Stephanie Hucke

The nuclear receptor PPARγ selectively inhibits Th17 differentiation in a T cell–intrinsic fashion and suppresses CNS autoimmunity

T helper cells secreting interleukin (IL)-17 (Th17 cells) play a crucial role in autoimmune diseases like multiple sclerosis (MS). Th17 differentiation, which is induced by a combination of transforming growth factor (TGF)-β/IL-6 or IL-21, requires expression of the transcription factor retinoic acid receptor–related orphan receptor γt (RORγt). We identify the nuclear receptor peroxisome proliferator–activated receptor γ (PPARγ) as a key negative regulator of human and mouse Th17 differentiation. PPARγ activation in CD4+ T cells selectively suppressed Th17 differentiation, but not differentiation into Th1, Th2, or regulatory T cells. Control of Th17 differentiation by PPARγ involved inhibition of TGF-β/IL-6–induced expression of RORγt in T cells. Pharmacologic activation of PPARγ prevented removal of the silencing mediator for retinoid and thyroid hormone receptors corepressor from the RORγt promoter in T cells, thus interfering with RORγt transcription. Both T cell–specific PPARγ knockout and endogenous ligand activation revealed the physiological role of PPARγ for continuous T cell–intrinsic control of Th17 differentiation and development of autoimmunity. Importantly, human CD4+ T cells from healthy controls and MS patients were strongly susceptible to PPARγ-mediated suppression of Th17 differentiation. In summary, we report a PPARγ-mediated T cell–intrinsic molecular mechanism that selectively controls Th17 differentiation in mice and in humans and that is amenable to pharmacologic modulation. We therefore propose that PPARγ represents a promising molecular target for specific immunointervention in Th17-mediated autoimmune diseases such as MS.

Sodium chloride promotes pro-inflammatory macrophage polarization thereby aggravating CNS autoimmunity

The increasing incidence in Multiple Sclerosis (MS) during the last decades in industrialized countries might be linked to a change in dietary habits. Nowadays, enhanced salt content is an important characteristic of Western diet and increased dietary salt (NaCl) intake promotes pathogenic T cell responses contributing to central nervous system (CNS) autoimmunity. Given the importance of macrophage responses for CNS disease propagation, we addressed the influence of salt consumption on macrophage responses in CNS autoimmunity. We observed that EAE-diseased mice receiving a NaCl-high diet showed strongly enhanced macrophage infiltration and activation within the CNS accompanied by disease aggravation during the effector phase of EAE. NaCl treatment of macrophages elicited a strong pro-inflammatory phenotype characterized by enhanced pro-inflammatory cytokine production, increased expression of immune-stimulatory molecules, and an antigen-independent boost of T cell proliferation. This NaCl-induced pro-inflammatory macrophage phenotype was accompanied by increased activation of NF-kB and MAPK signaling pathways. The pathogenic relevance of NaCl-conditioned macrophages is illustrated by the finding that transfer into EAE-diseased animals resulted in significant disease aggravation compared to untreated macrophages. Importantly, also in human monocytes, NaCl promoted a pro-inflammatory phenotype that enhanced human T cell proliferation. Taken together, high dietary salt intake promotes pro-inflammatory macrophages that aggravate CNS autoimmunity. Together with other studies, these results underline the need to further determine the relevance of increased dietary salt intake for MS disease severity.

Licensing of myeloid cells promotes central nervous system autoimmunity and is controlled by peroxisome proliferator-activated receptor γ

