PRKAG2.2 is essential for FoxA1+ regulatory T cell differentiation and metabolic rewiring distinct from FoxP3+ regulatory T cells.

Forkhead box A1 (FoxA1)+ regulatory T cells (Tregs) exhibit distinct characteristics from FoxP3+ Tregs while equally effective in exerting anti-inflammatory properties. The role of FoxP3+ Tregs in vivo has been challenged, motivating a better understanding of other Tregs in modulating hyperactive immune responses. FoxA1+ Tregs are generated on activation of the transcription factor FoxA1 by interferon-ß (IFNß), an anti-inflammatory cytokine. T cell activation, expansion, and function hinge on metabolic adaptability. We demonstrated that IFNß promotes a metabolic rearrangement of FoxA1+ Tregs by enhancing oxidative phosphorylation and mitochondria clearance by mitophagy. In response to IFNß, FoxA1 induces a specific transcription variant of adenosine 5'-monophosphate-activated protein kinase (AMPK) γ2 subunit, PRKAG2.2. This leads to the activation of AMPK signaling, thereby enhancing mitochondrial respiration and mitophagy by ULK1-BNIP3. This IFNß-FoxA1-PRKAG2.2-BNIP3 axis is pivotal for their suppressive function. The involvement of PRKAG2.2 in FoxA1+ Treg, not FoxP3+ Treg differentiation, underscores the metabolic differences between Treg populations and suggests potential therapeutic targets for autoimmune diseases.

Mitochondrial DNA damage triggers spread of Parkinson's disease-like pathology.

In the field of neurodegenerative diseases, especially sporadic Parkinson's disease (sPD) with dementia (sPDD), the question of how the disease starts and spreads in the brain remains central. While prion-like proteins have been designated as a culprit, recent studies suggest the involvement of additional factors. We found that oxidative stress, damaged DNA binding, cytosolic DNA sensing, and Toll-Like Receptor (TLR)4/9 activation pathways are strongly associated with the sPDD transcriptome, which has dysregulated type I Interferon (IFN) signaling. In sPD patients, we confirmed deletions of mitochondrial (mt)DNA in the medial frontal gyrus, suggesting a potential role of damaged mtDNA in the disease pathophysiology. To explore its contribution to pathology, we used spontaneous models of sPDD caused by deletion of type I IFN signaling (Ifnb-/-/Ifnar-/- mice). We found that the lack of neuronal IFNß/IFNAR leads to oxidization, mutation, and deletion in mtDNA, which is subsequently released outside the neurons. Injecting damaged mtDNA into mouse brain induced PDD-like behavioral symptoms, including neuropsychiatric, motor, and cognitive impairments. Furthermore, it caused neurodegeneration in brain regions distant from the injection site, suggesting that damaged mtDNA triggers spread of PDD characteristics in an "infectious-like" manner. We also discovered that the mechanism through which damaged mtDNA causes pathology in healthy neurons is independent of Cyclic GMP-AMP synthase and IFNß/IFNAR, but rather involves the dual activation of TLR9/4 pathways, resulting in increased oxidative stress and neuronal cell death, respectively. Our proteomic analysis of extracellular vesicles containing damaged mtDNA identified the TLR4 activator, Ribosomal Protein S3 as a key protein involved in recognizing and extruding damaged mtDNA. These findings might shed light on new molecular pathways through which damaged mtDNA initiates and spreads PD-like disease, potentially opening new avenues for therapeutic interventions or disease monitoring.

Neuronal TNFα, Not α-Syn, Underlies PDD-Like Disease Progression in IFNß-KO Mice.

