15 research outputs found
Mécanismes cellulaires et moléculaires des fonctions tolérogÚniques et immunogéniques des cellules dendritiques dans les réponses auto-immunes
La contribution des DCs dans lâinitiation et la perpĂ©tuation des maladies auto-immunes est bien Ă©tablie. Chez la souris Non ObĂšse DiabĂ©tique (NOD), modĂšle spontanĂ© du diabĂšte de type 1 (DT1), plusieurs travaux ont rapportĂ©s des anomalies phĂ©notypiques et fonctionnelles des DCs. Les DCs sont parmi les premiĂšres cellules qui infiltrent les ilots pancrĂ©atiques, produisent des quantitĂ©s excessives de cytokines pro-inflammatoires et contribuent Ă lâactivation des lymphocytes T auto-rĂ©actifs (Teff). Cette capacitĂ© accrue des DCs Ă activer les Teffs est rĂ©gulĂ©e par plusieurs voies de signalisation intracellulaire. STAT5 est parmi les facteurs de transcription critiques dans la rĂ©gulation des gĂšnes associĂ©s au dĂ©veloppement, la maturation et les fonctions des DCs. La prĂ©disposition au DT1 chez la NOD est dĂ©terminĂ©e par plusieurs rĂ©gions de susceptibilitĂ©s au diabĂšte (idd1-20). De façon intĂ©ressante, le gĂšne Stat5b est localisĂ© dans la rĂ©gion de susceptibilitĂ© idd4 chez la souris NOD suggĂ©rant son implication dans le dĂ©veloppement du diabĂšte. En effet des Ă©tudes rĂ©centes ont identifiĂ©s un dysfonctionnement dans la voie de signalisation JAK-STAT5 chez les souris NOD, y compris la prĂ©sence dâune mutation (L327M) au niveau du domaine de liaison Ă lâADN de Stat5b qui altĂšre sa liaison Ă lâADN. Par ailleurs, les Ă©tudes rĂ©alisĂ©es dans notre laboratoire ont montrĂ© que le conditionnement des DCs au GM-CSF ou Ă la TSLP, qui activent la voie de signalisation Jak-Stat5, constitue une voie potentielle dâimmunothĂ©rapie chez la souris NOD. Ces donnĂ©es suggĂšrent un rĂŽle central de Stat5b dans la rĂ©gulation des fonctions tolĂ©rogĂšnes des cellules du systĂšme immunitaire.
Nous avons gĂ©nĂ©rĂ© un modĂšle de souris NOD transgĂ©niques (NOD.CD11cStat5b-CA) exprimant de façon constitutive la forme active de STAT5B de la souris C57BL/6 spĂ©cifiquement dans les DCs. Nos rĂ©sultats ont montrĂ© que ces souris transgĂ©niques ont dĂ©veloppĂ©es une protection totale contre le diabĂšte auto-immun. Cette rĂ©sistance au diabĂšte Ă long terme est associe Ă lâacquisition des fonctions tolĂ©rogĂšnes par les Stat5b-CA.DCs qui se manifestent par un phĂ©notype mature tolĂ©rogĂšne, marquĂ©es par une forte expression de molĂ©cules immunorĂ©gulatrices (PD-L1 et PD-L2) et une grande production de cytokines anti-inflammatoire (TGF-ÎČ) et une baisse significative de la production de cytokines pro-inflammatoires (IL-12p70, TNF-α et dâIL-23). Par ailleurs, nous avons mis en Ă©vidence le rĂŽle de STAT5B dans la rĂ©gulation Ă la hausse dâIRF4 et lâimplication du complexe STAT5B/EZH2 dans le contrĂŽle de la rĂ©gulation Ă la baisse dâIRF8. En effet, cette rĂ©gulation diffĂ©rentielle de lâexpression des gĂšnes Irf4 et Irf8 est accompagnĂ©e du dĂ©veloppement dâune sous population CD11c+ CD11b+ DCs. Nos Ă©tudes ont dĂ©montrĂ© que le potentiel tolĂ©rogĂšne des Stat5b-CA.DCs Ă rĂ©tablir et Ă maintenir la tolĂ©rance pĂ©riphĂ©rique du systĂšme immunitaire vis-Ă -vis des auto-antigĂšnes est associe Ă leur grande capacitĂ© dâinduire la conversion et lâexpansion des Tregs ainsi que la diffĂ©rentiation de deux populations cellulaires rĂ©gulatrices Th2 et Tc2. Nous avons aussi dĂ©montrĂ© in vivo quâune injection intraveineuse unique de Stat5-CA.DCs (splĂ©niques ou gĂ©nĂ©rĂ©s de la moelle osseuse) ou de Tregs des souris transgĂ©niques NOD.CD11cStat5b-CA a induit une protection totale contre le diabĂšte chez les souris NOD receveuses. Notre Ă©tude apporte donc une Ă©vidence claire que la correction du dĂ©faut de la voie de signalisation Jak-Stat5b au sein des DC chez la souris NOD induit une protection Ă long terme contre le diabĂšte. Finalement, cette voie de signalisation peut constituer une cible thĂ©rapeutique Ă©ventuelle non seulement dans le contexte du diabĂšte de type 1 mais Ă©galement dans dâautres maladies auto-immunes.The contribution of DCs in the initiation and progression of autoimmune diseases is well established. Several studies have reported that phenotypic and functional abnormalities of DCs, in Non Obese Diabetic (NOD), contribute to spontaneous