Mécanismes cellulaires et moléculaires des fonctions tolérogèniques et immunogéniques des cellules dendritiques dans les réponses auto-immunes

La contribution des DCs dans l’initiation et la perpétuation des maladies auto-immunes est bien établie. Chez la souris Non Obèse Diabétique (NOD), modèle spontané du diabète de type 1 (DT1), plusieurs travaux ont rapportés des anomalies phénotypiques et fonctionnelles des DCs. Les DCs sont parmi les premières cellules qui infiltrent les ilots pancréatiques, produisent des quantités excessives de cytokines pro-inflammatoires et contribuent à l’activation des lymphocytes T auto-réactifs (Teff). Cette capacité accrue des DCs à activer les Teffs est régulée par plusieurs voies de signalisation intracellulaire. STAT5 est parmi les facteurs de transcription critiques dans la régulation des gènes associés au développement, la maturation et les fonctions des DCs. La prédisposition au DT1 chez la NOD est déterminée par plusieurs régions de susceptibilités au diabète (idd1-20). De façon intéressante, le gène Stat5b est localisé dans la région de susceptibilité idd4 chez la souris NOD suggérant son implication dans le développement du diabète. En effet des études récentes ont identifiés un dysfonctionnement dans la voie de signalisation JAK-STAT5 chez les souris NOD, y compris la présence d’une mutation (L327M) au niveau du domaine de liaison à l’ADN de Stat5b qui altère sa liaison à l’ADN. Par ailleurs, les études réalisées dans notre laboratoire ont montré que le conditionnement des DCs au GM-CSF ou à la TSLP, qui activent la voie de signalisation Jak-Stat5, constitue une voie potentielle d’immunothérapie chez la souris NOD. Ces données suggèrent un rôle central de Stat5b dans la régulation des fonctions tolérogènes des cellules du système immunitaire. Nous avons généré un modèle de souris NOD transgéniques (NOD.CD11cStat5b-CA) exprimant de façon constitutive la forme active de STAT5B de la souris C57BL/6 spécifiquement dans les DCs. Nos résultats ont montré que ces souris transgéniques ont développées une protection totale contre le diabète auto-immun. Cette résistance au diabète à long terme est associe à l’acquisition des fonctions tolérogènes par les Stat5b-CA.DCs qui se manifestent par un phénotype mature tolérogène, marquées par une forte expression de molécules immunorégulatrices (PD-L1 et PD-L2) et une grande production de cytokines anti-inflammatoire (TGF-β) et une baisse significative de la production de cytokines pro-inflammatoires (IL-12p70, TNF-α et d’IL-23). Par ailleurs, nous avons mis en évidence le rôle de STAT5B dans la régulation à la hausse d’IRF4 et l’implication du complexe STAT5B/EZH2 dans le contrôle de la régulation à la baisse d’IRF8. En effet, cette régulation différentielle de l’expression des gènes Irf4 et Irf8 est accompagnée du développement d’une sous population CD11c+ CD11b+ DCs. Nos études ont démontré que le potentiel tolérogène des Stat5b-CA.DCs à rétablir et à maintenir la tolérance périphérique du système immunitaire vis-à-vis des auto-antigènes est associe à leur grande capacité d’induire la conversion et l’expansion des Tregs ainsi que la différentiation de deux populations cellulaires régulatrices Th2 et Tc2. Nous avons aussi démontré in vivo qu’une injection intraveineuse unique de Stat5-CA.DCs (spléniques ou générés de la moelle osseuse) ou de Tregs des souris transgéniques NOD.CD11cStat5b-CA a induit une protection totale contre le diabète chez les souris NOD receveuses. Notre étude apporte donc une évidence claire que la correction du défaut de la voie de signalisation Jak-Stat5b au sein des DC chez la souris NOD induit une protection à long terme contre le diabète. Finalement, cette voie de signalisation peut constituer une cible thérapeutique éventuelle non seulement dans le contexte du diabète de type 1 mais également dans d’autres 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-β) 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 audienceGeosmin‐associated gpe1 gene of Penicillium expansum displayed ≥99% similarity to cytochrome P450 gene of geosmin‐producing P. restrictum, but ≤40% similarities to geosmin biosynthesis, non‐cytochromic gene of Streptomyces avermitilis and cytochrome P450 genes of non‐geosmin‐producing Neotyphodium lolii, Phoma betae and P. paxilli. Serial 10‐fold dilutions of P. expansum's DNA was subjected to a previously reported qPCR assay (Atoui et al. 2007), utilizing gpe1 specific primer pair ‘SNgpe1F/SNgpe1R’. A linear relationship between DNA quantity and Cycle Threshold (Ct), with strong correlative coefficient, was observed. Using the available physico‐chemical 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 μl−1 Penicillium's DNA corresponds to geosmin level below the permitted intensity limit i.e. 4, for ‘Flavour Profile Analysis’

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 TGF&beta; but low amounts of TNF&alpha; 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 PPARγ 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 10⁶ cells/mL) were loaded with [³H]-cholesterol (2 μCi/mL) for 24 h and were incubated with or without cAMP (0.3 mM) for 12 h. The cells were subsequently incubated with 50 μL/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 10⁶ cells/mL) were loaded with [³H]-cholesterol (2 μCi/mL) for 24 h. Following a 12-h pre-incubation phase, the cells were incubated with 50 μg/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>[³H]-cholesterol-loaded J774 macrophages were injected intraperitoneally into WT and PON1-Tg mice. [³H]-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 [³H]-cholesterol distribution in the plasma, <b>(B) [</b>³H]-cholesterol recovery in the liver after 48 h, and (<b>C) [</b>³H]-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 PPARγ protein expression by J774 macrophages was determined by Western blot. (<b>B)</b> PON1 transfected J774 macrophages and control macrophages (CTRL) were loaded with [³H]-cholesterol (2 μCi/mL) for 24 h followed by incubation with 50 μg/mL of apoA-1 for 4 h. Data are expressed as means ± SEM, n = 3. **p<0.005.</p