René Riedel

The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor–mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell–mediated immune responses and open new therapeutic possibilities.

IL-17 and GM-CSF Expression Are Antagonistically Regulated by Human T Helper Cells

GM-CSF–producing T helper cells in humans follow a distinct regulation program as compared to TH17 cells and are associated with multiple sclerosis. Cytokine Rivalry In patients with autoimmune diseases, cytokines—secreted immune mediators—are a crucial cause of tissue damage. However, the interplay between different cytokines and their individual roles in disease aggravation and resolution remain poorly defined, especially in humans. Noster et al. report that T helper (TH) cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) may play a pathogenic role in the brain of patients with multiple sclerosis (MS). They found that TH17-related cytokines—thought from mouse studies to be critical for pathogenesis—actually prevented induction of GM-CSF, whereas TH1-type cytokines promoted GM-CSF. These data provide a rationale for decreasing GM-CSF in patients with MS and suggest that, for MS at least, human may know best. Although T helper 17 (TH17) cells have been acknowledged as crucial mediators of autoimmune tissue damage, the effector cytokines responsible for their pathogenicity still remain poorly defined, particularly in humans. In mouse models of autoimmunity, the pathogenicity of TH17 cells has recently been associated with their production of granulocyte-macrophage colony-stimulating factor (GM-CSF). We analyzed the regulation of GM-CSF expression by human TH cell subsets. Surprisingly, the induction of GM-CSF expression by human TH cells is constrained by the interleukin-23 (IL-23)/ROR-γt/TH17 cell axis but promoted by the IL-12/T-bet/TH1 cell axis. IL-2–mediated signal transducer and activator of transcription 5 (STAT5) signaling induced GM-CSF expression in naïve and memory TH cells, whereas STAT3 signaling blocked it. The opposite effect was observed for IL-17 expression. Ex vivo, GM-CSF+ TH cells that coexpress interferon-γ and T-bet could be distinguished by differential chemokine receptor expression from a previously uncharacterized subset of GM-CSF–only–producing TH cells that did not express TH1, TH2, and TH17 signature cytokines or master transcription factors. Our findings demonstrate distinct and counterregulatory pathways for the generation of IL-17– and GM-CSF–producing cells and also suggest a pathogenic role for GM-CSF+ T cells in the inflamed brain of multiple sclerosis (MS) patients. This provides not only a scientific rationale for depleting T cell–derived GM-CSF in MS patients but also multiple new molecular checkpoints for therapeutic GM-CSF suppression, which, unlike in mice, do not associate with the TH17 but instead with the TH1 axis.

ICOS maintains the T follicular helper cell phenotype by down-regulating Krüppel-like factor 2

ICOS signaling is required for inhibition of the transcription factor Klf2, which controls expression of genes expressed by follicular T helper (Tfh) cells. When ICOS signaling is blocked, Tfh cells lose expression of characteristic Tfh genes and revert to an effector phenotype, resulting in disruption of the germinal center response.

Memory CD8+ T cells colocalize with IL-7+ stromal cells in bone marrow and rest in terms of proliferation and transcription

It is believed that memory CD8+ T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL‐7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8+ T cells individually colocalize with IL‐7+ reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4‐1BB (CD137), IL‐2, or IFN‐γ, despite the expression of CD69 on about 30% of the cells. Ninety‐five percent of the memory CD8+ T cells in BM are in G0 phase of cell cycle and do not express Ki‐67. Less than 1% is in S/M/G2 of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8+ memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8+ memory T cells. Taken together, the present results suggest that CD8+ memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL‐7 receptor signaling.

Vitamin D Receptor Activation Improves Allergen-Triggered Eczema in Mice

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that has increased in prevalence over the last several decades in industrialized countries. AD is a multifactorial, heterogenous disease with a variety of defects in the immune system, in antimicrobial defense mechanisms and epidermal barrier integrity, which collectively contribute to the risk and severity of AD development. Vitamin D receptor (VDR) signaling has been shown to be important not only in the immune system but also in the skin and in particular keratinocytes to regulate skin homeostasis and epidermal barrier function. However, this work aimed to analyze the role and clinical efficiency of VDR activation by a VDR agonist without calcium-mobilizing activity in a mouse model of allergen-triggered eczema. We show that the systemic administration of the low-calcemic VDR agonist significantly improved the allergen-triggered eczema. Thereby, forkhead box P3 (Foxp3)-expressing regulatory T cells, revealed to have a role in AD, were selectively increased in the skin of VDR agonist-treated mice. Moreover, our results demonstrate a marked induction of skin barrier gene and antimicrobial peptide gene expression in skin lesions of VDR agonist-treated mice. Thus, our study provides evidence that systemic VDR agonist treatment may improve allergen-triggered eczema in vivo.

miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T‐bet induce expression of microRNA‐148a (miR‐148a). miR‐148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR‐148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR‐148a antagomir‐treated cells restores viability of the Th1 cells, demonstrating that miR‐148a controls survival by regulating Bim expression. Thus, Twist1 and T‐bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

Nuclear factor of activated T cells regulates the expression of interleukin-4 in Th2 cells in an all-or-none fashion.

