Molecular mechanisms regulating pathogenic pathways in B cells and plasmacytoid dendritic cells in lupus

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease characterized by autoantibody production, inflammation and end organ damage resulting from an overactivation of the immune system through mechanisms that are still not completely understood. One critical component of this pernicious cycle is a loss of tolerance to self-antigen that precedes disease manifestations. Importantly, there has only been one FDA approved treatment for SLE in the last 60 years, though an additional treatment has recently been approved for lupus nephritis. Therefore, there is a need to understand how pathogenic pathways are regulated in the context of SLE. This thesis focused on not only further understanding how interferon regulatory factor 5 (IRF5) drives pathogenicity in a mouse of model of lupus but also discerning the molecular pathways important for blood dendritic cell antigen 2 (BDCA2) inhibition of type I interferon (IFN-I) release from plasmacytoid dendritic cells (pDCs). Gain-of-function polymorphisms in the transcription factor IRF5 are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. This work shows that monoallelic IRF5 deficiency in B cells markedly reduces disease in the FcγRIIB−/−Yaa mouse model of lupus. Mechanistically, B cell receptor and TLR7 signaling synergize to promote IRF5 phosphorylation through IRAK4, and also increase IRF5 protein expression, with these processes being independently regulated. This synergy increases B cell-intrinsic IL-6 and TNF- production, both key requirements for germinal center responses, with IL-6 and TNF- production in vitro and in vivo being substantially lower with loss of one allele of IRF5. Moreover, this thesis further strengthens the notion of IRF5 as a target for the treatment of SLE by therapeutically targeting IRF5 in the FcγRIIB−/−Yaa mouse lupus model. This work shows that tamoxifen driven deletion of IRF5 after disease associated manifestations have already developed confers protection in the FcγRIIB−/−Yaa mouse model. IFN-I is a family of pleotropic cytokines that are thought to be pathogenic in the context of SLE. PDCs are a major source of IFN-I. As such, they have been shown to play a major role in anti-viral immunity and are thought to be pathogenic in context of SLE. I used the anti-BDCA2 antibody 24F4A to ligate the receptor in a human pDC cell line (Gen 2.2) and applied a variety of proteomics methods, including profiling of post-translational modifications, to evaluate signaling downstream of BDCA2. I found that phosphorylation of phosphoinositide 3-kinase adapter protein 1 (PIK3AP1)/ B cell adaptor protein (BCAP) was increased after BDCA2 engagement by 24F4A. Deletion of BCAP from Gen2.2 cells reversed BDCA2 mediated AKT phosphorylation and IFN inhibition, suggesting that PI3 kinase (PI3K) may be important for BDCA2 mediated IFN inhibition. Treatment of human pDCs with PI3kinase inhibitor followed by TLR9 stimulation confirmed that PI3K activity is critical for BDCA2 mediated IFN inhibition. This work describes a critical threshold of IRF5 expression in B cells necessary for the development of the lupus like disease in the FcγRIIB−/−Yaa mice and that systemic deletion of IRF5 after disease onset reduces the disease manifestations in the same mouse model. Additionally, this work demonstrated that BCAP and PI3K are important pathways to mediate the IFN inhibition observed with engagement of the BDCA2 pathway. This work supports IRF5 as a therapeutic target in SLE and furthermore, describes a molecular pathway important for BDCA2 mediated IFN-I inhibition from pDCs

MYC is essential for the formation and maintenance of germinal centers

Germinal centers (GC) are sites of intense B cell proliferation, central for T cell dependent antibody responses. However, the role of MYC, a key cell cycle regulator, in this process has been questioned. Here, we identified MYC positive B cell subpopulations in immature and mature GCs, and show through genetic ablation of Myc that they play indispensable roles in GC formation and maintenance. The identification of these functionally critical cellular subsets has important implications for human B cell lymphomagenesis, which mostly originates from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the MYC positive GC subpopulations may be at a particularly high risk for malignant transformation

Monoallelic IRF5 deficiency in B cells prevents murine lupus

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduced disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils did not reduce disease severity. B cell receptor and TLR7 signaling synergized to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increased B cell-intrinsic IL-6 and TNF-alpha production, both key requirements for germinal center (GC) responses, with IL-6 and TNF-alpha production in vitro and in vivo being substantially lower with loss of 1 allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation was reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression was dynamically regulated in vivo with increased expression in GC B cells compared with non-GC B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells was required to promote disease in murine lupus

Anti-Apoptotic and Anti-Inflammatory Role of Trans ε-Viniferin in a Neuron-Glia Co-Culture Cellular Model of Parkinson's Disease