During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical for disease progression and ultimately organ damage. Here, we demonstrate that local cross-talk between invading autoreactive T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. This T cell-mediated licensing of central nervous system myeloid cells triggered astrocytic CCL2-release and promoted recruitment of inflammatory CCR2(+)-monocytes, which are the main effectors of disease progression. By employing a cell-specific knockout model, we identify the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in myeloid cells as key regulator of their disease-determining interactions with autoreactive T cells and brain-resident cells, respectively. LysM-PPARγ(KO) mice exhibited disease exacerbation during the effector phase of experimental autoimmune encephalomyelitis characterized by enhanced activation of central nervous system myeloid cells accompanied by pronounced local CCL2 production and inflammatory monocyte invasion, which finally resulted in increased demyelination and neuronal damage. Pharmacological PPARγ activation decreased antigen-specific T cell-mediated licensing of central nervous system myeloid cells, reduced myeloid cell-mediated neurotoxicity and hence dampened central nervous system autoimmunity. Importantly, human monocytes derived from patients with multiple sclerosis clearly responded to PPARγ-mediated control of proinflammatory activation and production of neurotoxic mediators. Furthermore, PPARγ in human monocytes restricted their capacity to activate human astrocytes leading to dampened astrocytic CCL2 production. Together, interference with the disease-promoting cross-talk between central nervous system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease progression and lesion development during ongoing central nervous system autoimmunity.

Salt-dependent chemotaxis of macrophages

Besides their role in immune system host defense, there is growing evidence that macrophages may also be important regulators of salt homeostasis and blood pressure by a TonEBP-VEGF-C dependent buffering mechanism. As macrophages are known to accumulate in the skin of rats fed under high salt diet conditions and are pivotal for removal of high salt storage, the question arose how macrophages sense sites of high sodium storage. Interestingly, we observed that macrophage-like RAW264.7 cells, murine bone marrow-derived macrophages and peritoneal macrophages recognize NaCl hypertonicity as a chemotactic stimulus and migrate in the direction of excess salt concentration by using an in vitro transwell migration assay. While RAW264.7 cells migrated toward NaCl in a dose-dependent fashion, no migratory response toward isotonic or hypotonic media controls, or other osmo-active agents, e.g. urea or mannitol, could be detected. Interestingly, we could not establish a specific role of the osmoprotective transcription factor TonEBP in regulating salt-dependent chemotaxis, since the specific migration of bone marrow-derived macrophages following RNAi of TonEBP toward NaCl was not altered. Although the underlying mechanism remains unidentified, these data point to a thus far unappreciated role for NaCl-dependent chemotaxis of macrophages in the clearance of excess salt, and suggest the existence of novel NaCl sensor/effector circuits, which are independent of the TonEBP system.

Fingolimod treatment promotes regulatory phenotype and function of B cells

To evaluate the influence of Fingolimod treatment on B‐cell subset composition and function in multiple sclerosis patients and its potential clinical relevance.

Increased Antigen Cross-Presentation but Impaired Cross-Priming after Activation of Peroxisome Proliferator-Activated Receptor γ Is Mediated by Up-Regulation of B7H1

Dendritic cells are able to take up exogenous Ags and present Ag-derived peptides on MHC class I molecules, a process termed cross-presentation. The mannose receptor (MR), an endocytic receptor expressed on a variety of APCs, has been demonstrated to target soluble Ags exclusively toward cross-presentation. In this study, we investigated the role of the murine nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor with immunomodulatory properties, in MR-mediated endocytosis and cross-presentation of the model Ag OVA. We could demonstrate both in vitro and in vivo that activation of PPARγ resulted in increased MR expression, which in consequence led to enhanced MR-mediated endocytosis and elevated cross-presentation of soluble OVA. Concomitantly, activation of PPARγ in dendritic cells induced up-regulation of the coinhibitory molecule B7H1, which, despite enhanced cross-presentation, caused an impaired activation of naive OVA-specific CD8+ T cells and the induction of T cell tolerance. These data provide a mechanistic basis for the immunomodulatory action of PPARγ which might open new possibilities in the development of therapeutic approaches aimed at the control of excessive immune responses, e.g., in T cell-mediated autoimmunity.

Implications of dietary salt intake for multiple sclerosis pathogenesis

In recent years it has become increasingly clear that, alongside genetic risk factors, environmental factors strongly influence the incidence and severity of multiple sclerosis (MS). Based on observations from epidemiological studies, the potential contribution of dietary habits has lately been a matter of debate. Recently it was shown that high salt conditions promote pathogenic T-cell responses and aggravate autoimmunity in an animal model of MS, suggesting that high dietary salt intake might promote central nervous system (CNS) autoimmunity. However, so far, not much is known about the influence of dietary salt intake on MS disease pathology. Here, we discuss the association of dietary salt levels and MS with a special focus on the mechanisms of salt-mediated modulation of the different cell types critically involved in the pathophysiology of MS.

