Shohreh Issazadeh‐Navikas

<h2>Summary</h2> Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-β (IFN-β) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking <i>Ifnb</i> function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-β signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-β promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-β overexpression prevented dopaminergic neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-β in neuronal homeostasis and validate <i>Ifnb</i> mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.

Since the basic mechanisms behind the beneficial effects of IFN-beta in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-beta gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN-beta knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-gamma in response to recall Ag. Consequently, we addressed the effect of IFN-beta on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-beta KO mice acquired a higher incidence and augmented EAE regardless of the source of T cells. This shows that the anti-inflammatory effect of endogenous IFN-beta is predominantly exerted on the effector phase of the disease. Histopathological investigations of CNS in the effector phase revealed an extensive microglia activation and TNF-alpha production in IFN-beta KO mice; this was virtually absent in wt littermates. This coincided with an increase in effector functions of T cells in IFN-beta KO mice, as measured by IFN-gamma and IL-4 production. We suggest that lack of endogenous IFN-beta in CNS leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits.

gamma-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could be activated by the low (nM-1 microM), physiological concentrations of GABA present around neurons in the brain. The cells expressed the alpha1, alpha4, beta2, beta3, gamma1 and delta GABAA channel subunits and formed functional, extrasynaptic-like GABA channels that were activated by 1 microM GABA. 100 nM and higher GABA concentrations decreased T cell proliferation. The results are consistent with GABA being immunomodulatory.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. In general, tumor growth requires disruption of the tissue microenvironment, yet how this affects glioma progression is unknown. We studied program death-ligand (PD-L)1 in neurons and gliomas in tumors from GBM patients and associated the findings with clinical outcome. Remarkably, we found that upregulation of PD-L1 by neurons in tumor-adjacent brain tissue (TABT) associated positively with GBM patient survival, whereas lack of neuronal PD-L1 expression was associated with high PD-L1 in tumors and unfavorable prognosis. To understand the molecular mechanism of PD-L1 signaling in neurons, we investigated PD-L1 function in cerebellar and cortical neurons and its impact on gliomas. We discovered that neuronal PD-L1-induced caspase-dependent apoptosis of glioma cells. Because interferon (IFN)-β induces PD-L1 expression, we studied the functional consequences of neuronal <i>Ifnb</i> gene deletion on PD-L1 signaling and function. <i>Ifnb</i>^−/− neurons lacked PD-L1 and were defective in inducing glioma cell death; this effect was reversed on PD-L1 gene transfection. <i>Ifnb</i>^−/− mice with intracerebral isografts survived poorly. Similar to the observations in GBM patients, better survival in wild-type mice was associated with high neuronal PD-L1 in TABT and downregulation of PD-L1 in tumors, which was defective in <i>Ifnb</i>^−/− mice. Our data indicated that neuronal PD-L1 signaling in brain cells was important for GBM patient survival. Reciprocal PD-L1 regulation in TABT and tumor tissue could be a prognostic biomarker for GBM. Understanding the complex interactions between tumor and adjacent stromal tissue is important in designing targeted GBM therapies.

After stroke, brain inflammation in the ischemic hemisphere hampers brain tissue reorganization and functional recovery. Housing rats in an enriched environment (EE) dramatically improves recovery of lost neurologic functions after experimental stroke. We show here that rats housed in EE after stroke induced by permanent occlusion of the middle cerebral artery (pMCAO), showed attenuated levels of proinflammatory cytokines in the ischemic core and the surrounding peri-infarct area, including a significant reduction in the stroke-induced chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor-1 (CXCL12). To mimic beneficial effects of EE, we studied the impact of inhibiting CXCL12 action on functional recovery after transient MCAO (tMCAO). Rats treated with the specific CXCL12 receptor antagonist 1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclo-tetradecan (AMD3100) showed improved recovery compared with saline-treated rats after tMCAO, without a concomitant reduction in infarct size. This was accompanied by a reduction of infiltrating immune cells in the ischemic hemisphere, particularly cluster of differentiation 3-positive (CD3(+)) and CD3(+)/CD4(+) T cells. Spleen atrophy and delayed death of splenocytes, induced by tMCAO, was prevented by AMD3100 treatment. We conclude that immoderate excessive activation of the CXCL12 pathway after stroke contributes to depression of neurologic function after stroke and that CXCR4 antagonism is beneficial for the recovery after stroke.

Common dietary components including vitamins A and D, omega-3 and probiotics are now widely accepted to be essential to protect against many diseases with an inflammatory nature. On the other hand, high-fat diets are documented to exert multiple deleterious effects, including fatty liver diseases. Here we discuss the effect of dietary components on regulatory T cell (Treg) homeostasis, a central element of the immune system to prevent chronic tissue inflammation. Accordingly, evidence on the impact of dietary components on diseases in which Tregs play an influential role will be discussed. We will review chronic tissue-specific autoimmune and inflammatory conditions such as inflammatory bowel disease, type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis and allergies among chronic diseases where dietary factors could have a direct influence via modulation of Tregs homeostasis and functions.

IFNB1/interferon (IFN)-β belongs to the type I IFNs and exerts potent antiproliferative, proapoptotic, antiangiogenic and immunemodulatory functions. Despite the beneficial effects of IFNB1 in experimental breast cancers, clinical translation has been disappointing, possibly due to induction of survival pathways leading to treatment resistance. Defects in autophagy, a conserved cellular degradation pathway, are implicated in numerous cancer diseases. Autophagy is induced in response to cancer therapies and can contribute to treatment resistance. While the type II IFN, IFNG, which in many aspects differs significantly from type I IFNs, can induce autophagy, no such function for any type I IFN has been reported. We show here that IFNB1 induces autophagy in MCF-7, MDAMB231 and SKBR3 breast cancer cells by measuring the turnover of two autophagic markers, MAP1LC3B/LC3 and SQSTM1/p62. The induction of autophagy in MCF-7 cells occurred upstream of the negative regulator of autophagy MTORC1, and autophagosome formation was dependent on the known core autophagy molecule ATG7 and the IFNB1 signaling molecule STAT1. Using siRNA-mediated silencing of several core autophagy molecules and STAT1, we provide evidence that IFNB1 mediates its antiproliferative effects independent of autophagy, while the proapoptotic function of IFNB1 was strongly enhanced in the absence of autophagy. This suggests that autophagy induced by IFNB1 promoted survival, which might contribute to tumor resistance against IFNB1 treatment. It may therefore be clinically relevant to reconcile a role for IFNB1 in the treatment of breast cancer with concomitant inhibition of autophagy.

The Type I interferons (IFN), beta (IFN-β) and the alpha family (IFN-α), act through a common receptor and have anti-inflammatory effects. IFN-β is used to treat multiple sclerosis (MS) and is effective against experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Mice with EAE show elevated levels of Type I IFNs in the central nervous system (CNS), suggesting a role for endogenous Type I IFN during inflammation. However, the therapeutic benefit of Type I IFN produced in the CNS remains to be established. The aim of this study was to examine whether experimentally induced CNS-endogenous Type I IFN influences EAE. Using IFN-β reporter mice, we showed that direct administration of polyinosinic-polycytidylic acid (poly I:C), a potent inducer of IFN-β, into the cerebrospinal fluid induced increased leukocyte numbers and transient upregulation of IFN-β in CD45/CD11b-positive cells located in the meninges and choroid plexus, as well as enhanced IFN-β expression by parenchymal microglial cells. Intrathecal injection of poly I:C to mice showing first symptoms of EAE substantially increased the normal disease-associated expression of IFN-α, IFN-β, interferon regulatory factor-7 and IL-10 in CNS, and disease worsening was prevented for as long as IFN-α/β was expressed. In contrast, there was no therapeutic effect on EAE in poly I:C-treated IFN receptor-deficient mice. IFN-dependent microglial and astrocyte response included production of the chemokine CXCL10. These results show that Type I IFN induced within the CNS can play a protective role in EAE and highlight the role of endogenous type I IFN in mediating neuroprotection.

Abstract 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 IFNβ or IFNAR1, the receptor for IFNα/β, causes pathological and behavioral changes resembling PDD, prompting us to hypothesize that dysregulated genes in IFNβ-IFNAR signaling pathway predispose one to sPD. By transcriptomic analysis, we found defective neuronal IFNβ-IFNAR signaling, including particularly elevated PIAS2 associated with sPDD. With meta-analysis of GWASs, we identified sequence variants in IFNβ-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.

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 IFNβ/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 IFNβ/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.