open7siFinanziato da NSERC (National Science and Engineering Research Council) of Canada, Discovery grantThe polyphenol trans-epsilon-viniferin (viniferin) is a dimer of resveratrol, reported to hold antioxidant and anti-inflammatory properties. The aims of our study were to evaluate the neuroprotective potential of viniferin in the nerve growth factor (NGF)-differentiated PC12 cells, a dopaminergic cellular model of Parkinson's disease (PD) and assess its anti-inflammatory properties in a N9 microglia-neuronal PC12 cell co-culture system. The neuronal cells were pre-treated with viniferin, resveratrol or their mixture before the administration of 6-hydroxydopamine (6-OHDA), recognized to induce parkinsonism in rats. Furthermore, N9 microglia cells, in a co-culture system with neuronal PC12, were pre-treated with viniferin, resveratrol or their mixture to investigate whether these polyphenols could reduce lipopolysaccharide (LPS)-induced inflammation. Our results show that viniferin as well as a mixture of viniferin and resveratrol protects neuronal dopaminergic cells from 6-OHDA-induced cytotoxicity and apoptosis. Furthermore, when viniferin, resveratrol or their mixture was used to pre-treat microglia cells in our co-culture system, they reduced neuronal cytotoxicity induced by glial activation. Altogether, our data highlight a novel role for viniferin as a neuroprotective and anti-inflammatory molecule in a dopaminergic cellular model, paving the way for nutraceutical therapeutic avenues in the complementary treatments of PD.openSergi, Domenico; Gélinas, Alex; Beaulieu, Jimmy; Renaud, Justine; Tardif-Pellerin, Emilie; Guillard, Jérôme; Martinoli, Maria-GraziaSergi, Domenico; Gélinas, Alex; Beaulieu, Jimmy; Renaud, Justine; Tardif-Pellerin, Emilie; Guillard, Jérôme; Martinoli, Maria-Grazi

Facteur de forme équivalent des pores: un outil pour la prédiction des types poreux dans les réservoirs carbonatés

International audienceThe equivalent pore aspect ratios (EPAR) provide a tool to detect pore types by combining P-and S-wave velocities, porosity, bulk density and mineralogical composition of carbonate rocks. The integration of laboratory measurements, well log data and petrographic analysis of 468 carbonate samples from various depositional and diagenetic settings (Lower Cretaceous pre-salt non-marine carbonates from offshore Brazil, Lower Cretaceous shallow-water platform carbonates from SE France and Upper Cretaceous deep-water carbonate from the Gargano Peninsula, Italy) allowed the acoustic signature of a wide range of pore types to be quantified. The equivalent pore aspect ratios are shown to be independent of pore volume and mineralogy. They can be therefore regarded as relevant parameters for discriminating selected pore network architectures in carbonate reservoirs displaying a wide range of porosity values and mineralogical compositions. Interparticle and intercrystalline micro-and macroporosity may include a variety of distinct pore network structures and a wide range of acoustic signatures depending on particle/crystal shape, nature of contacts, packing and cementation. The acoustic response of moldic pores appears to be mainly controlled by the shape of the dissolved allochems. The present work reveals also that correct pore type detection from acoustic and porosity measurements is highly dependent on the pore type classification used.Les facteurs de forme équivalents des pores (EPAR) fournissent un outil pour détecter les types de pores en combinant les vitesses des ondes P et S, la porosité, la densité apparente et la composition minéralogique des roches carbonatées. L'intégration de mesures en laboratoire, de diagraphies et d'analyses pétrographiques de 468 échantillons de carbonate provenant de divers dépôts et diagénétiques (carbonates non marins pré-salifères du Crétacé inférieur du Brésil, carbonates de plate-forme en eau peu profonde du Crétacé inférieur du SE France et du Crétacé supérieur carbonate d'eau de la péninsule du Gargano, Italie) a permis de quantifier la signature acoustique d'un large éventail de types de pores. Les rapports d'aspect des pores équivalents sont indépendants du volume des pores et de la minéralogie. Ils peuvent donc être considérés comme des paramètres pertinents pour discriminer des architectures de réseau de pores sélectionnées dans des réservoirs de carbonate présentant une large gamme de valeurs de porosité et de compositions minéralogiques. La microparticule et la macroporosité interparticulaire et intercristalline peuvent inclure une variété de structures de réseau de pores distinctes et une large gamme de signatures acoustiques en fonction de la forme des particules / cristaux, de la nature des contacts, de l'emballage et de la cimentation. La réponse acoustique des pores du moule semble être principalement contrôlée par la forme des allochems dissous. Le présent travail révèle également qu'une détection correcte du type de pores à partir de mesures acoustiques et de porosité dépend fortement de la classification du type de pores utilisée

Early functional and transcriptomic changes in the myocardium predict outcome in a long-term rat model of sepsis

Myocardial function is depressed in sepsis and is an important prognosticator in the human condition. Using echocardiography in a long-term fluid-resuscitated Wistar rat model of faecal peritonitis we investigated whether depressed myocardial function could be detected at an early stage of sepsis and, if so, whether the degree of depression could predict eventual outcome. At 6 h post-insult, a stroke volume <0.17 ml prognosticated 3-day mortality with positive and negative predictive values of 93 and 80%, respectively. Subsequent fluid loading studies demonstrated intrinsic myocardial depression with poor-prognosis animals tolerating less fluid than either good-prognosis or sham-operated animals. Cardiac gene expression analysis at 6 h detected 527 transcripts significantly up- or down-regulated by the septic process, including genes related to inflammatory and cell cycle pathways. Predicted mortality was associated with significant differences in transcripts of genes expressing proteins related to the TLR2/MyD88 (Toll-like receptor 2/myeloid differentiation factor 88) and JAK/STAT (Janus kinase/signal transducer and activator of transcription) inflammatory pathways, β-adrenergic signalling and intracellular calcium cycling. Our findings highlight the presence of myocardial depression in early sepsis and its prognostic significance. Transcriptomic analysis in heart tissue identified changes in signalling pathways that correlated with clinical dysfunction. These pathways merit further study to both better understand and potentially modify the disease process