Nuclear Receptor NR4A2 Orchestrates Th17 Cell-Mediated Autoimmune Inflammation via IL-21 Signalling

<div><p>IL-17-producing CD4⁺ T helper 17 (Th17) cells are pathogenic in a range of human autoimmune diseases and corresponding animal models. We now demonstrate that such T cells infiltrating the target organ during the induction of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveoretinitis (EAU) specifically express NR4A2. Further, we reveal a critical involvement of NR4A2 in Th17 cell functions and Th17 cell-driven autoimmune diseases. When NR4A2 expression was blocked with siRNA, full Th17 differentiation was prevented <i>in vitro</i>: although cells expressed the master Th17 regulator, RORγt, they expressed reduced levels of IL-23R and were unable to produce IL-17 and IL-21. Notably, Th17 differentiation in the absence of NR4A2 was restored by exogenous IL-21, indicating that NR4A2 controls full maturation of Th17 cells via autocrine IL-21 signalling. Preventing NR4A2 expression <i>in vivo</i> by systemic treatment with NR4A2-specific siRNA also reduced Th17 effector responses and furthermore protected mice from EAE induction. In addition, the lack of disease was associated with a reduction in autocrine IL-21 production and IL-23R expression. Similar modulation of NR4A2 expression was also effective as an intervention, reversing established autoimmune responses and ameliorating clinical disease symptoms. Thus, NR4A2 appears to control Th17 differentiation and so plays an essential role in the development of Th17-mediated autoimmune disease. As NR4A2 is also upregulated during human autoimmune disease, targeting NR4A2 may provide a new therapeutic approach in treating autoimmune disease.</p> </div

Autoimmune induction of NR4A2 in CD4⁺ T cells is associated with IL-17-secreting T cells.

<p>EAE or EAU was induced in C57BL/6 mice by immunization with MOG_35–55 or IRBP_1–20 peptide in CFA, respectively. CD4⁺ T cells were purified from spleen, blood, or target organ (CNS or retina) on the indicated days and RNA was isolated. <b>A</b> and <b>B:</b> NR4A2 expression was quantified by real time PCR relative to GAPDH for T cells from EAE (A) or EAU (B). Timepoints correspond to a minimum of 5 animals and data are representative of 3 independent experiments. CD4⁺ T cells from mice with EAE were restimulated with PMA/ionomycin for 3 hours and 4 populations of cytokine secreting cells (IL-17+IFN-γ-, IL-17+IFN-γ+, IL-17-IFN-γ+, and IL-17-IFN-γ-) were sorted by flow cytometry using IFN-γ and IL-17 cytokine secretion assay kits. <b>C:</b> NR4A2 expression by populations of cytokine-secreting CD4⁺ T cells was quantified by real time PCR at day 15 post-EAE induction for lymph nodes (LN) and CNS-infiltrating cells (CNS), and day 25 for blood T cells. <b>D</b> and <b>E:</b> NR4A2 expression by IL-17⁺IFN-γ⁻ or IL-17⁺IFN-γ⁺ CNS-infiltrating T cells (<b>D</b>) or blood T cells (<b>E</b>) was measured by real time PCR at a range of timepoints. Data are representative of 2 independent experiments. <b>F:</b> Th1-mediated diabetes was induced in C57BL/6 mice by 5 daily low dose STZ treatments. Other groups of C57BL/6 mice were immunized with peptides in CFA plus PTX either OVA_323–339 (OVA/CFA) or MOG_35–55 (MOG/CFA). On day 22, NR4A2 expression was assessed by real time PCR amongst CD4⁺ T cells from spleen and leukocytes isolated from the relevant target organ (ND, OVA/CFA; CNS, EAE; pancreas, STZ). Timepoints correspond to a minimum of 5 animals and data are representative of 2 independent experiments.</p

NR4A2 knockdown prevents IL-17 secretion but not RORγt upregulation.

<p>Naïve CD4⁺ T cells were transfected by electroporation with NR4A2-specific siRNA or scrambled control siRNA. Cells were then activated with 5 µg/ml plate-bound CD3-specific mAb and 0.5 µg/ml soluble CD28-specific mAb. <b>A:</b> IFN-γ production by cells activated in the presence or absence of 10 ng/ml IL-12 after 96 hours of culture. <b>B:</b> IL-17 production by cells activated in the presence of 20 ng/ml IL-6, 20 ng/ml IL-23, and TGF-β at a range of concentrations after 96 hours of culture. Significant differences between control and NR4A2 siRNA-treatments were tested with a student’s t-test, *p<0.05. <b>C:</b> IL-17 and IFN-γ intracellular cytokine staining for transfected T cells (control siRNA, left plots; NR4A2 siRNA, right plots) cultured for 96 hours in the presence of 10 ng/ml IL-12 (Th1 conditions, top row plots) or 20 ng/ml IL-6, 20 ng/ml IL-23, and 3 ng/ml TGF-β (Th17 conditions, bottom row plots). <b>D:</b> RORγt RNA expression as measured by real time PCR by activated T cells cultured under Th17 polarizing conditions at a range of timepoints. Data are representative of 5 independent experiments.</p

Absence of NR4A2 is associated with a lack of IL-21 production by Th17 cells.