Type II collagen (CII) is a candidate cartilage-specific autoantigen, which can become post-translationally modified by hydroxylation and glycosylation. T cell recognition of CII is essential for the development of murine collagen-induced arthritis (CIA) and also occurs in rheumatoid arthritis (RA). The common denominator of murine CIA and human RA is the presentation of an immunodominant CII-derived glycosylated peptide on murine Aq and human DR4 molecules, respectively. To investigate the importance of T cell recognition of glycosylated CII in CIA development after immunization with heterologous CII, we treated neonatal mice with different heterologous CII-peptides (non-modified, hydroxylated and galactosylated). Treatment with the galactosylated peptide (galactose at position 264) was superior in protecting mice from CIA. Protection was accompanied by a reduced antibody response to CII and by an impaired T cell response to the glycopeptide. To investigate the importance of glycopeptide recognition in an autologous CIA model, we treated MMC-transgenic mice, which express the heterologous CII epitope with a glutamic acid in position 266 in cartilage, with CII-peptides. Again, a strong vaccination potential of the glycopeptide was seen. Hence CII-glycopeptides may be the optimal choice of vaccination target in RA, since humans share the same epitope as the MMC mouse.

Article29 April 2021free access Transparent process IFN-β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 Emilie Tresse Emilie Tresse Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Lluís Riera-Ponsati Lluís Riera-Ponsati Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Elham Jaberi Elham Jaberi Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Wei Qi Guinevere Sew Wei Qi Guinevere Sew Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Karsten Ruscher Karsten Ruscher Laboratory for Experimental Brain Research and LUBIN Lab - Lunds Laboratorium för Neurokirurgisk Hjärnskadeforskning, Division of Neurosurgery, Department of Clinical Sciences, University of Lund, Lund, Sweden Search for more papers by this author Shohreh Issazadeh-Navikas Corresponding Author Shohreh Issazadeh-Navikas [email protected] orcid.org/0000-0003-0369-424X Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Emilie Tresse Emilie Tresse Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Lluís Riera-Ponsati Lluís Riera-Ponsati Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Elham Jaberi Elham Jaberi Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Wei Qi Guinevere Sew Wei Qi Guinevere Sew Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Karsten Ruscher Karsten Ruscher Laboratory for Experimental Brain Research and LUBIN Lab - Lunds Laboratorium för Neurokirurgisk Hjärnskadeforskning, Division of Neurosurgery, Department of Clinical Sciences, University of Lund, Lund, Sweden Search for more papers by this author Shohreh Issazadeh-Navikas Corresponding Author Shohreh Issazadeh-Navikas [email protected] orcid.org/0000-0003-0369-424X Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Emilie Tresse1, Lluís Riera-Ponsati1, Elham Jaberi1, Wei Qi Guinevere Sew1, Karsten Ruscher2 and Shohreh Issazadeh-Navikas *,1 1Faculty of Health and Medical Sciences, Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark 2Laboratory for Experimental Brain Research and LUBIN Lab - Lunds Laboratorium för Neurokirurgisk Hjärnskadeforskning, Division of Neurosurgery, Department of Clinical Sciences, University of Lund, Lund, Sweden *Corresponding author. Tel: +45 353 25649; E-mail: [email protected] The EMBO Journal (2021)40:e106868https://doi.org/10.15252/embj.2020106868 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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-β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN-β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN-β 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-β 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-β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN-β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism. SYNOPSIS How brain neurons cope with their high energy demands during periods of increased activity remains poorly understood. This genetic study reveals a role for IFN-β in the control of nerve cell mitochondrial dynamics, ensuring fission and protecting against mitochondrial dysfunction and neurodegeneration in vivo. Deficiency of neuronal Ifnb impairs mitochondrial fission, disrupting ATP production and oxidative stress response. IFN-β induces STAT5-dependent expression of PGAM5 phosphatase, that is required for S622 phosphorylation of the mitochondrial regulator Drp1. IFN-β rescues mitochondrial defects and neuronal survival in Parkinson disease mouse models. Introduction Using up to 20% of the energy that is consumed by the entire body, the brain has the highest metabolic rate of all organs in mammals. Neurons have specific energy requirements, due to their highly compartmentalized morphology and activities, such as restoration of the gradient across the membrane after neuronal depolarization (Crotty et al, 2006), neurotransmitter recycling (Attwell & Laughlin, 2001), and axonal transport (Maday et al, 2014). Mitochondria provide most neuronal energy through oxidative metabolism. They are highly dynamic organelles that can fuse (i.e., mitochondrial fusion), divide (fission), or migrate in response to metabolic and environmental changes. Defects in mitochondrial fusion or fission can result in gradual neurodegeneration (Burte et al, 2015). Disruptions in these processes have been implicated in familial and sporadic forms of Parkinson disease (PD) (Camilleri & Vassallo, 2014; Haelterman et al, 2014; Cieri et al, 2017), the most common progressive neurodegenerative movement disorder. Mitochondrial fusion mitigates oxidative stress by mixing the contents of partially damaged mitochondria as a form of complementation (Jahani-Asl et al, 2007). Fission is needed to create new or smaller mitochondria for transport to axonal extremities (Fukumitsu et al, 2016). Also, fission contributes to essential quality control processes by enabling the removal of damaged mitochondria by mitophagy (Buhlman et al, 2014) and regulating apoptosis during high levels of cellular stress (Germain et al, 2005). Several factors that are necessary for mitochondrial fusion and fission have been identified, including members of the guanosine triphosphatase (GTPase) family, such as mitofusins (MFN1 and MFN2) on the outer mitochondrial membrane (OMM), and optic atrophy 1 (OPA1), which lies on the inner mitochondrial membrane (IMM) and is involved in cristae remodeling [reviewed in Bertholet et al (2016)]. Dynamin-related protein 1 (Drp1) is another GTPase that is important in fission. On phosphorylation, Drp1 relocates to the mitochondrial surface and oligomerizes (Macdonald et al, 2014). The subsequent hydrolysis of GTP induces a conformational change in the Drp1 oligomers, generating traction forces that spawn contractile rings around mitochondria (Mears et al, 2011). Mitochondrial fission occurs at contact points with the endoplasmic reticulum (ER), which harbors the molecular machinery that is necessary for Drp1 maturation and complex formation, and coordinates actin polymerization to drive the assembly of Drp1 oligomers around mitochondria (Friedman et al, 2011; Hatch et al, 2014; Ji et al, 2015). Interferon-β (IFN-β) is a cytokine that is primarily associated with immune responses during viral infections. IFN-β signaling activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT)1/2 pathway, leading to the transcription of IFN-stimulated genes (ISGs). ISGs encode many antiviral effectors, controlling all of the steps in viral replication while limiting tissue damage and preventing autoimmunity (Schneider et al, 2014). Also, several members of the STAT family have nongenomic functions, such as STAT5 and its involvement in curvature of the ER membrane (Lee et al, 2013). IFN-β reduces the inflammatory response that is mediated by immune cell infiltration into the brain and is used to treat patients with multiple sclerosis (MS), a neuroinflammatory disease of the central nervous system (CNS) that is caused by immune cell-induced neuronal demyelination (Teige et al, 2003; Liu et al, 2014, 2017). Mitochondrial functions are linked to microbial infections and pathogen recognition responses, including recognition of microbial double-stranded DNA. Such events induce parallel antimicrobial immune responses through the activation of type I IFN pathway and response genes (West et al, 2015). These complementary pathways are well documented in immune cells, allowing them to fight infections. However, no direct function of the type I IFN, in particular IFN-β in regulating mitochondrial homeostasis in general or in neurons has been described. We have reported that Ifnb–/– mice experience age-related motor and cognitive deficits, aggregation of α-synuclein in intraneuronal Lewy bodies (LBs), and spontaneous neurodegeneration—collectively resembling the clinical features of PD with dementia (PDD) (Ejlerskov et al, 2015). Because dysfunctional mitochondria are a significant pathogenic factor in PD, we examined whether and how the absence of Ifnb affects neuronal mitochondrial homeostasis. Our results demonstrate that mitochondrial