Lior Mayo

Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor

Astrocytes have important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-Is) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from patients with multiple sclerosis (MS). IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and the suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered interferon (IFN)-β are partly mediated by AHR. Dietary tryptophan is metabolized by the gut microbiota into AHR agonists that have an effect on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate, indole-3-propionic acid and indole-3-aldehyde, or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AHR agonists were decreased. These findings suggest that IFN-Is produced in the CNS function in combination with metabolites derived from dietary tryptophan by the gut flora to activate AHR signaling in astrocytes and suppress CNS inflammation.

IL-27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the immunoregulatory molecule CD39

Dendritic cells (DCs) control the balance between effector T cells and regulatory T cells in vivo. Hence, the study of DCs might identify mechanisms of disease pathogenesis and guide new therapeutic approaches for disorders mediated by the immune system. We found that interleukin 27 (IL-27) signaling in mouse DCs limited the generation of effector cells of the TH1 and TH17 subsets of helper T cells and the development of experimental autoimmune encephalomyelitis (EAE). The effects of IL-27 were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs. IL-27-induced CD39 decreased the extracellular concentration of ATP and downregulated nucleotide-dependent activation of the NLRP3 inflammasome. Finally, therapeutic vaccination with IL-27-conditioned DCs suppressed established relapsing-remitting EAE. Thus, IL-27 signaling in DCs limited pathogenic T cell responses and the development of autoimmunity.

Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation

Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non–cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony–stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders.

The innate immune system in demyelinating disease

Summary:  Demyelinating diseases such as multiple sclerosis are chronic inflammatory autoimmune diseases with a heterogeneous clinical presentation and course. Both the adaptive and the innate immune systems have been suggested to contribute to their pathogenesis and recovery. In this review, we discuss the role of the innate immune system in mediating demyelinating diseases. In particular, we provide an overview of the anti‐inflammatory or pro‐inflammatory functions of dendritic cells, mast cells, natural killer (NK) cells, NK‐T cells, γδ T cells, microglial cells, and astrocytes. We emphasize the interaction of astroctyes with the immune system and how this interaction relates to the demyelinating pathologies. Given the pivotal role of the innate immune system, it is possible that targeting these cells may provide an effective therapeutic approach for demyelinating diseases.

RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS

Axonal pathology and necroptosis in ALS Necroptosis, a non–caspase-dependent form of cell death, can be reduced in disease states by inhibiting a kinase called RIPK1. Until now, no human mutations have been linked to necroptosis. Ito et al. show that loss of optineurin, which is encoded by a gene that has been implicated in the human neurodegenerative disorder ALS (amyotrophic lateral sclerosis), results in sensitivity to necroptosis and axonal degeneration. When RIPK1-kinase dependent signaling is disrupted in mice that lack optineurin, necroptosis is inhibited and axonal pathology is reversed. Science, this issue p. 603 Inflammatory and cell death mechanisms underlie axonal pathology in amyotrophic lateral sclerosis. Mutations in the optineurin (OPTN) gene have been implicated in both familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of this protein in the central nervous system (CNS) and how it may contribute to ALS pathology are unclear. Here, we found that optineurin actively suppressed receptor-interacting kinase 1 (RIPK1)–dependent signaling by regulating its turnover. Loss of OPTN led to progressive dysmyelination and axonal degeneration through engagement of necroptotic machinery in the CNS, including RIPK1, RIPK3, and mixed lineage kinase domain–like protein (MLKL). Furthermore, RIPK1- and RIPK3-mediated axonal pathology was commonly observed in SOD1G93A transgenic mice and pathological samples from human ALS patients. Thus, RIPK1 and RIPK3 play a critical role in mediating progressive axonal degeneration. Furthermore, inhibiting RIPK1 kinase may provide an axonal protective strategy for the treatment of ALS and other human degenerative diseases characterized by axonal degeneration.

Dual Role of CD38 in Microglial Activation and Activation-Induced Cell Death

Microglia, the resident immune cells of the CNS, are normally quiescent but become activated after infection or injury. Their properties then change, and they promote both repair and damage processes. The extent of microglial activation is regulated, in part, by activation-induced cell death (AICD). Although many apoptotic aspects of the microglial AICD mechanism have been elucidated, little is known about the connection between the activation step and the death process. Using mouse primary microglial cultures, we show that the ectoenzyme CD38, via its calcium-mobilizing metabolite cyclic-ADP-ribose (cADPR), helps promote microglial activation and AICD induced by LPS plus IFN-γ (LPS/IFN-γ), suggesting that CD38 links the two processes. Accordingly, CD38 expression and activity, as well as the intracellular calcium concentration ([Ca2+]i) in the primary microglia were increased by LPS/IFN-γ treatment. Moreover, CD38 deficiency or treatment with cADPR antagonists conferred partial resistance to LPS/IFN-γ-induced AICD and also reduced [Ca2+]i. Microglial activation, indicated by induced expression of NO synthase-2 mRNA and production of NO, secretion and mRNA expression of TNF-α and IL-12 p40, and expression of IL-6 mRNA, was attenuated by CD38 deficiency or cADPR-antagonist treatment. The observed effects of CD38 on microglial activation are probably mediated via a cADPR-dependent increase in [Ca2+]i and the effect on AICD by regulation of NO production. Our results thus suggest that CD38 significantly affects regulation of the amount and function of activated microglia, with important consequences for injury and repair processes in the brain.

