Jean-Marie Bach

MicroRNA-29b Modulates Innate and Antigen-Specific Immune Responses in Mouse Models of Autoimmunity

In addition to important regulatory roles in gene expression through RNA interference, it has recently been shown that microRNAs display immune stimulatory effects through direct interaction with receptors of innate immunity of the Toll-like receptor family, aggravating neuronal damage and tumour growth. Yet no evidence exists on consequences of microRNA immune stimulatory actions in the context of an autoimmune disease. Using microRNA analogues, we here show that pancreatic beta cell-derived microRNA sequences induce pro-inflammatory (TNFa, IFNa, IL-12, IL-6) or suppressive (IL-10) cytokine secretion by primary mouse dendritic cells in a sequence-dependent manner. For miR-29b, immune stimulation in RAW264.7 macrophages involved the endosomal Toll-like receptor-7, independently of the canonical RNA interference pathway. In vivo, the systemic delivery of miR-29b activates CD11b+B220− myeloid and CD11b-B220+ plasmacytoid dendritic cells and induces IFNa, TNFa and IL-6 production in the serum of recipient mice. Strikingly, in a murine model of adoptive transfer of autoimmune diabetes, miR-29b reduces the cytolytic activity of transferred effector CD8+ T-cells, insulitis and disease incidence in a single standalone intervention. Endogenous miR-29b, spontaneously released from beta-cells within exosomes, stimulates TNFa secretion from spleen cells isolated from diabetes-prone NOD mice in vitro. Hence, microRNA sequences modulate innate and ongoing adaptive immune responses raising the question of their potential role in the breakdown of tolerance and opening up new applications for microRNA-based immune therapy.

Diabetes acceleration by cyclophosphamide in the non-obese diabetic mouse is associated with differentiation of immunosuppressive monocytes into immunostimulatory cells

Cyclophosphamide (CTX) was previously shown to induce the recruitment of immunosuppressive myeloid cells in mouse. In the non-obese diabetic (NOD) mouse, which develops spontaneously type I diabetes, CTX is widely known to accelerate the autoimmune process. Our data demonstrated that CTX actually did mobilize an immunosuppressive myeloid CD11b(+) Ly-6G(-) population in the NOD mouse spleen in addition to a well-identified neutrophil CD11b(+) Ly-6G(+) population. CD11b(+) Ly-6G(-) cells, in contrast with CD11b(+) Ly-6G(+) cells, were able to inhibit in vitro mitogen-induced syngeneic T cell proliferation. CD11b(+) Ly-6G(-) cells represented a heterogeneous population mainly made of CD31(hi) cells and Ly-6C(+) monocytes. Only these last ones supported the immunosuppressive in vitro activity and resembled circulating inflammatory monocytes according to flow cytometry, cytology and RT-PCR data. Although CD11b(+) Ly-6G(-) Ly-6C(+) cells exhibited immunosuppressive function in vitro, they were not able to control the autoimmune response following CTX injection. Our data show that these CTX-induced immunosuppressive myeloid cells actually behaved as very plastic cells in vitro. Likewise, in the model of prediabetic NOD/SCID mice, CD11b(+) Ly-6G(-) Ly-6C(+) were able to differentiate into CD11c+ cells after i.v. injection. Herein, we described a new mechanism by which CTX might induce diabetes acceleration in the NOD mouse. In summary, recruited immunosuppressive cells might participate in the immunopotentiating effect of CTX on the autoimmune response by their further differentiation into immunostimulatory cells.

Type 1 Diabetes Prone NOD Mice Have Diminished Cxcr1 mRNA Expression in Polymorphonuclear Neutrophils and CD4+ T Lymphocytes

In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD) mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis.

β2-adrenoreceptor stimulation dampens the LPS-induced M1 polarization in pig macrophages

