principalement sur la β-oxydation. Methodes Nous avons construit par recombinaison homologue chez C. lusitaniae 11 mutants nuls (simples, doubles ou triples) afin de bloquer la β-oxydation peroxysomale (genes FOX1 , FOX2 , FOX3 ), mais aussi en amont l’import peroxysomal des acides gras a longue chaine carbonee (gene PXA1 ) et en aval le cycle du glyoxylate (gene ICL1 ). La caracterisation du phenotype des mutants obtenus a compris l’etude : – de l’assimilation des sources de carbone ; – de la consommation d’un substrat radioactif de la β-oxydation par des extraits bruts et fractions cellulaires ; – de l’immunolocalisation de la proteine Fox2 (western blot sur fractions cellulaires, microscopie electronique) ; – de la resistance in vitro a la phagocytose macrophagique (modele developpe au laboratoire) ; – de leur virulence dans un modele murin de candidemie. Resultats Contrairement a ce qui est rapporte pour C. albicans , l’inactivation du gene FOX2 (ou des genes FOX1 ou FOX3 ) ne se traduit pas par un blocage du catabolisme des acides gras chez C. lusitaniae . Nos travaux ont permis d’expliquer ce phenotype original par l’existence de 3 voies pour le catabolisme des acides gras chez C. lusitaniae et de demontrer la colocalisation inedite chez les Eucaryotes de la proteine Fox2 au peroxysome et a la mitochondrie. A 6 h d’infection, la survie des macrophages infectes par le mutant fox2Δ etait comprise entre celle des macrophages infectes par la souche sauvage (la plus faible) et celle des macrophages infectes par le mutant icl1Δ (la plus forte). Les mutants icl1Δ et fox2Δ se multipliaient moins que la souche sauvage. La virulence in vivo du mutant icl1Δ etait attenuee, alors que celle du mutant fox2Δ n’etait pas differente de la souche sauvage. Conclusion Le cycle peroxysomal de l’acide glyoxylique est indispensable pour la virulence de C. lusitaniae , et l’acetyl-CoA qui l’alimente ne provient pas seulement de la s-oxydation des acides gras.

BACKGROUND. The trigger of ventricular tachyarrhythmias after myocardial infarction (MI) has been attributed to increased incidence of electrogenic Ca2+ waves in cardiac Purkinje cells (Pcells). A change in Ca2+ release channel (CRC) gating properties and overload of intracellular Ca2+ store are 2 potential causes of this increase. We analysed through a model of intracellular Ca2+ dynamics the spontaneous Ca2+ transients of Pcells after MI. Regional protein expression was assessed for potential remodelling associated with ischemia.METHODS. Pcells were dispersed enzymatically from dog, pig, sheep, and human Purkinje fibers dissected from normal hearts and from hearts with MI. Spontaneous local Ca2+ transients were studied by high resolution confocal fluorescence microscopy techniques. Specific antibodies were used to map proteins involved in Ca2+ release/uptake functions (ERCs, SERCAs).RESULTS. In Pcells of MI heart, the density and firing rate of Ca2+-release sites were increased by 20% [Normal: 3.0 ± 0.3 sites /25µm (14 scans; 12 cells) vs MI: 3.6 ± 0.2 sites /25µm (9 cells); P

A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rβ2 in ECM development. C57BL/6 mice deficient for IL-12Rβ2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rβ2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rβ2–deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rβ2–deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rβ2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rβ2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis.

Ventricular arrhythmias are frequent in patients with repaired tetralogy of Fallot but their underlying mechanisms remain unclear. In this study, ventricular electrical and structural remodelling was assessed in an animal model that mimics postoperative tetralogy of Fallot. Piglets underwent a tetralogy of Fallot repair-like surgery (rTOF N=6) or were sham-operated (Sham N=5). Following cardiac function assessment in vivo by MRI 3-4 months after surgery, pigs were euthanized and their hearts rapidly excised. Electrophysiological properties of right (RV) and left ventricles (LV) were obtained by optical mapping. Fibrosis was assessed histologically. RV dysfunction was evident while LV function remained unaltered in rTOF pigs. LV action potential duration (APD) was significantly longer on the epicardium (Sham 280±50ms; rTOF 390±76ms) and endocardium (Sham 301±20ms; rTOF 403±34ms) of rTOF animals (P