fission is altered in Ifnb–/– brains—a model of spontaneous PDD—and that in other PD models, induced by 6OH-DA or by overexpression of human SCNA in the midbrain, IFN-β regulates mitochondrial fission and mitophagy, processes that are critical in PD, thereby mitigating mitochondrial damage and preventing dopaminergic neuronal cell death. Results Accumulated mitochondria in Ifnb–/– neurons have enlarged and multibranched morphology By 2D electron microscopy of thalamic sections, we observed an increase in mitochondrial accumulation in Ifnb–/– neurons, accompanied by large aggregates, none of which was detected in wild-type neurons (Ifnb+/+) in the brain (Fig EV1A and B) (Ejlerskov et al, 2015). These findings were confirmed by the rise in MitoTracker Green (MTG) staining in primary cultures of Ifnb–/– versus Ifnb+/+ cortical neurons (CNs) (Fig EV1C and D). Treatment with recombinant IFN-β (rIFN-β) significantly reduced the mitochondrial accumulation in Ifnb–/– neurons (Fig EV1D). Click here to expand this figure. Figure EV1. Mitochondrial dysfunction in cultured primary cortical neurons TEM of thalami in 12-month-old Ifnb+/+ and Ifnb–/– mice. White arrows indicate inclusions. M indicates mitochondria. N denotes nuclei. Scale bars equal 2 μm. Mitochondrial mass, quantified as total mitochondrial area per cell body. MitoTracker staining in DIV6 Ifnb+/+ and Ifnb–/– CNs. Scale bars equal 50 μm. Total mitochondrial mass in Ifnb+/+ and Ifnb–/– primary cortical neuronal cultures, untreated or complemented with 30 U/ml rIFN-β. Quantification was performed using MTG integrated density. Error bars mean + SEM from 3 independent cultures. Total cellular volume of CNs, quantified by β-3 tubulin staining, matching the Tom-20 staining in Fig 1. Mitochondrial volume, quantified from 3D imaging of Tom20 immunolabeling in Ifnb+/+ and Ifnb–/– MEFs. Scale bars equal 10 μm. Data information: For all graphs, error bars mean SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by t-test (B; E; F) or two-way ANOVA (D) with t-test as post-hoc. Download figure Download PowerPoint We then determined the morphological changes that occur when Ifnb is genetically deleted. By 3D immunofluorescence of the OMM protein Tom20, we verified the rise in total mitochondrial mass in Ifnb–/– primary CNs (Fig 1A and B) and found that this phenomenon was due to an increase in the size of individual mitochondria, wherein 40% of neurons had a mean mitochondrial volume above 10 μm3 (Fig 1C and D), despite the overall size of the cells remaining constant (Fig EV1E); these large mitochondria were found in cell bodies and axons. Further, MEFs that were derived from Ifnb–/– mice had nearly twice the mean volume of individual mitochondria compared with wild-type Ifnb+/+ MEFs (Fig EV1F). Figure 1. Mitochondria are enlarged and multibranched in Ifnb–/– CNs and thalamus tissue A–C. Immunostaining of Tom20 (mitochondria) in cells positive for β3-tubulin in DIV6 Ifnb+/+ and Ifnb–/– CNs with or without rIFN-β for 24 h. (A) Projection of 3D image of Tom20. Scale bars equal 20 μm. (B) Quantification of total mitochondrial mass per neuron. (C) Mean individual mitochondria volume in Ifnb+/+ and Ifnb–/– CNs. D–G. 3D electron microscopy of thalamus in 12-month-old Ifnb+/+ and Ifnb–/– mice. (D) Volume rendering of mitochondria (left panel) and corresponding mitochondria in 2D electron microscopy image (right panel) in cell bodies. Each individual mitochondrion is colored differently. Scale bars equal 2 μm. Voxel size equals 0.00878908x0.00878908x0.03 μm for the Ifnb+/+ neuron and 0.00439453x0.00438453x0.04 μm for Ifnb–/–. Nuclei are in gray and marked with “N’. (E) Reconstituted axonal mitochondria. Images extracted from Movies EV1 and EV2. (F) Size of individual mitochondria in cell bodies and nonmyelinated and myelinated axons. N > 30; error bars are SD. (G) Number of branches per mitochondrion. N > 30; error bars are SD. Data information: For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Download figure Download PowerPoint To confirm these findings, we reconstructed whole neurons, including axons, from the thalami of 1-year-old Ifnb–/– and Ifnb+/+ mice by 3D electron microscopy (EM) using serial block-face technology (Fig 1D and E and Movies EV1 and EV2), allowing us to quantify mitochondrial size and structure at a resolution of approximately 5 nm/pixel. We observed a significant increase in mitochondrial size in all compartments of neurons in the Ifnb–/– brain, including myelinated axons (Fig 1F and G and Movies EV1 and EV2), in addition to multibranched mitochondria—i.e., mitochondria with multiple extensions and several axes. In contrast, wild-type brains harbored few such mitochondria in nonmyelinated axons and none in myelinated axons (Fig 1G). These results demonstrate that neuronal IFN-β is essential for regulating mitochondrial structure and dynamics. Defective mitochondria accumulate in neurons in Ifnb–/– brains To determine whether these large mitochondria (Fig 1) were functional, we measured ATP and reactive oxygen species (ROS) levels in CNs and brain tissue. In the midbrains of Ifnb+/+ and Ifnb–/– mice, we quantified the ATP that was generated by these cells by luminescence-based assay. We observed a slight increase in ATP content in 3-day-old Ifnb–/– brains, which declined significantly at age 3 months and decreased further at age 1 year (Fig 2A). We then measured the ATP content in CNs to determine whether this alteration in ATP production specifically affected neurons. Notably, young Ifnb–/– neurons—i.e., 6 days old [days in vitro (DIV)6]—contained approximately 1.5-fold more ATP than wild-type cells (Fig EV2A). But the ATP content decreased gradually in Ifnb–/– neurons on aging at DIV18 by roughly 70% compared with their initial content at DIV6 and was approximately 60% lower than in aged wild-type neurons (Figs 2B and EV2A). Next, we determined whether the increase in mitochondrial mass caused the rise in ATP production. To this end, we quantified the ATP that was produced per mitochondrial unit. At DIV6, mitochondria from Ifnb–/– CNs generated approximately 40% less ATP than those from Ifnb+/+ CNs (Fig 2C). These findings demonstrate that mitochondrial deficiencies already exist in very young Ifnb−/− neurons (DIV6) and accumulate with age. Figure 2. Deficient mitochondrial metabolism and function in Ifnb–/– CNs A. ATP quantification on aging from 3 days to 1 year in Ifnb+/+ and Ifnb–/– midbrains. Error bars are mean ± SEM from 6 mice. B. ATP amounts in DIV18 CNs, untreated or complemented with 30 U/ml rIFN-β. Error bars equal SEM from 5 (untreated cells) or 3 (treated cells) independent experiments. C. ATP quantification on total mitochondrial mass in DIV6 CNs. Error bars show mean + SEM from 6 independent experiments. D, E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb–/– whole brains. OCR measurements under Complex I substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP, and rotenone/antimycin A to capture S3, S4o, S3 u, and nonmitochondrial respiration. N = 3–4 mice per group; bar graphs show mean + SE. F. Quantification of the respiratory control ratio (RCR), calculated as the ratio between S3 and S4o. G. ROS levels, based on mean fluorescence intensity of DCFDA in DIV18 Ifnb+/+ and Ifnb–/– CNs, cultured with or without 30 U/mL rIFN-β. Error bars equal SEM from 3 independent experiments. H. DCFDA quantifications in δIfnb N2A cells on treatment with the mitochondria-specific antioxidant S3qet and MitoTEMPO. I. MitoSox quantification in DIV6 Ifnb+/+ and Ifnb–/– CNs. Rotenone was used as a positive control for the assay. J. Expression of oxidative stress response genes by qPCR in DIV6 Ifnb+/+ and Ifnb–/– CNs. K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 μm. L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5–3 months) and old (aged 6–12 months) Ifnb+/+ and Ifnb–/– mice. M. Immunoblot for optineurin in 6-week-old midbrains of Ifnb+/+ and Ifnb–/– mice. N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb–/– CNs with or without 30 U/ml rIFN-β. Data information: For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Mitochondria are dysfunctional in Ifnb−/− neuron and brain, but this is not due to alterations of complex II function A. Quantification of ATP on aging from 6 to 18 days in Ifnb+/+ and Ifnb–/– CNs. Error bars are mean + SEM from 3 independent experiments. B. Monolayer of mitochondria after isolation, labeled with MitoTracker Green and TMRE, showing intact and polarized mitochondria. C, D. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb–/– whole brains. OCR measurements under Complex II substrate (succinate supplemented with rotenone to inhibit Complex 1 activity) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP, and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N = 3–4 mice per group. Bar graphs show mean + SEM. E. ROS levels based on mean fluorescence intensity of DCFDA in DIV6 Ifnb+/+ and Ifnb–/– CNs. F. Immunofluorescence of 8OHdG in DIV6 CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 μm. G. Expression of optineurin mRNA in DIV6 Ifnb+/+ and Ifnb–/– CNs, quantified by qPCR. H. Expression of optineurin mRNA in Ifnb+/+ and Ifnb–/– brainstems, quantified by qPCR. Data information: For all graphs, error bars mean SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by t-test (A; G; H) or two-way ANOVA (E) with t-test as post-hoc. Download figure Download PowerPoint We tested whether rIFN-β restored the defects in Ifnb–/– neurons and whether it could preserve mitochondrial function on aging. We quantified the ATP in aged CNs, at DIV18, and noted that rIFN-β rescued the mitochondrial dysfunction in Ifnb–/– CNs by enhancing ATP production by approximately 1.5-fold (Fig 2B). Treatment of wild-type neurons with rIFN-β increased ATP production to the levels in DIV6 (Fig 2B). To determine the cause of the decrease in ATP levels in the neurons and brain of Ifnb−/− mice, we performed Seahorse assay using isolated mitochondria from the brain (Figs 2D–F and EV2C and D). Seahorse coupling assay can be used to assess critical mitochondrial parameters, including coupled and uncoupled oxygen consumption rates (OCRs) and the respiratory control ratio (RCR), the ratio of oxidation rates in the presence of excess substrate and adenosine diphosphate (State 3) versus after ADP has been phosphorylated to a steady-state concentration (State 4) (Brand & Nicholls, 2011). We observed that basal respiration from Complex I (Fig 2D and E), activated by the downstream glycolysis products pyruvate and malate, decreased slightly in Ifnb−/− while rising modestly in CII (Fig EV2C and D). ADP was then added to the mitochondria to induce coupled respiration (State S3) by prompting ATP synthesis. The ability of Ifnb−/− mitochondria to stimulate this ATP synthesis declined by two-thirds. Uncoupled respiration that was induced by the mitochondrial uncoupler FCCP (State S3 u), which circumvents Complex V, was unaffected in mitochondria from Ifnb−/− brains. Finally, the RCR (Fig 2F), representing the effectiveness of oxidative phosphorylation, decreased by over 25% in isolated mitochondria from Ifnb−/− brains, demonstrating a defect in ATP production. The impairments in ATP production correlated inversely with the rise in ROS levels in Ifnb–/– neurons, based on staining with the ROS-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) in DIV6 (Fig EV2E) and DIV18 CNs (Fig 2G). Long-term rIFN-β treatment (30 U/ml added every third day of culture) or treatment with the mitochondria-specific antioxidants S3qel and MitoTempo significantly lowered ROS in Ifnb–/– neurons (Figs 2G and H and EV2E). Further, by MitoSox staining in CN cultures at DIV6, we observed a rise in mitochondrial superoxide (SOX) and the ability of rIFN-β to counterbalance the mild increase in SOX that was induced by rotenone (Fig 2I). To confirm the increase in ROS levels, we examined the oxidative stress response genes catalase (encoding catalase), gsr (glutathione reductase), park7 (the protein deglycase DJ-1), ucp2 (mitochondrial uncoupler 2; UCP-2), and sod1 and sod2 (superoxide dismutase 1 and 2; SOD1 and SOD2) by qPCR, of which gsr, sod2, and ucp2 were significantly upregulated in Ifnb–/– CNs (Fig 2J). To determine whether the rise in oxidative stress was causing damage to neurons, we stained cortical neurons for 8-hydroxy-2’deoxyguanosine (8OHdG), one of the major base lesions that are induced by oxidative stress and which has been linked to PD (Abe et al, 2003). In DIV18 primary CN cultures, we observed a robust increase in Ifnb–/– compared with wild-type neurons (Fig 2K). A moderate increase was already present at DIV6 (Fig EV2F). To determine whether excessive ROS were also produced in vivo, we measured 8OHdG in brain lysates of young (aged 6 weeks to 3 months) and old mice (aged 6 months to 1 year) by ELISA. As a result, we found a 50% increase in 8OHdG in young Ifnb–/– mice, rising further on aging (Fig 2L). Finally, we observed the accumulation of optineurin, a mitophagy adaptor that recognizes damaged mitochondria (Wong & Holzbaur, 2014), in CNs and brain tissue (Fig 2M and N). This change was not due to altered optineurin mRNA expression (Fig EV2H). These results suggest that IFN-β is essential for mitochondrial homeostasis, energy production, and the prevention of excessive oxidative stress and accumulation of damaged mitochondria. IFN-β promotes mitochondrial fission by inducing Drp1 phosphorylation on serine 622 and its localization to mitochondria Our results demonstrate that neuronal IFN-β is essential for regulating mitochondrial morphology and activity. The large size of mitochondria and their multibranched structure—as opposed to an increase in the number of smaller mitochondria—indicates a defect in fission in neurons that lack Ifnb. To determine whether and how IFN-β regulates mitochondrial fission, we first labeled the key fission protein Drp1 by immunofluorescence in CRISPR/Cas9-engineered Ifnb-depleted (δIfnb) versus wild-type nontargeting control (NTC) N2A neuroblastoma cells (Fig 3A). Drp1 was found primarily in dense puncta that localized to the mitochondria, whereas the deletion of Ifnb excluded Drp1 from mitochondria. To quantify this difference, we fractionated mitochondria from the cytoplasmic contents of wild-type NTC and Ifnb N2A neurons. In wild-type and NTC N2A cells, Drp1 abound in the mitochondrial fractions, whereas the levels of mitochondria-associated Drp1 were substantially reduced on depletion of Ifnb in 2 independently generated clones (Fig 3B and C). These data suggest that IFN-β activates Drp1 for mitochondrial translocation and that the lack of IFN-β in neurons causes its exclusion from mitochondria. Figure 3. IFN-β promotes the mitochondrial localization and S622 phosphorylation of Drp1 Immunostaining of Tom20 and Drp1 in N2As depleted of Ifnb (δIfnb) by CRISPR/Cas9 and matching nontargeting control (NTC). Immunoblot of Drp1 from cell fractions of wild-type (WT), δIfnb, and NTC N2As. Purity of cytoplasmic (C) and mitochondrial fractions (M) were evaluated by immunoblotting of tubulin and Tom20, respectively. Quantification of (B). Quantification of anti-pDrp1 immunohistostains in different regions of the brain. Images are shown in Fig EV3. Immunoblot of phospho-S622 Drp1 in cortices and brainstem of 3-month-old Ifnb+/+ and Ifnb–/– mice and quantification. Phospho-S622-Drp1 immunoblot and quantification in Ifnb+/+ and Ifnb–/– CN cultures. Vinculin was used as a loading control. Error bars are SEM of 3 independent experiments. Kinetics of Drp1 phosphorylation on S622 on rIFN-β treatment in wild-type CN cultures. Error bars are SEM from 3 independent experiments. Kinetics of Drp1 dephosphorylation at S643 on rIFN-β treatment in wild-type CN cultures. PD-L1 was used as a positive control of rIFN-β activity. MFN2 and OPA1 immunoblots and quantification in Ifnb+/+ and Ifnb–/– CN cultures and quantification. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb–/– MEFs transfected with Drp1 or phosphomimetic Drp1. Mean mitochondrial section area in Ifnb+/+ and Ifnb–/– MEFs transfected with Drp1 or phosphomimetic Drp1. Error bars are SEM from 10 transfectants. Quantification of optineurin staining in (J). Data information: For all graphs, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Download figure Download PowerPoint The localization of Drp1 to mitochondria is central for mitochondrial fission(Smirnova et al, 2001). The lack of this localization in Ifnb–/– neurons could thus be a major failure event that impairs mitochondrial fission, explaining our observation of large multibranched mitochondria (Figs 1 and EV1) and indicating that mitochondria are incapable of completing fission. To determine the molecular basis of this impairment in fission, we examined whether IFN-β affects Drp1 activity directly by regulating its phosphorylation. Intracellular localization of Drp1 has been shown to be controlled in part by its phosphorylation on serine (S)622—the equivalent of serine 616 in the human homolog—which has been implicated as its active form during fission (Taguchi et al, 2007). Overall, phosphorylated S622Drp1 levels decreased in the brains of 6-week-old to 3-month-old Ifnb–/– mice, particularly in the brainstem region, which encompasses the striatum, thalamus, and midbrain (Figs 3D and E, and EV3A and B). In primary CNs, the phosphorylation pattern of Drp1 was altered, reflected by a 50% decline in the phosphorylation of Drp1 on S622 in Ifnb–/– neurons compared with their wild-type counterpart (Fig 3F). To determine whether IFN-β promoted the phosphorylation of Drp1, we treated cultured CNs with rIFN-β. rIFN-β significantly induced neuronal Drp1 phosphorylation at S622 (Figs 3G and EV3B), as it did in N2A cells (Fig EV3A), and substantially decreased its S643-phosphorylated form within 1 h of treatment (Fig 3H). The rIFN-β-induced phosphorylation of Drp1 on S622 in CNs peaked between 1 and 2 h post-treatment and then declined rapidly, reaching baseline levels at 4–6 h (Fig EV3C). The lack of Ifnb did not have a significant impact on Drp1 mRNA (Fig EV3D) or total protein levels (Fig 3E and F). Click here to expand this figure. Figure EV3. Drp1 phosphorylation on S622 is impaired in Ifnb–/– cells Phospho-S622-Drp1 immunostaining in Ifnb+/+ and Ifnb–/–whole brains. Scale bars equal 1 mm. Control indicates incubation with secondary antibodies only. Phospho-S622-Drp1 immunoblots and quantification in Ifnb+/+ and Ifnb–/– CN cultures. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments. Kinetics of S622 phosphorylation on treatment with rIFN-β. Drp1 mRNA levels in Ifnb+/+ and Ifnb–/– CN cultures by qPCR. Error bars equal SEM from 5 independent ex

NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells suppressed a range of in vivo inflammatory conditions, including delayed-type hypersensitivity, antigen-induced airway inflammation, collagen-induced arthritis, and EAE, which were all ameliorated by mCII707-721 vaccination. The findings presented here offer new insight into the intrinsic roles of NKT cells in health and disease. Given the results, endogenous collagen peptide activators of NKT cells may offer promise as novel therapeutics in tissue-specific autoimmune and inflammatory diseases.

Interferon (IFN)-β exerts anti-inflammatory effects, coupled to remarkable neurological improvements in multiple sclerosis, a neuroinflammatory condition of the central nervous system. Analogously, it has been hypothesized that IFN-β, by limiting inflammation, decreases neuronal death and promotes functional recovery after stroke. However, the core actions of endogenous IFN-β signaling in stroke are unclear. To address this question, we used two clinically relevant models of focal cerebral ischemia, transient and permanent middle cerebral artery occlusion, and two genetically modified mouse lines, lacking either IFN-β or its receptor, the IFN-α/β receptor. Subsets of inflammatory and immune cells isolated from the brain, blood, and spleen were studied using flow cytometry. Sensorimotor deficits were assessed by a modified composite neuroscore, the rotating pole and grip strength tests, and cerebral infarct volumes were given by lack of neuronal nuclei immunoreactivity. Here, we report alterations in local and systemic inflammation in IFN-β knockout (IFN-βKO) mice over 8 days after induction of focal cerebral ischemia. Notably, IFN-βKO mice showed a higher number of infiltrating leukocytes in the brain 2 days after stroke. Concomitantly, in the blood of IFN-βKO mice, we found a higher percentage of total B cells but a similar percentage of mature and activated B cells, collectively indicating a higher proliferation rate. The additional differential regulation of circulating cytokines and splenic immune cell populations in wild-type and IFN-βKO mice further supports an important immunoregulatory function of IFN-β in stroke. Moreover, we observed a significant weight loss 2–3 days and a reduction in grip strength 2 days after stroke in the IFN-βKO group, while endogenous IFN-β signaling did not affect the infarct volume. We conclude that endogenous IFN-β signaling attenuates local inflammation, regulates peripheral immune cells, and, thereby, may contribute positively to stroke outcome.

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)-β were defective in generating FoxA1+Tregs in the brain. Here we show that lack of neuronal IFNβ 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 IFNβ-competent Tenc cells to mice lacking IFNβ 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. IFNβ 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.

To identify biomarkers associated with progressive phases of MS and with neuroprotective potential.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).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).These results point to a role of SERPINA3 as a biomarker associated with the progressive forms of MS, particularly PPMS.

We have previously investigated the physiological effects of IFN-beta on chronic CNS inflammation and shown that IFN-beta(-/-) mice develop a more severe experimental autoimmune encephalomyelitis than their IFN-beta(+/-) littermates. This result was shown to be associated with a higher activation state of the glial cells and a higher T cell cytokine production in the CNS. Because this state suggested a down-regulatory effect of IFN-beta on CNS-specific APCs, these results were investigated further. We report that IFN-beta pretreatment of astrocytes and microglia (glial cells) indeed down-modulate their capacity to activate autoreactive Th1 cells. First, we investigated the intrinsic ability of glial cells as APCs and report that glial cells prevent autoreactive Th1 cells expansion while maintaining Ag-specific T cell effector functions. However, when the glial cells are treated with IFN-beta before coculture with T cells, the effector functions of T cells are impaired as IFN-gamma, TNF-alpha, and NO productions are decreased. Induction of the T cell activation marker, CD25 is also reduced. This suppression of T cell response is cell-cell dependent, but it is not dependent on a decrease in glial expression of MHC class II or costimulatory molecules. We propose that IFN-beta might exert its beneficial effects mainly by reducing the Ag-presenting capacity of CNS-specific APCs, which in turn inhibits the effector functions of encephalitogenic T cells. This affect is of importance because activation of encephalitogenic T cells within the CNS is a prerequisite for the development of a chronic progressive CNS inflammation.

Plasmacytoid dendritic cells (pDCs) exhibit both innate and adaptive functions. In particular they are the main source of type I IFNs and directly impact T cell responses through antigen presentation. We have previously demonstrated that during experimental autoimmune encephalomyelitis (EAE) initiation, myelin-antigen presentation by pDCs is associated with suppressive Treg development and results in attenuated EAE. Here, we show that pDCs transferred during acute disease phase confer recovery from EAE. Clinical improvement is associated with migration of injected pDCs into inflamed CNS and is dependent on the subsequent and selective chemerin-mediated recruitment of endogenous pDCs to the CNS. The protective effect requires pDC pre-loading with myelin antigen, and is associated with the modulation of CNS-infiltrating pDC phenotype and inhibition of CNS encephalitogenic T cells. This study may pave the way for novel pDC-based cell therapies in autoimmune diseases, aiming at specifically modulating pathogenic cells that induce and sustain autoimmune inflammation.

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.

The existence of T cells restricted for the MHC I-like molecule CD1 is well established, but the function of these cells is still obscure; one implication is that CD1-dependent T cells regulate autoimmunity. In this study, we investigate their role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, using CD1-deficient mice on a C57BL/6 background. We show that CD1-/- mice develop a clinically more severe and chronic EAE compared with CD1+/+ C57BL/6 mice, which was histopathologically confirmed with increased demyelination and CNS infiltration in CD1-/- mice. Autoantigen rechallenge in vitro revealed similar T cell proliferation in CD+/+ and CD1-/- mice but an amplified cytokine response in CD1-/- mice as measured by both the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4. Investigation of cytokine production at the site of inflammation showed a CNS influx of TGF-beta1-producing cells early in the disease in CD1+/+ mice, which was absent in the CD1-/- mice. Passive transfer of EAE using an autoreactive T cell line induced equivalent disease in both groups, which suggested additional requirements for activation of the CD1-dependent regulatory pathway(s). When immunized with CFA before T cell transfer, the CD1-/- mice again developed an augmented EAE compared with CD1+/+ mice. We suggest that CD1 exerts its function during CFA-mediated activation, regulating development of EAE both through enhancing TGF-beta1 production and through limiting autoreactive T cell activation, but not necessarily via effects on the Th1/Th2 balance.

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.

Background Bevacizumab combination therapy is among the most frequently used treatments in recurrent glioblastoma and patients who achieve response to bevacizumab have improved survival as well as quality of life. Accordingly, the aim of this study was to identify predictive biomarkers for bevacizumab response in recurrent glioblastoma patients. Methods The study included a total of 82 recurrent glioblastoma patients treated with bevacizumab combination therapy whom were both response and biomarker evaluable. Gene expression of tumor tissue was analyzed by using a customized NanoString platform covering 800 genes. Candidate gene predictors associated with response were analyzed by multivariate logistic and Cox regression analysis. Results Two genes were independently associated with response: Low expression of angiotensinogen (2‐fold decrease in AGT; OR = 2.44; 95% CI: 1.45–4.17; P = 0.0009) and high expression of a HLA class II gene (2‐fold increase in HLA‐DQA1; OR = 1.22; 95% CI: 1.01–1.47; P = 0.04). These two genes were included in a model that is able predict response to bevacizumab combination therapy in clinical practice. When stratified for a validated prognostic index, the predictive model for response was significantly associated with improved overall survival. Conclusion Two genes (low angiotensinogen and high HLA‐class II expression) were predictive for bevacizumab response and were included in a predictive model for response. This model can be used in clinical practice to identify patients who will benefit from bevacizumab combination therapy.

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-β (IFN-β) 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.

Abstract Objective Clinical trials using interferon‐β (IFNβ) in the treatment of rheumatoid arthritis have shown conflicting results. We undertook this study to understand the mechanisms of IFNβ in arthritis at a physiologic level. Methods Collagen‐induced arthritis (CIA) was induced in IFNβ‐deficient and control mice. The role of IFNβ was investigated in both the priming and effector phases of the disease. The effect of IFNβ deficiency on synovial cells, macrophages, and fibroblasts from preimmunized mice was analyzed by flow cytometry, immunohistochemistry, and enzyme‐linked immunosorbent assay. Differences in osteoclast maturation were determined in situ by histology of arthritic and naive paws and by in vitro maturation studies of naive bone marrow cells. The importance of IFNβ‐producing fibroblasts was determined by transfering fibroblasts into mice at the time of CIA immunization. Results Mice lacking IFNβ had a prolonged disease with a higher incidence compared with control mice. IFNβ deficiency was found to influence the effector phase, but not the priming phase, of arthritis. Compared with control mice, IFNβ‐deficient mice had greater infiltration of CD11b+ cells and greater production of tumor necrosis factor α in vivo, and their macrophages and fibroblasts were both more activated in vitro. Moreover, IFNβ‐deficient mice generated a greater number of osteoclasts in vitro, and mice immunized to induce arthritis, but not naive mice, had a greater number of osteoclasts in vivo compared with control mice. Importantly, IFNβ‐competent fibroblasts were able to ameliorate arthritis in IFNβ‐deficient recipients. Conclusion Our data indicate that IFNβ is involved in regulating the activation state of osteoclasts and stromal cells, including macrophages and fibroblasts, but that it has little effect on T cells.