Background: Not every T helper type 2 (Th2) lymphocyte imprinted to express interleukin-4 (IL-4) does so when activated. Results: Preventing nuclear translocation of the nuclear factor of activated T cells (NFAT) reduces the number of Th2 lymphocytes reexpressing IL-4. Conclusion: NFAT is the limiting factor determining digital IL-4 expression in Th2 lymphocytes. Significance: This might help us to understand the regulation of immunopathology in allergy and asthma. Th2 memory lymphocytes have imprinted their Il4 genes epigenetically for expression in dependence of T cell receptor restimulation. However, in a given restimulation, not all Th cells with a memory for IL-4 expression express IL-4. Here, we show that in reactivated Th2 cells, the transcription factors NFATc2, NF-kB p65, c-Maf, p300, Brg1, STAT6, and GATA-3 assemble at the Il4 promoter in Th2 cells expressing IL-4 but not in Th2 cells not expressing it. NFATc2 is critical for assembly of this transcription factor complex. Because NFATc2 translocation into the nucleus occurs in an all-or-none fashion, dependent on complete dephosphorylation by calcineurin, NFATc2 controls the frequencies of cells reexpressing Il4, translates analog differences in T cell receptor stimulation into a digital decision for Il4 reexpression, and instructs all reexpressing cells to express the same amount of IL-4. This analog-to-digital conversion may be critical for the immune system to respond to low concentrations of antigens.

High-resolution microbiota flow cytometry reveals dynamic colitis-associated changes in fecal bacterial composition.

Using high‐resolution flow cytometry of bacterial shape (forward scatter) and DNA content (DAPI staining), we detected dramatic differences in the fecal microbiota composition during murine colitis that were validated using 16S rDNA sequencing. This innovative method provides a fast and inexpensive tool to interrogate the microbiota on the single‐cell level.

Direct uptake of Antagomirs and efficient knockdown of miRNA in primary B and T lymphocytes

Depending on their physiological expression level, microRNAs (miRNA) address different target genes, thus have different biological functions. To identify these, the physiological expression has to be blocked. Here, we describe the use of inhibitory cholesterol-modified oligonucleotides (Antagomirs) to inhibit miRNAs selectively in primary human and murine T and B lymphocytes. Due to their lipophilic cholesterol tag Antagomirs enter primary lymphocytes efficiently and quantitatively. We show here that at concentrations of 0.125 to 1 μM, Antagomirs selectively inhibit expression of their target miRNA up to 99.5% without affecting cell viability.

02.34 Enhanced conventional cd4+t cell proliferation in sle is associated with up-regulation of microrna-182 and increased il-7 receptor signalling

Background Recent reports have shown dysregulated microRNAs (miRNAs) in murine models of lupus, among them increased expression of microRNA-182 (miRNA-182), which has been demonstrated to target the transcription factor FOXO1 in activated murine CD4+ T cells, leading to spontaneous T cell activation and clonal expansion. Here we aimed to investigate the expression of miR-182 and FOXO1 in T cells from human SLE patients. Methods Expression levels of miR-182 were analysed with RT-PCR in purified peripheral blood CD4+ T cells from 9 patients with SLE and age/sex-matched healthy controls (HC). Multicolor flow cytometry was performed to analyse CD4+ T cell expression for FOXO1, Ki-67, Foxp3, the interleukin-7 receptor-α (CD127) and phosphorylated STAT-5a (pSTAT5). Analysis of serum IL-7 levels was performed with ELISA in 27 SLE patients and HC. Induction of miR-182 was assessed in vitro after polyclonal T cell stimulation in the presence of IL-7, and inhibition of T cell proliferation investigated using mir-182 antagomirs. Results MiRNA-182 was significantly upregulated in CD4+ T cells from SLE patients compared to HC, while the FOXO1 expression was significantly decreased. The percentage of proliferating Ki-67+ conventional Foxp3- CD4+ T cells TCON was significantly higher in SLE compared to HC (3.85% vs. 1.58%, p<0.001) and their basal pSTAT5 levels significantly enhanced, suggesting a recent stimulation with common gamma chain(γc)-signalling cytokines. SLE TCON displayed decreased expression levels for the FOXO1 target gene CD127 (MFI 2021 vs. 2553, p=0.049) and serum IL-7 levels were significantly higher in SLE compared to HC (17.0 pg/ml vs. 10.2 pg/ml, p=0.001). In vitro, miR-182 could be induced by IL-7, and specific inhibition of miR-182 inhibited T cell proliferation and survival. Conclusion Our data suggest that enhanced IL7R/STAT5 signalling mediates the induction of miR182 expression, which promotes the proliferation of conventional Foxp3- T cells SLE. Collectively, our data provide new insights in the pathophysiology of T cell hyperactivity in SLE and identifies miR-182 as a candidate target for future therapeutic approaches.