The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology.

Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.

PD-L1 positive astrocytes attenuate inflammatory functions of PD-1 positive microglia in models of autoimmune neuroinflammation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disorder of the central nervous system (CNS). Current therapies mainly target inflammatory processes during acute stages, but effective treatments for progressive MS are limited. In this context, astrocytes have gained increasing attention as they have the capacity to drive, but also suppress tissue-degeneration. Here we show that astrocytes upregulate the immunomodulatory checkpoint molecule PD-L1 during acute autoimmune CNS inflammation in response to aryl hydrocarbon receptor and interferon signaling. Using CRISPR-Cas9 genetic perturbation in combination with small-molecule and antibody-mediated inhibition of PD-L1 and PD-1 both in vivo and in vitro, we demonstrate that astrocytic PD-L1 and its interaction with microglial PD-1 is required for the attenuation of autoimmune CNS inflammation in acute and progressive stages in a mouse model of MS. Our findings suggest the glial PD-L1/PD-1 axis as a potential therapeutic target for both acute and progressive MS stages.

Regulation of the programmed cell death protein 1/programmed cell death ligand 1 axis in relapsing-remitting multiple sclerosis.

The programmed cell death protein 1/programmed cell death ligand 1 axis plays an important role in the adaptive immune system and has influence on neoplastic and inflammatory diseases, while its role in multiple sclerosis is unclear. Here, we aimed to analyse expression patterns of programmed cell death protein 1 and programmed cell death ligand 1 on peripheral blood mononuclear cells and their soluble variants in multiple sclerosis patients and controls, to determine their correlation with clinical disability and disease activity. In a cross-sectional study, we performed in-depth flow cytometric immunophenotyping of peripheral blood mononuclear cells and analysed soluble programmed cell death protein 1 and programmed cell death ligand 1 serum levels in patients with relapsing-remitting multiple sclerosis and controls. In comparison to control subjects, relapsing-remitting multiple sclerosis patients displayed distinct cellular programmed cell death protein 1/programmed cell death ligand 1 expression patterns in immune cell subsets and increased soluble programmed cell death ligand 1 levels, which correlated with clinical measures of disability and MRI activity over time. This study extends our knowledge of how programmed cell death protein 1 and programmed cell death ligand 1 are expressed in the membranes of patients with relapsing-remitting multiple sclerosis and describes for the first time the elevation of soluble programmed cell death ligand 1 in the blood of multiple sclerosis patients. The distinct expression pattern of membrane-bound programmed cell death protein 1 and programmed cell death ligand 1 and the correlation between soluble programmed cell death ligand 1, membrane-bound programmed cell death ligand 1, disease and clinical factors may offer therapeutic potential in the setting of multiple sclerosis and might improve future diagnosis and clinical decision-making.

Intranasal delivery of a small-molecule ErbB inhibitor promotes recovery from acute and late-stage CNS inflammation.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the CNS that is characterized by demyelination and axonal degeneration. Although several established treatments reduce relapse burden, effective treatments to halt chronic progression are scarce. Single-cell transcriptomic studies in MS and its animal models have described astrocytes and their spatial and functional heterogeneity as important cellular determinants of chronic disease. We combined CNS single-cell transcriptome data and small-molecule screens in primary mouse and human astrocytes to identify glial interactions, which could be targeted by repurposing FDA-approved small-molecule modulators for the treatment of acute and late-stage CNS inflammation. Using hierarchical in vitro and in vivo validation studies, we demonstrate that among selected pathways, blockade of ErbB by the tyrosine kinase inhibitor afatinib efficiently mitigates proinflammatory astrocyte polarization and promotes tissue-regenerative functions. We found that i.n. delivery of afatinib during acute and late-stage CNS inflammation ameliorates disease severity by reducing monocyte infiltration and axonal degeneration while increasing oligodendrocyte proliferation. We used unbiased screening approaches of astrocyte interactions to identify ErbB signaling and its modulation by afatinib as a potential therapeutic strategy for acute and chronic stages of autoimmune CNS inflammation.

The Aryl Hydrocarbon Receptor-Dependent TGF-α/VEGF-B Ratio Correlates With Disease Subtype and Prognosis in Multiple Sclerosis.

OBJECTIVE: To evaluate the aryl hydrocarbon receptor (AHR)-dependent transforming growth factor alpha (TGF-α)/vascular endothelial growth factor B (VEGF-B) ratio, which regulates the effects of metabolic, dietary, and microbial factors on acute and chronic CNS inflammation, as a potential marker in multiple sclerosis (MS). METHODS: TGF-α, VEGF-B, and AHR agonistic activity were determined in serum of 252 patients with relapsing-remitting (RR) MS, primary and secondary progressive MS, as well as during active disease (clinically isolated syndrome [CIS] and RRMS relapse). RESULTS: The TGF-α/VEGF-B ratio and AHR agonistic activity were decreased in all MS subgroups with a stable disease course as compared to controls. During active CNS inflammation in CIS and RRMS relapse, the TGF-α/VEGF-B ratio and AHR agonistic activity were increased. Conversely, in patients with minimal clinical impairment despite long-standing disease, the TGF-α/VEGF-B ratio and AHR agonistic activity were unaltered. Finally, the TGF-α/VEGF-B ratio and AHR agonistic activity correlated with neurologic impairment and time to conversion from CIS to MS. CONCLUSIONS: The AHR-dependent TGF-α/VEGF-B ratio is altered in a subtype, severity, and disease activity-specific manner and correlates with time to conversion from CIS to MS. It may thus represent a novel marker and serve as additive guideline for immunomodulatory strategies in MS. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that serum levels of AHR, TGF-α, and VEGF-B distinguish subtypes of MS and predict the severity and disease activity of MS.