OBJECTIVE: Parkinson's disease (PD) manifests in motor dysfunction, non-motor symptoms, and eventual dementia (PDD). Neuropathological hallmarks include nigrostriatal neurodegeneration, Lewy body (LB) pathology, and neuroinflammation. Alpha-synuclein (α-syn), a primary component of LBs, is implicated in PD pathogenesis, accumulating, and aggregating in both familial and sporadic PD. However, as α-syn pathology is often comorbid with amyloid-beta (Aß) plaques and phosphorylated tau (pTau) tangles in PDD, it is still unclear whether α-syn is the primary cause of neurodegeneration in sporadic PDD. We aimed to determine how the absence of α-syn would affect PDD manifestation. METHODS: IFN-ß knockout (Ifnb-/- ) mice spontaneously develop progressive behavior abnormalities and neuropathology resembling PDD, notably with α-syn+ LBs. We generated Ifnb/Snca double knockout (DKO) mice and evaluated their behavior and neuropathology compared with wild-type (Wt), Ifnb-/- , and Snca-/- mice using immunohistochemistry, electron microscopy, immunoblots, qPCR, and modification of neuronal signaling. RESULTS: Ifnb/Snca DKO mice developed all clinical PDD-like behavioral manifestations induced by IFN-ß loss. Independently of α-syn expression, lack of IFN-ß alone induced Aß plaques, pTau tangles, and LB-like Aß+ /pTau+ inclusion bodies and neuroinflammation. IFN-ß loss caused significant elevated glial and neuronal TNF-α and neuronal TNFR1, associated with neurodegeneration. Restoring neuronal IFN-ß signaling or blocking TNFR1 rescued caspase 3/t-BID-mediated neuronal-death through upregulation of c-FLIPS and lowered intraneuronal Aß and pTau accumulation. INTERPRETATION: These findings increase our understanding of PD pathology and suggest that targeting α-syn alone is not sufficient to mitigate disease. Targeting specific aspects of neuroinflammation, such as aberrant neuronal TNF-α/TNFR1 or IFN-ß/IFNAR signaling, may attenuate disease. ANN NEUROL 2021;90:789-807.

Transcriptome and Function of Novel Immunosuppressive Autoreactive Invariant Natural Killer T Cells That Are Absent in Progressive Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine whether natural killer T (NKT) cells, including invariant (i) NKT cells, have clinical value in preventing the progression of multiple sclerosis (MS) by examining the mechanisms by which a distinct self-peptide induces a novel, protective invariant natural killer T cell (iNKT cell) subset. METHODS: We performed a transcriptomic and functional analysis of iNKT cells that were reactive to a human collagen type II self-peptide, hCII707-721, measuring differentially induced genes, cytokines, and suppressive capacity. RESULTS: We report the first transcriptomic profile of human conventional vs novel hCII707-721-reactive iNKT cells. We determined that hCII707-721 induces protective iNKT cells that are found in the blood of healthy individuals but not progressive patients with MS (PMS). By transcriptomic analysis, we observed that hCII707-721 promotes their development and proliferation, favoring the splicing of full-length AKT serine/threonine kinase 1 (AKT1) and effector function of this unique lineage by upregulating tumor necrosis factor (TNF)-related genes. Furthermore, hCII707-721-reactive iNKT cells did not upregulate interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-13, or IL-17 by RNA-seq or at the protein level, unlike the response to the glycolipid alpha-galactosylceramide. hCII707-721-reactive iNKT cells increased TNFα only at the protein level and suppressed autologous-activated T cells through FAS-FAS ligand (FAS-FASL) and TNFα-TNF receptor I signaling but not TNF receptor II. DISCUSSION: Based on their immunomodulatory properties, NKT cells have a potential value in the treatment of autoimmune diseases, such as MS. These significant findings suggest that endogenous peptide ligands can be used to expand iNKT cells, without causing a cytokine storm, constituting a potential immunotherapy for autoimmune conditions, including PMS.

PIAS2-mediated blockade of IFN-ß signaling: a basis for sporadic Parkinson disease dementia.