A protective and anti-inflammatory role for CD1d-dependent NKT cells (NKTs) has been reported in experimental and human autoimmune diseases. However, their role in arthritis has been unclear, with conflicting reports of CD1d-dependent NKTs acting both as regulatory and disease-promoting cells in arthritis. These differing modes of action might be due to genetic differences of inbred mice and incomplete backcrossing of gene-modified mice. We therefore put special emphasis on controlling the genetic backgrounds of the mice used. Additionally, we used two different murine arthritis models, Ag-induced arthritis (AIA) and collagen-induced arthritis (CIA), to evaluate acute and chronic arthritis in CD1d knockout mice and mice depleted of NK1.1(+) cells. CD1d-deficient mice developed more severe AIA compared with wild-type littermates, with a higher degree of inflammation and proteoglycan depletion. Chronic arthritis in CIA was also worse in the absence of CD1d-dependent NKTs. Elevated levels of Ag-specific IFN-gamma production accompanied these findings rather than changes in IL-17alpha. Depletion of NK1.1(+) cells supported these findings in AIA and CIA. This report provides support for CD1d-dependent NKTs being suppressor cells in acute and chronic arthritis, likely via inhibition of arthritogenic Th1 cells. These results make CD1d-dependent NKTs an attractive target for therapeutic intervention.

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-β (IFN-β), IFN-β had no significant therapeutic effects, whereas etomoxir treatment starting at day 1 and 5 significantly improved the clinical scores compared to the IFN-β 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.

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.

Bevacizumab combined with chemotherapy produces clinical durable response in 25–30% of recurrent glioblastoma patients. This group of patients has shown improved survival and quality of life. The aim of this study was to investigate changes in gene expression associated with response and resistance to bevacizumab combination therapy. Recurrent glioblastoma patients who had biomarker-accessible tumor tissue surgically removed both before bevacizumab treatment and at time of progression were included. Patients were grouped into responders (n = 7) and non-responders (n = 14). Gene expression profiling of formalin-fixed paraffin-embedded tumor tissue was performed using RNA-sequencing. By comparing pretreatment samples of responders with those of non-responders no significant difference was observed. In a paired comparison analysis of pre- and posttreatment samples of non-responders 1 gene was significantly differentially expressed. In responders, this approach revealed 256 significantly differentially expressed genes (72 down- and 184 up-regulated genes at the time of progression). Genes differentially expressed in responders revealed a shift towards a more proneural and less mesenchymal phenotype at the time of progression. Bevacizumab combination treatment demonstrated a significant impact on the transcriptional changes in responders; but only minimal changes in non-responders. This suggests that non-responding glioblastomas progress chaotically without following distinct gene expression changes while responding tumors adaptively respond or progress by means of the same transcriptional changes. In conclusion, we hypothesize that the identified gene expression changes of responding tumors are associated to bevacizumab response or resistance mechanisms.

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.

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors involved in regulating type I IFN genes and other genes participating in the early antiviral host response. To better understand the mechanisms involved in virus-induced central nervous system (CNS) inflammation, we studied the influence of IRF1, -3, -7, and -9 on the transcriptional activity of key genes encoding antiviral host factors in the CNS of mice infected with lymphocytic choriomeningitis virus (LCMV). A key finding is that neither IRF3 nor IRF7 is absolutely required for induction of a type I IFN response in the LCMV-infected CNS, whereas concurrent elimination of both factors markedly reduces the virus-induced host response. This is unlike the situation in the periphery, where deficiency of IRF7 almost eliminates the LCMV-induced production of the type I IFNs. This difference is seemingly related to the local environment, as peripheral production of type I IFNs is severely reduced in intracerebrally (i.c.) infected IRF7-deficient mice, which undergo a combined infection of the CNS and peripheral organs, such as spleen and lymph nodes. Interestingly, despite the redundancy of IRF7 in initiating the type I IFN response in the CNS, the response is not abolished in IFN-β-deficient mice, as might have been expected. Collectively, these data demonstrate that the early type I IFN response to LCMV infection in the CNS is controlled by a concerted action of IRF3 and -7. Consequently this work provides strong evidence for differential regulation of the type I IFN response in the CNS versus the periphery during viral infection.

Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. "Classical" protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the relevance of considering all phosphatase families when mining for potentially druggable targets.

BackgroundPoint 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.MethodsWe 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.FindingsUsing 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.InterpretationMitoSV-seq identifies disease-relevant mtDNA mutations in single cells with high resolution, rendering it a potential drug screening platform in neurodegenerative diseases and cancers.FundingThe Lundbeck Foundation, Danish Council for Independent Research-Medicine, and European Union Horizon 2020 Research and Innovation Programme.

Vitamin A may have some influence on the immune system, but the role in allergy modulation is still unclear.To clarify whether high levels of retinoic acid (RA) affects allergic response in vivo, we used a murine experimental model of airway allergic disease.Ovalbumin (OVA)-immunization/OVA-challenge (OVA/OVA) and house dust mite (HDM)-immunization/HDM-challenge (HDM/HDM) experimental murine models of allergic airway disease, using C57Bl.10/Q groups of mice (n = 10) treated subcutaneously with different concentrations of all-trans RA (0, 50, 500 and 2,500 ug) every 2-days were used to assess the allergic immune response.Levels of total and specific-IgE in sera were increased in all groups of RA treated OVA/OVA and HDM/HDM mice. Percentage and total amount of recruited eosinophil in airways by bronchoalveolar lavage fluid (BALF) were significantly enhanced in groups treated with 50, 500 and 2,500 ug of RA compared to non-treated mice. However, the group of mice treated with 2,500 ug had less eosinophil recruitment than the other two groups (50 and 500 ug). In parallel, levels of IL-5 and total IgE in BALF were also significantly diminished in the group treated with 2,500 ug compared to the other 2 groups (50 and 500 ug). Finally, total lung resistance was decreased in group treated with 2,500 ug compared to non-treated mice.Our results suggest that retinoic acid directly enhances allergic response in vivo, but in higher doses may produce of immune suppression.

Complex immune responses have evolved to protect multicellular organisms against the invasion of pathogens. This has exerted strong developmental pressure for specialized functions that can also limit damage to self-tissue. Two arms of immunity, the innate and adaptive immune systems, have evolved for quick, non-specific immune responses to pathogens and more efficient, long-lasting ones upon specific recognition of recurrent pathogens. Specialized cells have arisen as the sentinels of these functions, including macrophages, natural killer (NK), and T and B-lymphocytes. Interestingly, a population of immune cells that can exert both of these complex functions, NKT cells, not only share common functions but also exhibit shared cell surface markers of cells of both arms of the immune system. These features, in combination with sophisticated maintenance of immune homeostasis, will be discussed. The recent finding of self-peptide reactivity of NKT cells in the context of CD1d, with capacity to regulate multiple autoimmune and inflammatory conditions, motivates the current proposal that self-reactive NKT cells might be the ancestral link between present NK and T cells. Their parallel selection through evolution by higher vertebrates could be related to their central function as master regulators of immune homeostasis that in part is shared with regulatory T cells. Hypothetical views on how self-reactive NKT cells secure such a central role will also be proposed.

NKT cells are a subgroup of T cells, which express a restricted TCR repertoire and are critical for the innate immune responses to viral infections. Activation of NKT cells depends on the major histocompatibility complex-related molecule CD1d, which presents bioactive lipids to NKT cells. The marine sponge derived lipid αGalCer has recently been demonstrated as a specific agonist for activation of human and murine NKT cells. In the present study we investigated the applicability of αGalCer pre-treatment for immune protection against intra-vaginal HSV-2 infection. We found that C57BL/6 WT mice that received local pre-treatment with αGalCer prior to intra-vaginal HSV-2 infection had a lower mean disease score, mortality and viral load in the vagina following infection, compared to mice that did not receive αGalCer pre-treatment. Further, we found increased numbers of CD45 and NK1.1 positive cells in vaginal tissue and elevated levels of IFN-γ in the vaginal tissue and in vaginal fluids 24 h after αGalCer pre-treatment. Collectively our data demonstrate a protective effect of αGalCer induced activation of NKT cells in the innate immune protection against viral infection.