Vitamin D deficiency exacerbates UV/endorphin and opioid addiction.

The current opioid epidemic warrants a better understanding of genetic and environmental factors that contribute to opioid addiction. Here we report an increased prevalence of vitamin D (VitD) deficiency in patients diagnosed with opioid use disorder and an inverse and dose-dependent association of VitD levels with self-reported opioid use. We used multiple pharmacologic approaches and genetic mouse models and found that deficiencies in VitD signaling amplify exogenous opioid responses that are normalized upon restoration of VitD signaling. Similarly, physiologic endogenous opioid analgesia and reward responses triggered by ultraviolet (UV) radiation are repressed by VitD signaling, suggesting that a feedback loop exists whereby VitD deficiency produces increased UV/endorphin-seeking behavior until VitD levels are restored by cutaneous VitD synthesis. This feedback may carry the evolutionary advantage of maximizing VitD synthesis. However, unlike UV exposure, exogenous opioid use is not followed by VitD synthesis (and its opioid suppressive effects), contributing to maladaptive addictive behavior.

Aryl Hydrocarbon Receptor Plasma Agonist Activity Correlates With Disease Activity in Progressive MS.

OBJECTIVE: The relationship between serum aryl hydrocarbon receptor (AHR) agonistic activity levels with disease severity, its modulation over the course of relapsing-remitting MS (RRMS), and its regulation in progressive MS (PMS) are unknown. Here, we report the analysis of AHR agonistic activity levels in cross-sectional and longitudinal serum samples of patients with RRMS and PMS. METHODS: In a cross-sectional investigation, a total of 36 control patients diagnosed with noninflammatory diseases, 84 patients with RRMS, 35 patients with secondary progressive MS (SPMS), and 41 patients with primary progressive MS (PPMS) were included in this study. AHR activity was measured in a cell-based luciferase assay and correlated with age, sex, the presence of disease-modifying therapies, Expanded Disability Status Scale scores, and disease duration. In a second longitudinal investigation, we analyzed AHR activity in 13 patients diagnosed with RRMS over a period from 4 to 10 years and correlated AHR agonistic activity with white matter atrophy and lesion load volume changes. RESULTS: In RRMS, AHR ligand levels were globally decreased and associated with disease duration and neurologic disability. In SPMS and PPMS, serum AHR agonistic activity was decreased and correlated with disease severity. Finally, in longitudinal serum samples of patients with RRMS, decreased AHR agonistic activity was linked to progressive CNS atrophy and increased lesion load. CONCLUSIONS: These findings suggest that serum AHR agonist levels negatively correlate with disability in RRMS and PMS and decrease longitudinally in correlation with MRI markers of disease progression. Thus, serum AHR agonistic activity may serve as novel biomarker for disability progression in MS.

Inhibition of FGF and TGF-ß Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor.

The fibroblast growth factor (FGF) and the transforming growth factor-ß (TGF-ß) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24-48 h) of FGF and TGF-ß inhibition in hESCs, identifying direct and indirect target genes. In this study, we focused on the earliest transcriptome changes occurring between 3 and 9 h after FGF and TGF-ß inhibition to identify direct target genes only. Our analysis clearly shows that only a handful of target transcripts are common to both pathways. This is surprising in light of the previous literature, and has implications for models of cell signaling in human pluripotent cells. In addition, we identified STOX2 as a novel primary target of the TGF-ß signaling pathway. We show that STOX2 might act as a novel SMAD2/4 cofactor. Taken together, our results provide insights into the effect of cell signaling on the transcription profile of human pluripotent cells.

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology.

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9-mediated genome engineering to pinpoint the function of serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified cyclin-dependent kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.

Comparative analysis of differential gene expression tools for RNA sequencing time course data.

RNA sequencing (RNA-seq) has become a standard procedure to investigate transcriptional changes between conditions and is routinely used in research and clinics. While standard differential expression (DE) analysis between two conditions has been extensively studied, and improved over the past decades, RNA-seq time course (TC) DE analysis algorithms are still in their early stages. In this study, we compare, for the first time, existing TC RNA-seq tools on an extensive simulation data set and validated the best performing tools on published data. Surprisingly, TC tools were outperformed by the classical pairwise comparison approach on short time series (<8 time points) in terms of overall performance and robustness to noise, mostly because of high number of false positives, with the exception of ImpulseDE2. Overlapping of candidate lists between tools improved this shortcoming, as the majority of false-positive, but not true-positive, candidates were unique for each method. On longer time series, pairwise approach was less efficient on the overall performance compared with splineTC and maSigPro, which did not identify any false-positive candidate.