Familial Parkinson disease (PD) is associated with rare genetic mutations, but the etiology in most patients with sporadic (s)PD is largely unknown, and the basis for its progression to dementia (sPDD) is poorly characterized. We have identified that loss of IFNß or IFNAR1, the receptor for IFNα/ß, causes pathological and behavioral changes resembling PDD, prompting us to hypothesize that dysregulated genes in IFNß-IFNAR signaling pathway predispose one to sPD. By transcriptomic analysis, we found defective neuronal IFNß-IFNAR signaling, including particularly elevated PIAS2 associated with sPDD. With meta-analysis of GWASs, we identified sequence variants in IFNß-IFNAR-related genes in sPD patients. Furthermore, sPDD patients expressed higher levels of PIAS2 mRNA and protein in neurons. To determine its function in brain, we overexpressed PIAS2 under a neuronal promoter, alone or with human α-synuclein, in the brains of mice, which caused motor and cognitive impairments and correlated with intraneuronal phosphorylated (p)α-synuclein accumulation and dopaminergic neuron loss. Ectopic expression of neuronal PIAS2 blocked mitophagy, increased the accumulation of senescent mitochondrial and oxidative stress, as evidenced by excessive oxDJ1 and 8OHdG, by inactivating ERK1/2-P53 signaling. Conversely, PIAS2 knockdown rescued the clinicopathological manifestations of PDD in Ifnb-/- mice on restoring mitochondrial homeostasis, oxidative stress, and pERK1/2-pP53 signaling. The regulation of JAK-STAT2-PIAS2 signaling was crucial for neurite outgrowth and neuronal survival and excitability and thus might prevent cognitive impairments. Our findings provide insights into the progression of sPD and dementia and have implications for new therapeutic approaches.

IFN-ß rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1.

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource-demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN-ß is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN-ß induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN-ß signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria-endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN-ß in the Ifnb-/- model of Parkinson disease (PD) disrupts STAT5-PGAM5-Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN-ß rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN-ß activates mitochondrial fission in neurons through the pSTAT5/PGAM5/S622 Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.

CSF SERPINA3 Levels Are Elevated in Patients With Progressive MS.

OBJECTIVE: To identify biomarkers associated with progressive phases of MS and with neuroprotective potential. METHODS: Combined analysis of the transcriptional and proteomic profiles obtained in CNS tissue during chronic progressive phases of experimental autoimmune encephalomyelitis (EAE) with the transcriptional profile obtained during the differentiation of murine neural stem cells into neurons. Candidate biomarkers were measured by ELISA in the CSF of 65 patients with MS (29 with relapsing-remitting MS [RRMS], 20 with secondary progressive MS, and 16 with primary progressive MS [PPMS]) and 30 noninflammatory neurologic controls (NINCs). RESULTS: Integrative analysis of gene and protein expression data identified 2 biomarkers, the serine protease inhibitor Serpina3n and the calcium-binding protein S100A4, which were upregulated in chronic progressive EAE and whose expression was induced during neuronal differentiation. Immunofluorescence studies revealed a primarily neuronal expression of S100A4 and Serpina3n during EAE. CSF levels of SERPINA3, the human ortholog of murine Serpina3n, and S100A4 were increased in patients with MS compared with NINCs (SERPINA3: 1,320 vs 838.6 ng/mL, p = 0.0001; S100A4: 1.6 vs 0.8 ng/mL, p = 0.02). Within the MS group, CSF SERPINA3 levels were significantly elevated in patients with progressive forms, mainly patients with PPMS compared with patients with RRMS (1,617 vs 1,129 ng/mL, p = 0.02) and NINCs (1,617 vs 838.6 ng/mL, p = 0.0001). Of interest, CSF SERPINA3 levels significantly correlated with CSF neurofilament light chain levels only in the PPMS group (r = 0.62, p = 0.01). CONCLUSION: These results point to a role of SERPINA3 as a biomarker associated with the progressive forms of MS, particularly PPMS.

Effects of a single dose of psilocybin on behaviour, brain 5-HT2A receptor occupancy and gene expression in the pig.

Psilocybin has in some studies shown promise as treatment of major depressive disorder and psilocybin therapy was in 2019 twice designated as breakthrough therapy by the U.S. Food and Drug Administration (FDA). A very particular feature is that ingestion of just a single dose of psilocybin is associated with lasting changes in personality and mood. The underlying molecular mechanism behind its effect is, however, unknown. In a translational pig model, we here present the effects of a single dose of psilocybin on pig behaviour, receptor occupancy and gene expression in the brain. An acute i.v. injection of 0.08 mg/kg psilocybin to awake female pigs induced characteristic behavioural changes in terms of headshakes, scratching and rubbing, lasting around 20 min. A similar dose was associated with a cerebral 5-HT2A receptor occupancy of 67%, as determined by positron emission tomography, and plasma psilocin levels were comparable to what in humans is associated with an intense psychedelic experience. We found that 19 genes were differentially expressed in prefrontal cortex one day after psilocybin injection, and 3 genes after 1 week. Gene Set Enrichment Analysis demonstrated that multiple immunological pathways were regulated 1 week after psilocybin exposure. This provides a framework for future investigations of the lasting molecular mechanisms induced by a single dose of psilocybin. In the light of an ongoing debate as to whether psilocybin is a safe treatment for depression and other mental illnesses, it is reassuring that our data suggest that any effects on gene expression are very modest.

ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation.

A disintegrin and metalloproteinase (ADAM)12 was previously found to be expressed in T cells in the inflamed brain. However, the function of ADAM12 in T-cell responses in general and in tissue inflammation has not been examined. Here, we studied the role of ADAM12 in T-cell responses, fate determination on activation, and its functions in T cells to mediate tissue inflammation. We identified ADAM12 as a costimulatory molecule that is expressed on naive T cells and downregulated on stimulation. ADAM12 mimics CD28 costimulatory signaling to activate and induce the proliferation of T-helper 1 (Th1) cells. Monoclonal ADAM12 Fab antibodies trigger T-cell activation by amplifying TCR signaling to stimulate T-bet-mediated IFNγ production. Lack of genomic ADAM12 and its knockdown in T cells diminished T-bet and IFNγ production in Th1 cells, whereas other T cells, including Th17 cells, were unaffected. ADAM12 had similar functions in vivo on myelin antigen (MOG35-55)-induced T-cell activation. We found that genetic loss of ADAM12 profoundly alleviated Th1-mediated neuroinflammation and thus disease severity in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Transcriptomic profiling of MOG35-55-specific ADAM12-/- T cells revealed differentially expressed genes that are important for T-cell activation, proliferation, and costimulatory signaling and Th1 pathogenicity, consistent with their inability to cause T-cell-mediated skin inflammation in a model of adoptive delayed-type hypersensitivity. We conclude that ADAM12 is a T-cell costimulatory molecule that contributes to the pathogenesis of tissue inflammation and a potential target for the treatment of Th1-mediated diseases.

A mutation in DOP1B identified as a probable cause for autosomal recessive Peters anomaly in a consanguineous family.

Purpose: Peters anomaly (PA) is a heterogeneous developmental disorder characterized by central corneal opacity and iridocorneal or corneolenticular adhesions. Although many causative genes have been identified, most screened patients do not have mutations in the known genes. We aimed to identify the genetic cause of Peters anomaly in a pedigree with three affected individuals. Methods: Slit-lamp biomicroscopy and ultrasound biomicroscopy were performed for definitive diagnosis. Exome sequencing was conducted on the DNA of all three patients. After identification of a candidate causative gene, expression of the gene was assessed with real-time PCR in various ocular tissues of three human embryos and three adults. Results: The patients were affected with isolated PA. The parents of the patients were related to one another. Inheritance of PA was autosomal recessive. After appropriate filtering of the exome data, a homozygous variation in DOP1B remained as the only candidate genetic cause of PA in the pedigree. The variant segregated with disease status in the pedigree and was absent among 800 control Iranians. The variant has been reported in various databases at frequencies of 0.006 or less only in the heterozygous state in some cohorts of African origin. The p.Val1660 amino acid affected by the mutation is completely conserved in mammals and birds during evolution. Expression of DOP1B was shown in all adult and embryonic lens, iris, cornea, sclera, and retina tissues that were tested. Conclusions: DOP1B that encodes DOP1 leucine zipper like protein B was identified as the putative PA-causing gene in pedigree PA-101. As DOP1B is positioned within the Down syndrome chromosomal region on chromosome 21, until now this gene has mostly been studied with respect to brain functions. However, members of the Dopey gene family have been shown to have roles in development in other organisms. Evidence of the expression of DOP1B in various PA-relevant eye tissues, which, to the best of our knowledge, is shown here for the first time, is to be noted. However, this finding does not necessarily implicate a specific role for DOP1B in eye development as the gene is expressed in many tissues. Ultimately, definitive assessment of the contribution of DOP1B to PA pathology awaits identification of mutations in the gene in unrelated patients with PA and functional studies.

