Zain Fanek

Identification of a unique TGF-β–dependent molecular and functional signature in microglia

Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1–deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.

Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice.

To investigate miR‐155 in the SOD1 mouse model and human sporadic and familial amyotrophic lateral sclerosis (ALS).

Activation of microglia by retroviral infection correlates with transient clearance of prions from the brain but does not change incubation time

Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrPC) into a misfolded isoform (PrPSc) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrPSc‐clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion‐only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.

REMOVED: Identification of a unique molecular and functional microglia signature in health and disease

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Targeting miR-155 restores dysfunctional microglia and ameliorates disease in the SOD1 model of ALS

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Corrigendum: Identification of a unique TGF-β–dependent molecular and functional signature in microglia

Corrigendum: Identification of a unique TGF-β–dependent molecular and functional signature in microglia

Dysregulation of the APOE-TGFb pathway leads to loss of the microglial homeostatic signature in neurologic diseases including MS, ALS and AD

the disease. We are now interested in translating these findings to the human system. Therefore, we analyzed human Th17 cells in MS patients and healthy persons. Furthermore, we established Th17 cultures from human peripheral blood mononuclear cells (PBMCs). Methods: Cytokine producers were analyzed in patients with a highly active disease (treated with fingolimod or natalizumab) and ageand sex-matched controls. PBMCs were isolated from the blood of both natalizumab-treated patients and healthy donors via gradient density centrifugation. The cells were stimulated for 4 h and a magnetic bead based IL-17 secretion assay was performed on the cells. The cells were cultured and enriched for 6 days and characterized or co-cultured with either murine or human neurons for 24 h. The cell death rate was determined via TUNEL staining. Results: The cytokine profiles of 40 patients treated with natalizumab show very significant differences in lymphocyte numbers and cytokines compared to healthy donors. While natalizumab-treated patients show higher lymphocyte numbers and larger absolute amounts of IL-17 producing cells per milliliter of blood, only very few lymphocytes in fingolimod-treated patients were detectable. Ex vivo derived Th17 cells could be successfully expanded in vitro. Th17 cultures from patients and healthy controls showed similarly stable IL-17 expression concomitant with the cytokines IFN-γ, IL-22 and IL-4. The pathogenicity of these cells were analyzed in different cellular assays and compared with control T cells. Furthermore, Th17 absolute numbers were correlated with disease activity and MRI data in patients after stopping therapy with natalizumab. Conclusion: Differences in cytokine profiles and numbers in the patient group versus controls could be shown. Higher IL-17 producer numbers in natalizumab-treated patients suggest that Th17 cells are sequestered in the blood.

Dysregulation of the homeostatic microglia signature in germ-free mice

responses during autoimmunity, we investigated the role of Nur77 in modulation of human and murine T cell responses in vitro and characterised autoreactive T cell responses in vivo making use of the animal model of Multiple Sclerosis (MS), i.e. experimental autoimmune encephalomyelitis (EAE). We observed that Nur77 was expressed within hours after T cell receptor triggering. Upon T cell receptormediated activation, Nur77-deficient T cells proliferated stronger than wildtype T cells and exhibited increased potential to differentiate into pathogenic TH1 and TH17 cells — both, in an antigen-independent setting and upon antigen-specific activation. Also in vivo, antigenspecific T cell activation by Nur77-competent dendritic cells resulted in enhanced proliferation and cytokine production when T cells lacked Nur77. After induction of EAE, Nur77-deficient animals exhibited an earlier onset of disease and a significantly aggravated clinical score, which was accompanied by enhanced MOG35–55-specific TH1 and TH17 cell responses both in the periphery and within the CNS. Importantly, also the transfer of MOG35–55-specific Nur77-deficient T cells into healthy wildtype recipients induced a more aggravated EAE disease course than transfer of MOG35–55-specific wildtype T cells, which further demonstrated the importance of Nur77 for restriction of pathogenic T cell responses. In human T cells we observed dysregulated Nur77 expression after TCR triggering when T cells were derived from MS patients, thus indicating that Nur77 could also be important for control of human autoreactive T cell responses. Taken together, Nur77 limits T cell responses in the context of CNS autoimmunity by restricting T cell proliferation and differentiation into TH1 and TH17 effector cells. Hence, Nur77 dysregulation might contribute to enhanced T cell activation in T cell-mediated autoimmune diseases such as MS.

Erratum: Identification of a unique TGF-β-dependent molecular and functional signature in microglia (Nature Neuroscience (2014) 17 (131-143))

Corrigendum: Identification of a unique TGF-β–dependent molecular and functional signature in microglia

Erratum: Corrigendum: Identification of a unique TGF-β–dependent molecular and functional signature in microglia

Corrigendum: Identification of a unique TGF-β–dependent molecular and functional signature in microglia