International audience; Rationale: IL-22 has both proinflammatory and antiinflammatory properties. Its role in allergic lung inflammation has not been explored. Objectives: To investigate the expression and roles of IL-22 in the onset and resolution of experimental allergic asthma and its cross-talk with IL-17A. Methods: IL-22 expression was assessed in patient samples and in the lung of mice immunized and challenged with ovalbumin. IL-22 functions in allergic airway inflammation were evaluated using mice deficient in IL-22 or anti-IL-22 neutralizing antibodies. Moreover, the effects of recombinant IL-22 and IL-17A neutralizing antibodies were investigated. Measurements and Main Results: Increased pulmonary IL-22 expression is found in the serum of patients with asthma and mice immunized and challenged with ovalbumin. Allergic lung inflammation is IL-22 dependent because eosinophil recruitment, Th2 cytokine including IL-13 and IL-33, chemokine production, airway hyperreactivity, and mucus production are drastically reduced in mice deficient in IL-22 or by IL-22 antibody neutralization during immunization of wild-type mice. By contrast, IL-22 neutralization during antigen challenge enhanced allergic lung inflammation with increased Th2 cytokines. Consistent with this, recombinant IL-22 given with allergen challenge protects mice from lung inflammation. Finally, IL-22 may regulate the expression and proinflammatory properties of IL-17A in allergic lung inflammation. Conclusions: IL-22 is required for the onset of allergic asthma, but functions as a negative regulator of established allergic inflammation. Our study reveals that IL-22 contributes to the proinflammatory properties of IL-17A in experimental allergic asthma.

BackgroundIdiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.MethodsThe contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.ResultsWe show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt(+) γδ T cells and to a lesser extent by CD4αβ(+) T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.ConclusionsOur findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.

International audience; Th17 cells are involved in host defense against several pathogens. Using interleukin (IL) 17RA-deficient mice, we demonstrated reduced ileitis with diminished neutrophil recruitment and inflammatory lesions in the ileum, in the regional lymph node, in the spleen, and in the liver at day 7 and prolonged survival after Toxoplasma gondii infection. In addition, IL-17A antibody neutralization reduced inflammation and enhanced survival in BL6 mice. Diminished inflammation is associated with augmented interferon (IFN) gamma serum levels and enhanced production of IL-10 and IFN-gamma in cultured splenocytes upon antigen restimulation. Finally, cyst load and inflammation in the brain at 40 days are greater in surviving BL6 mice than in IL-17RA-deficient mice. In conclusion, oral T. gondii infection increases IL-17 expression and contributes to the inflammatory response, and IL-17 neutralization has a partial protective effect against fatal T. gondii-associated inflammation.

Lung emphysema and fibrosis are severe complications of chronic obstructive pulmonary disease, and uncontrolled protease activation may be involved in the pathogenesis. Using experimental elastase-induced acute inflammation, we demonstrate here that inflammation and development of emphysema is IL-1R1 and Toll/IL-1R signal transduction adaptor MyD88 dependent; however, TLR recognition is dispensable in this model. Elastase induces IL-1β, TNF-α, keratinocyte-derived chemokine, and IL-6 secretion and neutrophil recruitment in the lung, which is drastically reduced in the absence of IL-1R1 or MyD88. Further, tissue destruction with emphysema and fibrosis is attenuated in the lungs of IL-1R1- and MyD88-deficient mice. Specific blockade of IL-1 by IL-1R antagonist diminishes acute inflammation and emphysema. Finally, IL-1β production and inflammation are reduced in mice deficient for the NALP3 inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and we identified uric acid, which is produced upon elastase-induced lung injury, as an activator of the NALP3/ASC inflammasome. In conclusion, elastase-mediated lung pathology depends on inflammasome activation with IL-1β production. IL-1β therefore represents a critical mediator and a possible therapeutic target of lung inflammation leading to emphysema.

International audience; RATIONALE: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1beta production, inflammation, and fibrosis remain poorly understood. OBJECTIVES: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung. Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid. MEASUREMENTS AND MAIN RESULTS: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1beta production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor. CONCLUSIONS: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1beta production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1beta production and chronic inflammatory lung pathology.

Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol anchor phosphatidyl-myo-inositol hexamannosides PIM(6) or PIM(2) exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12 (IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing adaptor protein inducing interferon beta (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced NFkappaB translocation. Synthetic PIM(1) and a PIM(2) mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM analogs represent powerful anti-inflammatory leads.

The molecular mechanisms of acute lung injury resulting in inflammation and fibrosis are not well established. Here we investigate the roles of the IL-1 receptor 1 (IL-1R1) and the common adaptor for Toll/IL-1R signal transduction, MyD88, in this process using a murine model of acute pulmonary injury. Bleomycin insult results in expression of neutrophil and lymphocyte chemotactic factors, chronic inflammation, remodeling, and fibrosis. We demonstrate that these end points were attenuated in the lungs of IL-1R1- and MyD88-deficient mice. Further, in bone marrow chimera experiments, bleomycin-induced inflammation required primarily MyD88 signaling from radioresistant resident cells. Exogenous rIL-1beta recapitulated a high degree of bleomycin-induced lung pathology, and specific blockade of IL-1R1 by IL-1 receptor antagonist dramatically reduced bleomycin-induced inflammation. Finally, we found that lung IL-1beta production and inflammation in response to bleomycin required ASC, an inflammasome adaptor molecule. In conclusion, bleomycin-induced lung pathology required the inflammasome and IL-1R1/MyD88 signaling, and IL-1 represented a critical effector of pathology and therapeutic target of chronic lung inflammation and fibrosis.