Identification of unique and shared mitochondrial DNA mutations in neurodegeneration and cancer by single-cell mitochondrial DNA structural variation sequencing (MitoSV-seq).

BACKGROUND: Point mutations and structural variations (SVs) in mitochondrial DNA (mtDNA) contribute to many neurodegenerative diseases. Technical limitations and heteroplasmy, however, have impeded their identification, preventing these changes from being examined in neurons in healthy and disease states. METHODS: We have developed a high-resolution technique-Mitochondrial DNA Structural Variation Sequencing (MitoSV-seq)-that identifies all types of mtDNA SVs and single-nucleotide variations (SNVs) in single neurons and novel variations that have been undetectable with conventional techniques. FINDINGS: Using MitoSV-seq, we discovered SVs/SNVs in dopaminergic neurons in the Ifnar1-/- murine model of Parkinson disease. Further, MitoSV-seq was found to have broad applicability, delivering high-quality, full-length mtDNA sequences in a species-independent manner from human PBMCs, haematological cancers, and tumour cell lines, regardless of heteroplasmy. We characterised several common SVs in haematological cancers (AML and MDS) that were linked to the same mtDNA region, MT-ND5, using only 10 cells, indicating the power of MitoSV-seq in determining single-cancer-cell ontologies. Notably, the MT-ND5 hotspot, shared between all examined cancers and Ifnar1-/- dopaminergic neurons, suggests that its mutations have clinical value as disease biomarkers. INTERPRETATION: MitoSV-seq identifies disease-relevant mtDNA mutations in single cells with high resolution, rendering it a potential drug screening platform in neurodegenerative diseases and cancers. FUNDING: The Lundbeck Foundation, Danish Council for Independent Research-Medicine, and European Union Horizon 2020 Research and Innovation Programme.

Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by demyelination and inflammation. Dysregulated lipid metabolism and mitochondrial dysfunction are hypothesized to play a key role in MS. Carnitine Palmitoyl Transferase 1 (CPT1) is a rate-limiting enzyme for beta-oxidation of fatty acids in mitochondria. The therapeutic effect of pharmacological CPT1 inhibition with etomoxir was investigated in rodent models of myelin oligodendrocyte glycoprotein- and myelin basic protein-induced experimental autoimmune encephalitis (EAE). Mice receiving etomoxir showed lower clinical score compared to placebo, however this was not significant. Rats receiving etomoxir revealed significantly lower clinical score and lower body weight compared to placebo group. When comparing etomoxir with interferon-ß (IFN-ß), IFN-ß had no significant therapeutic effects, whereas etomoxir treatment starting at day 1 and 5 significantly improved the clinical scores compared to the IFN-ß and the placebo group. Immunohistochemistry and image assessments of brain sections from rats with EAE showed higher myelination intensity and decreased expression of CPT1A in etomoxir-treated rats compared to placebo group. Moreover, etomoxir mediated increased interleukin-4 production and decreased interleukin-17α production in activated T cells. In conclusion, CPT1 is a key protein in the pathogenesis of EAE and MS and a crucial therapeutic target for the treatment.

CHST6 mutations identified in Iranian MCD patients and CHST6 mutations reported worldwide identify targets for gene editing approaches including the CRISPR/Cas system.

PURPOSE: To identify CHST6 mutations in Iranians macular corneal dystrophy (MCD) patients and also to assess distribution of amino acids in the encoded protein that are affected by CHST6 mutations reported hitherto in various populations in order to predict gene regions that may be appropriate targets for gene editing approaches including the CRISPR/Cas system. The analysis will also reveal biologically and functionally important regions of the protein. METHODS: Mutation screening of CHST6 by sequencing was performed on 21 Iranian MCD-affected probands. Previously reported MCD causing CHST6 mutations were identified by searches in NCBI. RESULTS: Nineteen CHST6 mutations were found among the 21 Iranian patients, most of which were missense mutations and six of which were novel. Totally, 189 mutations among 375 MCD patients have been found worldwide, and 134 of these are missense mutations. The distribution of 88 amino acids affected by missense mutations along the length of the encoded protein was not random, and four regions of possible mutation clustering were noted. 25% of patients harbored mutations in a DNA region consisting of only 36 nucleotides. CONCLUSION: Similar to most populations, CHST6 mutations among Iranians are very heterogeneous as indicated by finding 19 different mutations among 21 MCD patients. Nevertheless, identification of four potential mutation clusters identifies regions that are most suitable for gene therapy targeting by the CRISPR/Cas approach. Additionally, the mutation clusters identify regions with potential structural and/or functional importance. Consistent with this, the amino acids in these regions are well conserved among various membrane-bound sulfotransferases.