Non-steroidal anti-inflammatory drug indometacin enhances endogenous remyelination

Multiple sclerosis is the most frequent demyelinating disease in the CNS that is characterized by inflammatory demyelinating lesions and axonal loss, the morphological correlate of permanent clinical disability. Remyelination does occur, but is limited especially in chronic disease stages. Despite effective immunomodulatory therapies that reduce the number of relapses the progressive disease phase cannot be prevented. Therefore, promotion of neuroprotective and repair mechanisms, such as remyelination, represents an attractive additional treatment strategy. A number of pathways have been identified that may contribute to impaired remyelination in MS lesions, among them the Wnt/β-catenin pathway. Here, we demonstrate that indometacin, a non-steroidal anti-inflammatory drug (NSAID) that has been also shown to modulate the Wnt/β-catenin pathway in colorectal cancer cells promotes differentiation of primary human and murine oligodendrocytes, myelination of cerebellar slice cultures and remyelination in cuprizone-induced demyelination. Our in vitro experiments using GSK3β inhibitors, luciferase reporter assays and oligodendrocytes expressing a mutant, dominant stable β-catenin indicate that the mechanism of action of indometacin depends on GSK3β activity and β-catenin phosphorylation. Indometacin might represent a promising treatment option to enhance endogenous remyelination in MS patients.

Targeting Different Monocyte/Macrophage Subsets Has No Impact on Outcome in Experimental Stroke

Background and Purpose— Peripheral immune cell infiltration contributes to neural injury after ischemic stroke. However, in contrast to lymphocytes and neutrophils, the role of different monocyte/macrophage subsets remains to be clarified. Therefore, we evaluated the effects of selective and unselective monocyte/macrophage depletion and proinflammatory (M1-) and anti-inflammatory (M2-) macrophage transfer on the outcome after experimental cerebral ischemia. Methods— To assess short-term effects of monocytes/macrophages in acute ischemic stroke, mice underwent transient middle cerebral artery occlusion and received either clodronate liposomes for unselective macrophage depletion, MC-21-antibody for selective depletion of proinflammatory Ly-6Chigh monocytes, or proinflammatory (M1-) or anti-inflammatory (M2-) macrophage transfer. In addition, the impact of MC-21-antibody administration and M2-macrophage transfer on long-term neural recovery was investigated after photothrombotic stroke. Neurobehavioral tests were used to analyze functional outcomes, infarct volumes were determined, and immunohistochemical analyses were performed to characterize the postischemic inflammatory reaction. Results— Selective and unselective monocyte/macrophage depletion and M1- and M2-macrophage transfer did not influence tissue damage and neurobehavioral outcomes in the acute phase after middle cerebral artery occlusion. Beyond, selective depletion of Ly-6Chigh monocytes and M2-macrophage transfer did not have an impact on neural recovery after photothrombotic stroke. Conclusions— Targeting different monocyte/macrophage subsets has no impact on outcome after ischemic stroke in mice. Altogether, our study could not identify monocytes/macrophages as relevant therapeutic targets in acute ischemic stroke.

Puma, but not noxa is essential for oligodendroglial cell death.

The mechanisms involved in oligodendroglial cell death in human demyelinating diseases are only partly understood. Here, we demonstrate that the BH3 only protein Puma, but not Noxa, is essential for oligodendroglial cell death in toxic demyelination induced by the copper chelator cuprizone. Primary oligodendrocytes derived from Noxa‐ or Puma‐deficient mice showed comparable differentiation to wild‐type cells, but Puma‐deficient oligodendrocytes were less susceptible to spontaneous, staurosporine, or nitric oxide‐induced cell death. Furthermore, Puma was expressed in oligodendrocytes in multiple sclerosis (MS) lesions and Puma mRNA levels were upregulated in primary human oligodendrocytes upon cell death induction by staurosporine. Our data demonstrate that Puma is pivotal for oligodendroglial cell death induced by different cell death stimuli and might play a role in oligodendroglial cell death in MS. GLIA 2013;61:1712–1723