type 1 diabetes (T1D) development. DCs are among the first cells that infiltrate the pancreatic islets, produce excessive amounts of pro-inflammatory cytokines, and contribute to the activation of T effector cells (Teff). This increased ability of DCs to activate Teff is regulated by several intracellular signaling pathways. STAT5 is among the critical transcription factors in the regulation of genes associated with the development, maturation and functions of DCs. The predisposition to T1D in NOD is determined by several regions of susceptibility to diabetes (idd1-20). Interestingly, the Stat5b gene is located in the idd4 susceptibility region in NOD mice suggesting its involvement in the development of diabetes. Recent studies have identified a dysfunction in the Jak-Stat5 signaling pathway in NOD mice, including the presence of a mutation (L327M) at the DNA-binding domain of Stat5b which alters its binding to DNA. Furthermore, previous studies from our laboratory have shown that the GM-CSF- or TSLP-conditioned DCs, which activate the Jak-Stat5 signaling pathway, is a potential pathway for immunotherapy in NOD mice. These data suggest a central role for Stat5b in the regulation of tolerogenic functions of the immune cells.
Here, we generated a transgenic NOD mouse model (NOD.CD11cStat5b-CA) that constitutively express the active form of STAT5B from the C57BL/6 mouse specifically in DCs. Our results showed that these transgenic mice are completely protected against autoimmune diabetes. This long-term diabetes protection is associated with the acquisition of tolerogenic functions by Stat5b-CA.DCs, that exhibit a mature tolerogenic phenotype, overexpression of immunoregulatory molecules (PD-L1 and PD-L2) and produce anti-inflammatory cytokines (TGF-ÎČ) and a significantly decrease their production of pro-inflammatory cytokines (IL-12p70, TNF-α and IL-23). Moreover, we have highlighted the role of STAT5B in the upregulation of IRF4 and also the involvement of the STAT5B/ EZH2 complex in downregulation of IRF8. This differential regulation of the Irf4 and Irf8 genes expression is accompanied by promoting the development of CD11c+CD11b+ DC subset. Furthermore, we demonstrated that the tolerogenic Stat5b-CA.DCs were able to restore and maintain peripheral immune tolerance to autoantigens, which is associated with their high ability to induce conversion and expansion Tregs and to promote Th2 and Tc2 immune deviation. We also demonstrated that a single intravenous injection of Stat5-CA.DCs (splenic or bone marrow derived dendritic cells) or Tregs from transgenic mice NOD.CD11cStat5b-CA halted ongoing diabetes in recipient NOD mice. Thus, our study provides clear evidence that the correction of the Jak-Stat5b signaling pathway defect in DC of NOD mice induces long-term protection against diabetes suggesting that signaling pathway can be a potential therapeutic target not only in the context of type 1 diabetes but also in other autoimmune diseases
Development of a novel quantitative PCR assay as a measurement for the presence of geosmin-producing fungi
International audienceGeosminâassociated gpe1 gene of Penicillium expansum displayed â„99% similarity to cytochrome P450 gene of geosminâproducing P. restrictum, but â€40% similarities to geosmin biosynthesis, nonâcytochromic gene of Streptomyces avermitilis and cytochrome P450 genes of nonâgeosminâproducing Neotyphodium lolii, Phoma betae and P. paxilli. Serial 10âfold dilutions of P. expansum's DNA was subjected to a previously reported qPCR assay (Atoui et al. 2007), utilizing gpe1 specific primer pair âSNgpe1F/SNgpe1Râ. A linear relationship between DNA quantity and Cycle Threshold (Ct), with strong correlative coefficient, was observed. Using the available physicoâchemical method, geosmin was quantified in 188 grape samples. Penicillium spp's DNA was quantified in these samples, utilizing the developed qPCR assay. A strong positive correlation (R2 = 0·97) between Penicillium's DNA and geosmin concentration was observed. Furthermore, <50 ng ÎŒlâ1 Penicillium's DNA corresponds to geosmin level below the permitted intensity limit i.e. 4, for âFlavour Profile Analysisâ
Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol efflux and reverse cholesterol transport.