Abstract The cross‐talk between sympatho‐adreno‐medullar axis and innate immunity players was mainly studied in rodents. In intensive husbandry, pigs are exposed to multiple stressors inducing repeated releases of catecholamines that bind to adrenoreceptors (AR) on target cells. Among adrenoreceptors, the &bgr;2‐AR is largely expressed by immune cells including macrophages. We report herein on the effects of catecholamines, through &bgr;2‐AR stimulation, on pig macrophage functions activated by LPS. &bgr;2‐AR stimulation of porcine macrophages prevented the LPS‐induced increase in TNF&agr; and IL‐8 secretion while increasing IL‐10 secretion. In contrast, treatment with a &bgr;2‐agonist had no effect on anti‐microbial functions. Lastly, &bgr;2‐AR stimulation of macrophages reduced the expression of genes up‐regulated by LPS. Altogether, we demonstrated that &bgr;2‐AR stimulation of porcine macrophages prevented polarization towards a pro‐inflammatory phenotype. Since porcine macrophages are a suitable model for human macrophages, our results might be relevant to appreciate catecholamine effects on human macrophages. HighlightsPig macrophages express different adrenoreceptors including ADR&bgr;2.Catecholamines and &bgr;2‐agonist modulate their cytokines secretion in response to LPS.&bgr;2‐AR stimulation of pig macrophages does not alter their anti‐microbial functions.&bgr;2‐agonist treatment limits LPS‐induced M1 gene expression in pig macrophages.

β2-adrenergic stimulation of dendritic cells favors IL-10 secretion by CD4 + T cells

Adrenergic receptor agonists and antagonists are extensively used as drugs in medicine for a broad spectrum of indications. We examined the consequences of β2-adrenergic stimulation of murine dendritic cells (DCs) on CD4+ T cell activation. We demonstrated in vitro that treatment of LPS-matured DCs with the β2-agonist salbutamol reduced their ability to trigger OT-II T cell proliferation specific for ovalbumin antigen. Salbutamol also induced a decrease in MHC class II molecule expression by DC through Gi protein activation. Co-culture of CD4+ T cells with salbutamol-conditioned mature DC impaired TNFα and IL-6 secretion while preserving IL-10 production by T cells. Using a vaccination protocol in mice, we showed that salbutamol favored IL-10-producing CD4+ T cells. None of these effects was observed when working with β2-adrenoreceptor deficient mice. Finally, we suggest that β2-adrenergic stimulation of DC could be an interesting way to shape CD4+ T cell responses for the purposes of immunotherapy.

Les microARNs : vers de nouvelles pistes thérapeutiques et de biomarqueurs dans le diabète de type 1

Introduction La predisposition polygenique complexe au diabete de type 1 (DT1) n’explique pas, seule, la forte progression de l’incidence de la maladie. Il est crucial de mieux comprendre les mecanismes physiopathologiques initiaux de l’agression des cellules beta pancreatiques afin d’identifier (1) des biomarqueurs originaux de cette agression lors du prediabete ou pour le suivi de protocole de recherche therapeutique et (2) de nouvelles cibles therapeutiques. Les microARNs (miRNAs) sont impliques dans le controle de toutes les fonctions physiologiques et cellulaires par un mecanisme d’interference avec l’ARNm. Abondants dans les fluides biologiques, ils constituent une nouvelle classe tres prometteuse de biomarqueurs non-invasifs. L’objectif de notre projet est (1) l’identification de profils de miRNAs seriques specifiques du DT1 et (2) l’etude de leur role sur la balance auto-immunite/tolerance a l’initiation de la pathogenie du DT1. Materiels et methodes/Resultats Nos resultats preliminaires chez la souris NOD (non obese diabetic) montrent que la quantite serique relative de certains miRNAs augmente ou diminue au cours de l’agression des cellules beta pancreatiques. De plus, nous venons d’identifier 3 sequences de miRNAs exprimees par les cellules beta pancreatiques capables de stimuler la secretion d’IFNa, de TNFa et d’IL-12 par des cellules dendritiques myeloides ou plasmacytoides. Cette stimulation semble impliquer le recepteur de l’immunite innee Toll Like Receptor 7 . Un de ces miRNAs montre des proprietes tolero-genes dans le transfert adoptif de diabete dans le modele murin transgenique CL4/Ins-HA. Les miRNAs et les recepteurs de l’immunite innee sont d’origine tres ancienne et bien conserves entre especes, c’est pourquoi nous etudions actuellement le potentiel immunostimulant des miRNAs sur les cellules immunitaires humaines. Conclusion L’exploitation de ce nouvel effet biologique potentiel des miRNAs (immunostimulation), en plus de leurs proprietes interferentes, pourrait ouvrir de nouvelles perspectives dans la modulation therapeutique des reponses T diabetogenes et du processus auto-immun. Chez l’homme, nous visons aussi l’identification de miR-IDD (miRNAs sont localises dans un locus IDDM) presentant un profil d’expression serique modifie et specifique du DT1. Leur interet comme biomarqueurs du DT1 chez l’homme est a l’etude.