Malaria pigment hemozoin was reported to activate the innate immunity by Toll-like receptor (TLR)-9 engagement. However, the role of TLR activation for the development of cerebral malaria (CM), a lethal complication of malaria infection in humans, is unknown. Using Plasmodium berghei ANKA (PbA) infection in mice as a model of CM, we report here that TLR9-deficient mice are not protected from CM. To exclude the role of other members of the TLR family in PbA recognition, we infected mice deficient for single TLR1, -2, -3, -4, -6, -7, or -9 and their adapter proteins MyD88, TIRAP, and TRIF. In contrast to lymphotoxin alpha-deficient mice, which are resistant to CM, all TLR-deficient mice were as sensitive to fatal CM development as wild-type control mice and developed typical microvascular damage with vascular leak and hemorrhage in the brain and lung, together with comparable parasitemia, thrombocytopenia, neutrophilia, and lymphopenia. In conclusion, the present data do not exclude the possibility that malarial molecular motifs may activate the innate immune system. However, TLR-dependent activation of innate immunity is unlikely to contribute significantly to the proinflammatory response to PbA infection and the development of fatal CM.

Objective The growing adult population with surgically corrected tetralogy of Fallot (TOF) is at risk of arrhythmias and sudden cardiac death. We sought to investigate the contribution of right ventricular (RV) structural and electrophysiological remodelling to arrhythmia generation in a preclinical animal model of repaired TOF (rTOF). Methods and results Pigs mimicking rTOF underwent cardiac MRI functional characterisation and presented with pulmonary regurgitation, RV hypertrophy, dilatation and dysfunction compared with Sham-operated animals (Sham). Optical mapping of rTOF RV-perfused wedges revealed a significant prolongation of RV activation time with slower conduction velocities and regions of conduction slowing well beyond the surgical scar. A reduced protein expression and lateralisation of Connexin-43 were identified in rTOF RVs. A remodelling of extracellular matrix-related gene expression and an increase in collagen content that correlated with prolonged RV activation time were also found in these animals. RV action potential duration (APD) was prolonged in the epicardial anterior region at early and late repolarisation level, thus contributing to a greater APD heterogeneity and to altered transmural and anteroposterior APD gradients in rTOF RVs. APD remodelling involved changes in Kv4.3 and MiRP1 expression. Spontaneous arrhythmias were more frequent in rTOF wedges and more complex in the anterior than in the posterior RV. Conclusion Significant remodelling of RV conduction and repolarisation properties was found in pigs with rTOF. This remodelling generates a proarrhythmic substrate likely to facilitate re-entries and to contribute to sudden cardiac death in patients with rTOF.

The right ventricular outflow tract (RVOT) has a distinct embryological origin from the rest of the right ventricle (RV) and is a frequent origin for idiopathic and disease-related arrhythmias. We hypothesised that heterogeneous action potential duration (APD) across the right ventricle (RV) may contribute to RVOT arrhythmia generation. Pigs were anesthetized and monophasic action potentials (MAPs) recorded in sinus rhythm from the epicardium of the RV free wall and RVOT. The RV was isolated and perfused via both right and left anterior coronary arteries. The preparation was paced (1-5Hz) and the electrical activity optically mapped (di-4-ANEPPS, 10μM) on both epicardial (EPI) and endocardial (ENDO) surfaces. The expression of potassium channels was assessed by RT-PCR. In vivo, MAP durations measured at 20% and 80% repolarization were both significantly shorter in the RVOT than in the RV free wall EPI (P

The right ventricular outflow tract (RVOT) has a distinct embryological origin and is a common anatomical source of arrhythmias in the healthy and diseased myocardium. We hypothesised that specific RVOT activation and conduction properties may underlie the preferential RVOT origin of arrhythmias. Pig right ventricular (RV) wedge preparations were perfused via the left anterior descending and right coronary arteries. Electrical activation and conduction properties were obtained by optical mapping of the epicardial surface (di-4- ANEPPS 10μM) upon electrical stimulation of the preparation. Transmural needles were inserted in the RV free wall and RVOT and unipolar electrograms (EGMs) were recorded. Fiber orientation was obtained by diffusion tensor MRI. Regional mRNA expression was determined by RT-PCR and fibrosis was assessed histologically. Longitudinal and transverse conduction velocities were significantly reduced in RVOT compared to RV free wall (P