This study was aimed to investigate the effect of human PON1 overexpression in mice on cholesterol efflux and reverse cholesterol transport. PON1 overexpression in PON1-Tg mice induced a significant 3-fold (p<0.0001) increase in plasma paraoxonase activity and a significant ~30% (p<0.0001) increase in the capacity of HDL to mediate cholesterol efflux from J774 macrophages compared to wild-type mice. It also caused a significant 4-fold increase (p<0.0001) in the capacity of macrophages to transfer cholesterol to apoA-1, a significant 2-fold (p<0.0003) increase in ABCA1 mRNA and protein expression, and a significant increase in the expression of PPARγ (p<0.0003 and p<0.04, respectively) and LXRα (p<0.0001 and p<0.01, respectively) mRNA and protein compared to macrophages from wild-type mice. Moreover, transfection of J774 macrophages with human PON1 also increased ABCA1, PPARγ and LXRα protein expression and stimulates macrophages cholesterol efflux to apo A1. In vivo measurements showed that the overexpression of PON1 significantly increases the fecal elimination of macrophage-derived cholesterol in PON1-Tg mice. Overall, our results suggested that the overexpression of PON1 in mice may contribute to the regulation of the cholesterol homeostasis by improving the capacity of HDL to mediate cholesterol efflux and by stimulating reverse cholesterol transport
Elucidating the Role of Ezh2 in Tolerogenic Function of NOD Bone Marrow-Derived Dendritic Cells Expressing Constitutively Active Stat5b
Tolerogenic dendritic cells (toDCs) are crucial to controlling the development of autoreactive T cell responses and the prevention of autoimmunity. We have reported that NOD.CD11cStat5b-CA transgenic mice expressing a constitutively active (CA) form of Stat5b under the control of a CD11c promoter are protected from diabetes and that Stat5b-CA-expressing DCs are tolerogenic and halt ongoing diabetes in NOD mice. However, the molecular mechanisms by which Stat5b-CA modulates DC tolerogenic function are not fully understood. Here, we used bone marrow-derived DCs (BMDCs) from NOD.CD11cStat5b-CA transgenic mice (Stat5b-CA.BMDCs) and found that Stat5b-CA.BMDCs displayed high levels of MHC class II, CD80, CD86, PD-L1, and PD-L2 and produced elevated amounts of TGFβ but low amounts of TNFα and IL-23. Stat5b-CA.BMDCs upregulated Irf4 and downregulated Irf8 genes and protein expression and promoted CD11c+CD11b+ DC2 subset differentiation. Interestingly, we found that the histone methyltransferase Ezh2 and Stat5b-CA bound gamma-interferon activated site (GAS) sequences in the Irf8 enhancer IRF8 transcription, whereas Stat5b but not Ezh2 bound GAS sequences in the Irf4 promoter to enhance IRF4 transcription. Injection of Stat5b-CA.BMDCs into prediabetic NOD mice halted progression of islet inflammation and protected against diabetes. Importantly, inhibition of Ezh2 in tolerogenic Stat5b-CA.BMDCs reduced their ability to prevent diabetes development in NOD recipient mice. Taken together, our data suggest that the active form of Stat5b induces tolerogenic DC function by modulating IRF4 and IRF8 expression through recruitment of Ezh2 and highlight the fundamental role of Ezh2 in Stat5b-mediated induction of tolerogenic DC function
PON1 overexpression in mice up-regulates PPARÎł protein and RNA expression by MPM.