Antiviral, Immunomodulatory and Antiproliferative Activities of Recombinant Soluble IFNAR2 without IFN-ß Mediation.

Soluble receptors of cytokines are able to modify cytokine activities and therefore the immune system, and some have intrinsic biological activities without mediation from their cytokines. The soluble interferon beta (IFN-ß) receptor is generated through alternative splicing of IFNAR2 and has both agonist and antagonist properties for IFN-ß, but its role is unknown. We previously demonstrated that a recombinant human soluble IFN-ß receptor showed intrinsic therapeutic efficacy in a mouse model of multiple sclerosis. Here we evaluate the potential biological activities of recombinant sIFNAR2 without the mediation of IFN-ß in human cells. Recombinant sIFNAR2 down-regulated the production of IL-17 and IFN-É£ and reduced the cell proliferation rate. Moreover, it showed a strong antiviral activity, fully protecting the cell monolayer after being infected by the virus. Specific inhibitors completely abrogated the antiviral activity of IFN-ß, but not that of the recombinant sIFNAR2, and there was no activation of the JAK-STAT signaling pathway. Consequently, r-sIFNAR2 exerts immunomodulatory, antiproliferative and antiviral activities without IFN-ß mediation, and could be a promising treatment against viral infections and immune-mediated diseases.

Interferon-ß-induced miR-1 alleviates toxic protein accumulation by controlling autophagy.

Appropriate regulation of autophagy is crucial for clearing toxic proteins from cells. Defective autophagy results in accumulation of toxic protein aggregates that detrimentally affect cellular function and organismal survival. Here, we report that the microRNA miR-1 regulates the autophagy pathway through conserved targeting of the orthologous Tre-2/Bub2/CDC16 (TBC) Rab GTPase-activating proteins TBC-7 and TBC1D15 in Caenorhabditis elegans and mammalian cells, respectively. Loss of miR-1 causes TBC-7/TBC1D15 overexpression, leading to a block on autophagy. Further, we found that the cytokine interferon-ß (IFN-ß) can induce miR-1 expression in mammalian cells, reducing TBC1D15 levels, and safeguarding against proteotoxic challenges. Therefore, this work provides a potential therapeutic strategy for protein aggregation disorders.

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses.

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα-chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll-like receptor (TLR) ligand activation of TCR-transgenic murine dNKT cells. IFN-γ production by dNKT cells required dendritic cells (DC), cell-to-cell contact and presence of TLR ligands. TLR-stimulated DC activated dNKT cells to secrete IFN-γ in a CD1d-, CD80/86- and type I IFN-independent manner. In contrast, a requirement for IL-12p40, and a TLR ligand-selective dependence on IL-18 or IL-15 was observed. TLR ligand/DC stimulation provoked early secretion of pro-inflammatory cytokines by both CD62L+ and CD62L- dNKT cells. However, proliferation was limited. In contrast, TCR/co-receptor-mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L- dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co-receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.

DNMT1 regulates expression of MHC class I in post-mitotic neurons.