A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).

Switch enhancers interpret TGF-ß and Hippo signaling to control cell fate in human embryonic stem cells.

A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the transforming growth factor ß (TGF-ß) pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex composed of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGF-ß pathway (SMAD2/3), and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements, and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch-enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development.

The TGFß superfamily in stem cell biology and early mammalian embryonic development.

BACKGROUND: Members of the Transforming Growth Factor-beta (TGFß) superfamily of cytokines are essential for early embryonic development and play crucial roles in pluripotency and differentiation of embryonic stem cells in vitro. SCOPE OF REVIEW: In this review, we discuss how TGFß family signals are read by cells and how they are modulated by the cellular context. Furthermore, we review recent advances in our understanding of TGFß function in embryonic stem cells and point out hot topics at the intersection of TGFß signaling and stem cell biology fields. MAJOR CONCLUSION: TGFß family signals are essential for early mammalian development and the importance of this pathway is reflected in pluripotent stem cells derived from the mammalian embryo. GENERAL SIGNIFICANCE: Understanding signaling pathways underlying pluripotency and cell fate specification holds promises for the advent of personalized regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP:

The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis.

The liver is frequently exposed to insults, including toxic chemicals and alcohol, viral infection or metabolic overload. Although it can fully regenerate after acute injury, chronic liver damage causes liver fibrosis and cirrhosis, which can result in complete liver failure. In this study, we demonstrate that the NF-E2-related factor 2 (Nrf2) transcription factor protects the liver from acute and chronic toxin-mediated damage. Repair of the liver injury that occurs after a single treatment with the hepatotoxin carbon tetrachloride (CCl(4)) was severely delayed in Nrf2-deficient mice. The defect in repair was accompanied by an enhanced and prolonged inflammatory and profibrotic response. After long-term CCl(4) treatment, liver fibrosis was strongly aggravated in the Nrf2 knockout mice and inflammation was enhanced. We demonstrate that these abnormalities are at least in part due to the reduced expression of known and novel Nrf2 target genes in hepatocytes, which encode enzymes involved in the detoxification of CCl(4) and its metabolites. These results suggest that activation of Nrf2 may be a novel strategy to prevent or ameliorate toxin-induced liver injury and fibrosis.

The cytoprotective Nrf2 transcription factor controls insulin receptor signaling in the regenerating liver.

The Nrf2 transcription factor is a crucial regulator of the cellular redox homeostasis through its capacity to induce the expression of enzymes, which detoxify reactive oxygen species, and of other antioxidant proteins. Therefore, it plays an important role in the protection from carcinogenesis induced by various insults. In addition, recent results identified a novel role of Nrf2 in tissue repair. In the liver, regeneration after partial hepatectomy was strongly delayed in the absence of Nrf2. This defect was shown to result from transient resistance to insulin and insulin-like growth factor 1 that was caused by chronic oxidative stress in hepatocytes. These results demonstrate a link between Nrf2 deficiency, oxidative stress and insulin resistance, and suggest that activation of this transcription factor could be a novel strategy to improve liver regeneration in patients with acute or chronic liver injury. In addition, it may help to alleviate oxidative stress-induced insulin resistance in the liver and potentially also in other organs.

Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance.

The liver is frequently challenged by surgery-induced metabolic overload, viruses or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signaling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. After partial hepatectomy, liver regeneration was significantly delayed. Using in vitro and in vivo studies, we identified oxidative stress-mediated insulin/insulin-like growth factor resistance as an underlying mechanism. This deficiency impaired the activation of p38 mitogen-activated kinase, Akt kinase and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signaling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress-induced defects in liver regeneration. These findings may provide the basis for the development of new strategies to improve regeneration in patients with acute or chronic liver damage.

Electrophilic chemicals but not UV irradiation or reactive oxygen species activate Nrf2 in keratinocytes in vitro and in vivo.

The NF-E2-related factor 2 (Nrf2) transcription factor is a potent inducer of cytoprotective genes, which encode--among others--enzymes that detoxify reactive oxygen species (ROS). As we demonstrated a crucial role of Nrf2 in the prevention of skin carcinogenesis, it is of interest to identify Nrf2-activating factors in keratinocytes. For this purpose, keratinocytes from mice transgenic for an Nrf2-responsive reporter gene were analyzed. Electrophilic compounds activated the reporter in keratinocytes, and induced nuclear translocation of Nrf2 and the expression of known Nrf2 target genes. This is biologically relevant, as we show that Nrf2-mediated gene expression protects keratinocytes from the toxicity of these substances. By contrast, hydrogen peroxide, glucose oxidase, UVA, and UVB irradiation had no effect, although these treatments strongly increased the levels of intracellular ROS. To verify these results in vivo, transgenic reporter mice with and without functional Nrf2 alleles were topically treated with electrophilic chemicals or irradiated with UVB. Electrophiles but not UVB activated the reporter in an Nrf2-dependent manner. These results provide the basis for the identification of novel Nrf2 activators in keratinocytes with therapeutic potential for skin tumor prevention.

Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing.

The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.