<p>PPARγ protein and RNA expression by MPM from WT and PON1-Tg mice were analyzed by <b>(A)</b> Western blotting (n = 5 mice/group) and <b>(B)</b> RT-PCR (n = 4 mice/group), respectively. Data are expressed as means ± SEM. *p = 0.0451, ***p = 0.0003.</p
PON1 overexpression up-regulates LXRα protein and RNA expression.
<p>LXRα protein and RNA expression by MPM from WT and PON1-Tg mice were determined by <b>(A)</b> Western blotting (n = 5 mice/group) and <b>(B)</b> RT-PCR (n = 4 mice/group), respectively. Data are expressed as means ± SEM. *p = 0.0127, ****p<0.0001.</p
PON1 overexpression in mice up-regulates ABCA1 protein and RNA expression by MPM.
<p>ABCA1 protein and RNA expression by MPM from WT and PON1-Tg mice were determined by <b>(A)</b> Western blotting (n = 5 mice/group) and <b>(B)</b> RT-PCR (n = 4 mice/group), respectively. Data are expressed as means ± SEM. ***p = 0.0009, ****p<0.0001.</p
PON1 overexpression in mice enhances macrophage-derived cholesterol efflux.
<p><b>(A)</b> Plasma was obtained from WT and PON1-Tg mice. Paraoxonase activity was determined by recording the increase in absorbance at 412 nm using paraoxon as the substrate. One unit of paraoxonase activity was defined as 1 nmole 4-nitrophenol formed per minute. <b>(B)</b> J774 macrophages (1 x 10<sup>6</sup> cells/mL) were loaded with [<sup>3</sup>H]-cholesterol (2 ΌCi/mL) for 24 h and were incubated with or without cAMP (0.3 mM) for 12 h. The cells were subsequently incubated with 50 ΌL/mL of plasma from WT or PON1-Tg mice, which was then precipitated with PEG. <b>(C)</b> MPM from WT and PON1-Tg mice (1 x 10<sup>6</sup> cells/mL) were loaded with [<sup>3</sup>H]-cholesterol (2 ΌCi/mL) for 24 h. Following a 12-h pre-incubation phase, the cells were incubated with 50 Όg/mL of Apo-A1 for 4 h. Data are expressed as means ± SEM. n = 14 mice/group. ****p<0.0001.</p
PON1 overexpression increases reverse cholesterol transport.
<p>[<sup>3</sup>H]-cholesterol-loaded J774 macrophages were injected intraperitoneally into WT and PON1-Tg mice. [<sup>3</sup>H]-cholesterol plasma levels were measured at 6, 24, and 48 h post-injection. Feces were collected continuously from 0 to 48 h post-injection. <b>(A)</b> Time course of [<sup>3</sup>H]-cholesterol distribution in the plasma, <b>(B) [</b><sup>3</sup>H]-cholesterol recovery in the liver after 48 h, and (<b>C) [</b><sup>3</sup>H]-cholesterol recovery in the feces as sterols and biliary acid. Data are expressed as means ± SEM. n = 14 mice/group. **p = 0.0013, ***0.0007</p
Transfection of J774 macrophages with human PON1 up-regulates ABCA1, LXRα and PPARγ protein expression and stimulates cholesterol efflux.
<p><b>(A)</b> ABCA1, LXRα and PPARÎł protein expression by J774 macrophages was determined by Western blot. (<b>B)</b> PON1 transfected J774 macrophages and control macrophages (CTRL) were loaded with [<sup>3</sup>H]-cholesterol (2 ÎŒCi/mL) for 24 h followed by incubation with 50 ÎŒg/mL of apoA-1 for 4 h. Data are expressed as means ± SEM, n = 3. **p<0.005.</p