Major Histocompability Complex I (MHC-I) molecules present cellularly derived peptides to the adaptive immune system. Generally MHC-I is not expressed on healthy post-mitotic neurons in the central nervous system, but it is known to increase upon immune activation such as viral infections and also during neurodegenerative processes. MHC-I expression is known to be regulated by the DNA methyltransferase DNMT1 in non-neuronal cells. Interestingly DNMT1 expression is high in neurons despite these being non-dividing. This suggests a role for DNMT1 in neurons beyond the classical re-methylation of DNA after cell division. We thus investigated whether DNMT1 regulates MHC-I in post-mitotic neurons. For this we used primary cultures of mouse cerebellar granule neurons (CGNs). Our results showed that knockdown of DNMT1 in CGNs caused upregulation of some, but not all subtypes of MHC-I genes. This effect was synergistically enhanced by subsequent IFNγ treatment. Overall MHC-I protein level was not affected by knockdown of DNMT1 in CGNs. Instead our results show that the relative MHC-I expression levels among the different MHC subtypes is regulated by DNMT1 activity. In conclusion, we show that while the mouse H2-D1/L alleles are suppressed in neurons by DNMT1 activity under normal circumstances, the H2-K1 allele is not. This finding is particularly important in two instances. One: in the context of CNS autoimmunity with epitope presentation by specific MHC-I subtypes where this allele specific regulation might become important; and two: in amyotropic lateral sclerosis (ALS) where H2-K but not H2-D protects motor neurons from ALS astrocyte-induced toxicity in a mouse model of ALS.

RhoA Drives T-Cell Activation and Encephalitogenic Potential in an Animal Model of Multiple Sclerosis.

T-cells are known to be intimately involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). T-cell activation is controlled by a range of intracellular signaling pathways regulating cellular responses such as proliferation, cytokine production, integrin expression, and migration. These processes are crucial for the T-cells' ability to mediate inflammatory processes in autoimmune diseases such as MS. RhoA is a ubiquitously expressed small GTPase well described as a regulator of the actin cytoskeleton. It is essential for embryonic development and together with other Rho GTPases controls various cellular processes such as cell development, shaping, proliferation, and locomotion. However, the specific contribution of RhoA to these processes in T-cells in general, and in autoreactive T-cells in particular, has not been fully characterized. Using mice with a T-cell specific deletion of the RhoA gene (RhoAfl/flLckCre+), we investigated the role of RhoA in T-cell development, functionality, and encephalitogenic potential in EAE. We show that lack of RhoA specifically in T-cells results in reduced numbers of mature T-cells in thymus and spleen but normal counts in peripheral blood. EAE induction in RhoAfl/flLckCre+ mice results in significantly reduced disease incidence and severity, which coincides with a reduced CNS T-cell infiltration. Besides presenting reduced migratory capacity, both naïve and autoreactive effector T-cells from RhoAfl/flLckCre+ mice show decreased viability, proliferative capacity, and an activation profile associated with reduced production of Th1 pro-inflammatory cytokines. Our study demonstrates that RhoA is a central regulator of several archetypical T-cell responses, and furthermore points toward RhoA as a new potential therapeutic target in diseases such as MS, where T-cell activity plays a central role.

Corrigendum: Neuronal IFN-beta-induced PI3K/Akt-FoxA1 signalling is essential for generation of FoxA1+Treg cells.

This corrects the article DOI: 10.1038/ncomms14709.

Neuronal IFN-beta-induced PI3K/Akt-FoxA1 signalling is essential for generation of FoxA1+Treg cells.

Neurons reprogramme encephalitogenic T cells (Tenc) to regulatory T cells (Tregs), either FoxP3+Tregs or FoxA1+Tregs. We reported previously that neuronal ability to generate FoxA1+Tregs was central to preventing neuroinflammation in experimental autoimmune encephalomyelitis (EAE). Mice lacking interferon (IFN)-ß were defective in generating FoxA1+Tregs in the brain. Here we show that lack of neuronal IFNß signalling is associated with the absence of programme death ligand-1 (PDL1), which prevents their ability to reprogramme Tenc cells to FoxA1+Tregs. Passive transfer-EAE via IFNß-competent Tenc cells to mice lacking IFNß and active induced-EAE in mice lacking its receptor, IFNAR, in the brain (NesCre:Ifnarfl/fl) result in defective FoxA1+Tregs generation and aggravated neuroinflammation. IFNß activates neuronal PI3K/Akt signalling and Akt binds to transcription factor FoxA1 that translocates to the nucleus and induces PDL1. Conversely, inhibition of PI3K/Akt, FoxA1 and PDL1 blocked neuronal ability to generate FoxA1+Tregs. We characterize molecular factors central for neuronal ability to reprogramme pathogenic T cells to FoxA1+Tregs preventing neuroinflammation.