Conflicting observations have been reported concerning the role of CD1d-dependent natural killer T (NKT) cells in contact hypersensitivity (CHS), supporting either a disease-promoting or downregulatory function. We studied the role of NKT cells in CHS by comparing the immune response in CD1d knockout (CD1d KO) and wild-type (Wt) mice after contact allergen exposure. For induction of CHS, C57BL/6 CD1d KO mice (n = 6) and C57BL/6 Wt mice (n = 6) were sensitised with 1% (w/v) dinitrochlorobenzene (DNCB) or vehicle for three consecutive days and subsequently challenged with a single dose of 0.5% DNCB (w/v) on the ears fifteen days later. We demonstrate that CD1d KO mice, as compared with Wt littermates, have more pronounced infiltration of mononuclear cells in the skin (29.1% increase; P < 0.001), lower frequencies of interleukin-10(+) B cells (B(regs) ) in the spleen (53.2% decrease; P < 0.05) and peritoneal cavity (80.8% decrease; P < 0.05) and increased production of interferon-γ (3-fold; P < 0.05) after DNCB sensitisation and challenge, which suggests an important regulatory and protective role of CD1d-dependent NKT cells in CHS in our model, at least in part via regulation of IL-10 producing B(regs) .

To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis.Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T cells expressing CCR5 and CXCR3.In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells.The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring their migration into the CNS.

IFN-beta has been shown to be effective as therapy for multiple sclerosis. Some reports attributed its beneficial effects to the capacity to induce a T(H)2 response. However, other studies have suggested that endogenous type I IFN might downregulate the allergic response in mice.We sought to define the differential role of endogenous IFN-beta in controlling the development of allergic inflammation.We assessed whether deletion of the gene encoding IFN-beta (IFNB) with knockout mice participated in the development of allergic response in ovalbumin (OVA)-sensitized and OVA-challenged mice.OVA-sensitized and OVA-challenged mice with lack of the IFNB gene had more severe pulmonary inflammation with increased lung local response, including IL-4, IL-5, IL-13, IgE, eosinophilia, and goblet cells, than their litter mates (IFN-beta+/-), whereas no differences were observed in regard to local levels of IFN-gamma. Moreover, systemic response with IgE production is also enhanced. Lack of IFN-beta also results in significantly higher antigen-specific T cells, with higher levels of IL-4, IL-5, and IL-13, whereas no significant differences in IFN-gamma response could be observed. We have also detected a higher ratio of CD4+/CD8+ T cells and increased expression of B7.1/B7.2 on B cells and antigen-presenting cells in IFNB knockout mice.These results demonstrate that IFN-beta plays an important role in immunoregulation of allergic response in mice. The stronger pulmonary inflammation could be a consequence of significantly expanded antigen-specific CD4+ T(H)2 cells as a result of efficient antigen presentation by antigen-presenting cells and hence increased production of IgE by B cells.

IFN-beta is anticipated to have an important function in mucosal tolerance, as it is one of the major cytokines produced by plasmacytoid dendritic cells, and has recently been suggested as central to the maintenance of mucosal homeostasis. Here, we have investigated whether oral tolerance is dependent on endogenous IFN-beta by feeding low-dose self-antigen myelin basic protein to IFN-beta(-/-) mice with subsequent induction of experimental autoimmune encephalomyelitis (EAE). Our study shows that oral tolerance was readily induced in IFN-beta(-/-) mice compared with their wild-type littermates (IFN-beta(+/+)). The non-self-antigen ovalbumin induced oral tolerance in both groups. These data indicate that endogenous IFN-beta is not required for induction of oral tolerance, whereas delivery of recombinant IFN-beta results in significant reduction in clinical score of EAE. Oral tolerance induction was associated with lower production of antigen-specific IFN-gamma, no shift toward antigen-specific Th2, Th17 or TGF-beta response was observed. Oral tolerance in IFN-beta(-/-) mice was also associated with the induction of regulatory and memory T cells in the mucosal-associated immune organs, however this was not a prerequisite for establishment of oral tolerance.

Interleukin-4 (IL-4) is a potent antiinflammatory cytokine. However its use in the clinic is hampered by side effects. We here describe the identification of a novel synthetic peptide, termed Ph8, derived from α-helix C of IL-4, which interacts with IL-4 receptor α (IL-4Rα). Employing various cultured genetically engineered cell lines and primary lymphocytes, surface plasmon resonance, qPCR, ELISA and immunoblotting techniques we found that Ph8 bound IL-4Rα and mimicked the anti-inflammatory effects of IL-4 by inhibiting TNF-α production by macrophages in vitro. It induced phosphorylation of STAT6 65kD but inhibited phosphorylation of STAT6 110 kD induced by IL-4 in a B-cell line that expressed the type I receptor. It also inhibited the IL-4-stimulated expression of a STAT6-inducible reporter gene in cells that expressed the type II receptor. Ph8 inhibited the proliferation of Th1/2 cells and downregulated the production of IFN-γ in stimulated Th1 cells. Moreover, Ph8 did not induce any shift in Th1/Th2 profile. This is a favorable effect and it is indicating that Ph8 could block general T cell activation and inflammatory responses without further inducing the side effects generally associated with IL-4 signaling. These data collectively show that Ph8 is only a partial agonist of IL-4 mimicking its desirable properties. In agreement, Ph8 treatment of rats with collagen-induced arthritis, a Th1- and antibody- mediated disease of joint, delayed the manifestation of chronic inflammation and reduced acute inflammation in carrageenan-induced edema. Our findings indicate that Ph8 is a promising potential drug candidate for the treatment of inflammatory diseases.

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-ɣ 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.

CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-beta in the immunomodulatory effects of CpG-ODN is not known.Here, we aimed to elucidate the role of IFN-beta in the anti-allergic effect of CpG motifs.We assessed the immune response in OVA-primed/OVA-challenged IFN-beta knockout (-/-) mice compared to wild type (WT) control, after intranasal and systemic treatment with synthetic CpG motifs.Vaccination with CpG-ODN reduced the number of cells in airways of OVA-sensitized WT but not IFN-beta-/- mice. Although airway eosinophilia was reduced in both treated groups, they were significantly higher in IFN-beta-/- mice. Other inflammatory cells, such as lymphocytes and macrophages were enhanced in airways by CpG treatment in IFN-beta-/- mice. The ratio of IFN-gamma/IL-4 cytokines in airways was significantly skewed to a Th1 response in WT compared to IFN-beta-/- group. In contrast, IL-4 and IgE were reduced with no differences between groups. Ag-specific T-cell proliferation, Th1-cytokines such as IFN-gamma, IL-2 and also IL-12 were significantly lower in IFN-beta-/- mice. Surprisingly, we discovered that intranasal treatment of mice with CpG-ODN results in mild synovitis particularly in IFN-beta-/- mice.Our results indicate that induction of Th1 response by therapy with CpG-ODN is only slightly and partially dependent on IFN-beta, while IFN-beta is not an absolute requirement for suppression of airway eosinophilia and IgE. Furthermore, our finding of mild synovitis is a warning for possible negative effects of CpG-ODN vaccination.

Abstract 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.

Collagen-induced arthritis (CIA) is induced in H-2(q) mice after immunization with rat type II collagen (CII). The immunodominant T cell epitope on heterologous CII has been located to CII256-270. We have previously shown that TSC transgenic mice, which express the heterologous epitope in type I collagen (CI), e.g. in skin, are tolerized against rat CII and resistant to CIA. In this study we transplanted skin from TSC transgenic mice onto non-transgenic CIA-susceptible littermates to investigate whether introduction of this epitope to a naïve immune system would lead to T cell priming and graft rejection or instead to tolerance and arthritis protection. Interestingly, TSC grafts were accepted and not even immunization of recipient mice with CII in adjuvant induced graft rejection. Instead, TSC skin recipients displayed a reduced T and B cell response to CII and were also protected from arthritis. However, additional priming could break arthritis protection and was accompanied by an increased T cell response to the grafted epitope. Strikingly, despite the regained T cell response, development of arthritis was not accompanied by graft rejection, showing that these immune-mediated inflammatory responses involve different mechanisms.