<p>Naïve CD4⁺ T cells were transfected by electroporation with NR4A2-specific siRNA or scrambled control siRNA and were activated with 5 µg/ml plate-bound CD3-specific mAb and 0.5 µg/ml soluble CD28-specific mAb in the presence of 20 ng/ml IL-6, 20 ng/ml IL-23, and 3 ng/ml TGF-β. <b>A:</b> RNA levels of IL-21, IL-23R, and IL-17 were quantified by real time PCR at the indicated timepoints following activation. Data are representative of 3 independent experiments. <b>B:</b> IL-21 supernatant concentration was measured by ELISA at 96 hours. Data are representative of 3 independent experiments. *p<0.05. <b>C:</b> RNA expression of c-maf quantified by real time PCR. <b>D:</b> RNA expression of IL-22 quantified by real time PCR. Data are representative of 2 independent experiments.</p

Systemic administration of NR4A2-specific siRNA reduces EAE severity.

<p>siRNA, either NR4A2-specific or control, was stabilized in a collagen matrix and administered <i>i.v.</i> to groups of C57BL/6 mice at the time of EAE induction. EAE was scored clinically (<b>A</b>) and at day 15 post-EAE induction, production of IL-17 and IFN-γ by CNS-infiltrating leukocytes restimulated with 20 µg/ml MOG peptide for 96 hours were assessed by ELISA (<b>B</b>). CNS-infiltrating T cells were also assessed for IL-17 production at a range of timepoints by intracellular flow cytometry (<b>C</b>). Data are representative of 3 independent experiments. Control or NR4A2-specific siRNA was applied to MOG-immunized mice at day 10 post-disease induction and disease was scored clinically (<b>D</b>). Timepoints correspond to a minimum of 5 animals and data are representative of 2 independent experiments. IL-21 and IL-23R expressions amongst CNS-infiltrating T cells were measured by real time PCR (<b>E&F</b>). Data are representative of 2 independent experiments. Clinical scores in panels A) and D) were tested with a two-way ANOVA test. *p<0.01, **p<0.001.</p

Additional file 1: Figure S1. of MicroRNA-101a regulates microglial morphology and inflammation

The expression level of miR-101a in MG6 cells cultured with each miRNA was analyzed by quantitative real-time PCR. Figure S2. Neither miR-101a nor its inhibitor exhibited any influence on the viability of MG6 cells as measured by MTT assay. (PPTX 52 kb

Additional file 2: Figure S3. of MicroRNA-101a regulates microglial morphology and inflammation

Predicted miR-101a target genes included in MAPK signaling pathway. Asterisks indicate miR-101a targets. (TIF 101 kb

A Population of Kisspeptin/Neurokinin B Neurons in the Arcuate Nucleus May Be the Central Target of the Male Effect Phenomenon in Goats

<div><p>Exposure of females to a male pheromone accelerates pulsatile gonadotropin-releasing hormone (GnRH) secretion in goats. Recent evidence has suggested that neurons in the arcuate nucleus (ARC) containing kisspeptin and neurokinin B (NKB) play a pivotal role in the control of GnRH secretion. Therefore, we hypothesized that these neurons may be the central target of the male pheromone. To test this hypothesis, we examined whether NKB signaling is involved in the pheromone action, and whether ARC kisspeptin/NKB neurons receive input from the medial nucleus of the amygdala (MeA)—the nucleus suggested to relay pheromone signals. Ovariectomized goats were implanted with a recording electrode aimed at a population of ARC kisspeptin/NKB neurons, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA) volleys, was measured. Pheromone exposure induced an MUA volley and luteinizing hormone (LH) pulse in control animals, whereas the MUA and LH responses to the pheromone were completely suppressed by the treatment with an NKB receptor antagonist. These results indicate that NKB signaling is a prerequisite for pheromone action. In ovariectomized goats, an anterograde tracer was injected into the MeA, and possible connections between the MeA and ARC kisspeptin/NKB neurons were examined. Histochemical observations demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA. These results suggest that the male pheromone signal is conveyed via the MeA to ARC kisspeptin neurons, wherein the signal stimulates GnRH pulse generator activity through an NKB signaling-mediated mechanism in goats.</p> </div

Multiple-unit activity (MUA) profiles and plasma luteinizing hormone concentrations in two representative ovariectomized goats.

<p>Goats were exposed to the male pheromone in the absence (upper panels) or presence (lower panels) of the NK3R antagonist SB222200. The timing of pheromone exposure is indicated by an arrow and dotted line. Vehicle (open arrowheads) or SB222200 (closed arrowheads) were injected intravenously twice (at the first and fourth blood sampling points) after the preceding MUA volley.</p

Photomicrographs of biotin dextran amine-labeled fibers.

<p>A, Labeled fibers forming a dense plexus in the bed nucleus of the stria terminalis. B, Labeled fibers coursing areas over the optic tract. C, Labeled fibers forming a dense plexus in the ventromedial nucleus of the hypothalamus. D, E, Labeled fibers distributed in the rostral (D) and middle region (E) of the arcuate nucleus. F, High magnification of the area indicated in E. Arrowheads indicate apparent terminal buttons. ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; LHA; lateral hypothalamic area; VMH, ventromedial nucleus of the hypothalamus; <i>fx</i>, fornix; <i>LV</i>, lateral ventricle; <i>ot</i>, optic tract; <i>pt</i>, par tuberalis; <i>3V</i>, third ventricle. Scale bar: A = 300 µm; B-E =150 µm; F= 20 µm.</p