Reduced Folate Carrier Gene Silencing in Multiple Antifolate-resistant Tumor Cell Lines Is Due to a Simultaneous Loss of Function of Multiple Transcription Factors but Not Promoter Methylation

The human reduced folate carrier (hRFC) is the major uptake route for antifolates used in cancer chemotherapy. Here we explored the molecular basis for the decrease or loss of hRFC gene expression in seventeen tumor cell lines with resistance to multiple antifolates due to impaired antifolate transport. We studied the role of various cis-acting elements including CRE/AP-1-like element and GC-box in hRFC promoters A and B, respectively, as well as AP-2, Mzf-1 and E-box that are contained within or near four tandemly repeated sequences upstream of promoter A. Decreased or abolished binding either to [32P]GC-box, Mzf-1, AP-1, E-box, or CRE oligonucleotides was detected in ∼50-80% of antifolate-resistant cell lines. Strikingly, ∼80% of the cell lines displayed a simultaneously decreased binding to three or more of these hRFC promoter elements, whereas normal AP-2 binding was retained. The possible contribution of promoter methylation to hRFC gene silencing was also explored. None of the antifolate-resistant cell lines, except for MDA-MB-231 cells, showed hRFC promoter methylation; consistently, MDA-MB-231 was the only cell line that retained binding to all six cis-acting elements. Western blot analysis demonstrated decreased expression of transcriptional activators (pCREB-1, pATF-1, USF-1, c-Fos, c-Jun, Sp1, and Sp3) and/or increased expression of repressors (short Sp3 isoforms), whereas normal AP2α levels were retained. Transient expression of the relevant transcription factors restored, at least partially, both promoter binding and hRFC gene expression. This is the first report that transcriptional silencing of the hRFC gene in multiple tumor cell lines with resistance to various novel antifolates is a result of a simultaneous loss of function of multiple transcription factors but not promoter methylation.

Characterization of LPS and interferon-gamma triggered activation-induced cell death in N9 and primary microglial cells: induction of the mitochondrial gateway by nitric oxide.

Characterization of LPS and interferon- γ triggered activation-induced cell death in N9 and primary microglial cells: induction of the mitochondrial gateway by nitric oxide

CD38 Facilitates Recovery from Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. It causes progressive tissue atrophy and consequent neurological dysfunctions. TBI is accompanied by neuroinflammation, a process mediated largely by microglia. CD38 is an ectoenzyme that promotes transmembrane signaling via the synthesis of potent calcium mobilizing agents or via its receptor activity. CD38 is expressed in the brain in various cell types including microglia. In previous studies, we showed that CD38 regulates microglial activation and response to chemokines. In view of the important role of neuroinflammation in TBI and the effects of CD38 on microglial responses, the present study examines the role of CD38 in the recovery of mice from closed head injury (CHI), a model of focal TBI. For this purpose, CD38-deficient and wild-type (WT) mice were subjected to a similar severity of CHI and the effect of the injury on neurobehavioral and cognitive functions was assessed by the Neurological Severity Score (NSS) and the Object Recognition Test, at various time points post-injury. The results show that recovery after CHI (as indicated by the NSS) was significantly lower in CD38-deficient mice than in WT mice and that the object recognition performance after injury was significantly impaired in injured CD38-deficient mice than in WT mice. In addition, we also observed that the amount of activated microglia/macrophages at the injury site was significantly lower in CD38-deficient mice compared with WT mice. Taken together, our findings indicate that CD38 plays a beneficial role in the recovery of mice from CHI and that this effect is mediated, at least in part, via the effect of CD38 on microglia responses.

Impaired glutamate recycling and GluN2B-mediated neuronal calcium overload in mice lacking TGF-β1 in the CNS

Transforming growth factor β1 (TGF‐β1) is a pleiotropic cytokine expressed throughout the CNS. Previous studies demonstrated that TGF‐β1 contributes to maintain neuronal survival, but mechanistically this effect is not well understood. We generated a CNS‐specific TGF‐β1‐deficient mouse model to investigate the functional consequences of TGF‐β1‐deficiency in the adult mouse brain. We found that depletion of TGF‐β1 in the CNS resulted in a loss of the astrocyte glutamate transporter (GluT) proteins GLT‐1 (EAAT2) and GLAST (EAAT1) and decreased glutamate uptake in the mouse hippocampus. Treatment with TGF‐β1 induced the expression of GLAST and GLT‐1 in cultured astrocytes and enhanced astroglial glutamate uptake. Similar to GLT‐1‐deficient mice, CNS‐TGF‐β1‐deficient mice had reduced brain weight and neuronal loss in the CA1 hippocampal region. CNS‐TGF‐β1‐deficient mice showed GluN2B‐dependent aberrant synaptic plasticity in the CA1 area of the hippocampus similar to the glutamate transport inhibitor DL‐TBOA and these mice were highly sensitive to excitotoxic injury. In addition, hippocampal neurons from TGF‐β1‐deficient mice had elevated GluN2B‐mediated calcium signals in response to extrasynaptic glutamate receptor stimulation, whereas cells treated with TGF‐β1 exhibited reduced GluN2B‐mediated calcium signals. In summary, our study demonstrates a previously unrecognized function of TGF‐β1 in the CNS to control extracellular glutamate homeostasis and GluN2B‐mediated calcium responses in the mouse hippocampus.