Collagen-induced arthritis is a mouse model of rheumatoid arthritis (RA) and is commonly induced after immunization with type II collagen (CII) of a non-mouse origin. T cell recognition of heterologous CII epitopes has been shown to be critical in development of arthritis, as mice with cartilage-restricted transgenic expression of the heterologous T cell epitope (MMC mice) are partially tolerized to CII. However, the mechanism responsible for tolerance and arthritis resistance in these mice is unclear. The present study investigated the regulatory mechanisms in naturally occurring self-tolerance in MMC mice. We found that expression of heterologous rat CII sequence in the cartilage of mice positively selects autoreactive CD4(+) T cells with suppressive capacity. Although CD4(+)CD25(+) cells did not play a prominent role in this suppression, CD152-expressing T cells played a crucial role in this tolerance. MMC CD4(+) T cells were able to suppress proliferation of wild-type cells in vitro where this suppression required cell-to-cell contact. The suppressive capability of MMC cells was also demonstrated in vivo, as transfer of such cells into wild-type arthritis susceptible mice delayed arthritis onset. This study also determined that both tolerance and disease resistance were CD152-dependent as demonstrated by Ab treatment experiments. These findings could have relevance for RA because the transgenic mice used express the same CII epitope in cartilage as humans and because autoreactive T cells, specific for this epitope, are present in transgenic mice as well as in patients with RA.

Efficient and specific knockdown of proteins in post-mitotic cells such as differentiated neurons can be difficult to achieve. Further, special care must be taken to maintain the health of neurons in vitro. We wanted to achieve knockdown in primary cerebellar granule neurons, which can be effectively grown in Neurobasal™ media. We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer. We observed a more specific knockdown of target in Neurobasal™ media, than in Accell Delivery media when using cerebellar granule neurons. Transfection efficiency and cell viability was comparable between the two media. Delivery of siRNA in Neurobasal™ media facilitates increased specificity of the knockdown compared to delivery in Accell Delivery media. The off-target effect observed in Accell Delivery media was not a secondary biological response to downregulation of target, but rather a mixture of specific and non-specific off-target effects. Specific knockdown of target can be achieved in primary cerebellar granule cells using Accell siRNAs in Neurobasal™ media. This method ensures specific knockdown in post-mitotic neurons without the need for biosafety level 2 laboratories, additional reagents, or instruments needed by other transfection

Anticancer immunotherapies are highly desired. Conversely, unwanted inflammatory or immune responses contribute to oncogenesis, tumor progression, and cancer-related death. For non-immunogenic therapies to inhibit tumor growth, they must promote, not prevent, the activation of anticancer immune responses. Here, the central immunoregulatory role of brain-specific stromal cells and neurons as well as their ability to maintain an immunological balance and prevent the development of glioblastoma is discussed.

Forkhead box A1 (FoxA1) + regulatory T cells (T regs ) exhibit distinct characteristics from FoxP3 + T regs while equally effective in exerting anti-inflammatory properties. The role of FoxP3 + T regs in vivo has been challenged, motivating a better understanding of other T regs in modulating hyperactive immune responses. FoxA1 + T regs are generated on activation of the transcription factor FoxA1 by interferon-β (IFNβ), an anti-inflammatory cytokine. T cell activation, expansion, and function hinge on metabolic adaptability. We demonstrated that IFNβ promotes a metabolic rearrangement of FoxA1 + T regs by enhancing oxidative phosphorylation and mitochondria clearance by mitophagy. In response to IFNβ, 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 IFNβ-FoxA1-PRKAG2.2-BNIP3 axis is pivotal for their suppressive function. The involvement of PRKAG2.2 in FoxA1 + T reg , not FoxP3 + T reg differentiation, underscores the metabolic differences between T reg populations and suggests potential therapeutic targets for autoimmune diseases.

This corrects the article DOI: 10.1038/ncomms14709.

Correction to: Immunology and Cell Biology advance online publication, 12 January 2010; doi:10.1038/icb.2009.111 Since the publication of the above article the authors have noticed a series of errors in the keys of some figures. The correct figures are reproduced below (Figures 1a, 2a and b, and 4a and d). The typesetters would like to apologize for this mistake. Figure 1a: The bottom two entries in the key incorrectly state ‘MBS’; it should be ‘MBP’. Figure 2a: The second-to-last entry in the key incorrectly states ‘MBS’; it should be ‘MBP’. Figure 2b: The second-to-last entry in the key incorrectly states ‘MBS’; it should be ‘MBP’. Figure 4a: The x-axis label for the third set of bars is incorrectly written ‘NLM’; it should be ‘MLN’. Figure 4d: The second-to-last entry in the key incorrectly states ‘MBS’; it should be ‘MBP’.

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.

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.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.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.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.

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 (Aβ) 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.IFN-β 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.Ifnb/Snca DKO mice developed all clinical PDD-like behavioral manifestations induced by IFN-β loss. Independently of α-syn expression, lack of IFN-β alone induced Aβ plaques, pTau tangles, and LB-like Aβ+ /pTau+ inclusion bodies and neuroinflammation. IFN-β loss caused significant elevated glial and neuronal TNF-α and neuronal TNFR1, associated with neurodegeneration. Restoring neuronal IFN-β signaling or blocking TNFR1 rescued caspase 3/t-BID-mediated neuronal-death through upregulation of c-FLIPS and lowered intraneuronal Aβ and pTau accumulation.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-β/IFNAR signaling, may attenuate disease. ANN NEUROL 2021;90:789-807.

To the Editor:We are grateful to Mr Vieth1.Vieth R Misuse of terminology to imply that 1,25-dihydroxy-vitamin D is a nutrient: there is no evidence for an association between vitamin D and allergy.J Allergy Clin Immunol. 2004; 113: 790-791Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar for paying attention to our recent article published in The Journal of Allergy and Clinical Immunology.2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Vitamin is a general term for a number of unrelated organic substances that occur in many foods in small amounts and that are necessary in trace amounts for the normal metabolic function of the body. “Vitamin D” is any one of several fat-soluble compounds, known commonly as calciferol. The recommendations of International Union of Pure and Applied Chemistry and International Union of Biochemistry and Molecular Biology, based on H. F. DeLuca's and other workers' proposal, say that the term vitamin D should be used as a general term to describe all steroids that exhibit qualitatively the biological activity of calciol.3.IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) Nomenclature of vitamin D. Recommendations 1981.Mol Cell Biochem. 1982; 49: 177-181PubMed Google Scholar Definitely, many compounds can be called vitamin D, and some of them4.Endres B Kato S DeLuca H.F Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo.Biochemistry. 2000; 39: 2123-2129Crossref PubMed Scopus (29) Google Scholar with activity in vitamin D receptor, but either 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2-vitamin D3) or 1,25-dihydroxycholecalciferol or calcitriol had been a more exact term in our paper.2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google ScholarDiscussing the effects of vitamin D on allergic inflammation in mice,2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Mr Vieth is claiming that it was not a nutritional study. We totally agree. The paper is a study of pharmacologic effects of 1α,25(OH)2-vitamin D3 (1,25-dihydroxycholecalciferol or calcitriol), an active metabolite of vitamin D, on the immune system in mice in short-term experiments.2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar This was not hidden, as Mr Vieth claims. Our aim was to investigate if it is possible to interfere with the TH1/TH2 balance through administration of an active vitamin D–metabolite.5.Overbergh L Decallonne B Waer M Rutgeerts O Valckx D Casteels K.M et al.1-Alpha,25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524-543).Diabetes. 2000; 49: 1301-1307Crossref PubMed Scopus (160) Google Scholar Calcitriol, given as subcutaneous injections every second day, was used6.Lemire J.M Adams J.S Sakai R Jordan S.C 1-Alpha,25dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells.J Clin Invest. 1984; 74: 657-661Crossref PubMed Scopus (388) Google Scholar, 7.Cantorna M.T Munsick C Bemiss C Mahon B.D 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease.J Nutr. 2000; 130: 2648-2652Crossref PubMed Scopus (438) Google Scholar to control the blood levels in all mice. Furthermore, the dose 100 ng of calcitriol is taken from the mouse immunology literature, there given either as intraperitoneal injections or as special diets.7.Cantorna M.T Munsick C Bemiss C Mahon B.D 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease.J Nutr. 2000; 130: 2648-2652Crossref PubMed Scopus (438) Google Scholar, 8.Overbergh L Decallonne B Branisteanu D.D Valckx D Kasran A Bouillon R et al.Acute shock induced by antigen vaccination in NOD mice.Diabetes. 2003; 52: 335-341Crossref PubMed Scopus (16) Google ScholarThe possibility that vitamin D status is an environmental factor affecting the prevalence of autoimmune diseases has been published elsewhere.9.Cantorna M.T Vitamin D and autoimmunity: is vitamin D status an environmental factor affecting autoimmune disease prevalence?.Proc Soc Exp Biol Med. 2000; 223: 230-233Crossref PubMed Scopus (205) Google Scholar The other side of the coin might be that vitamin D or analogues could contribute to the development of allergic diseases. Mr Vieth claims that our study provides no association between allergy and vitamin D. We think we have clearly demonstrated that in C57BL/10 mice, which are not a common strain of mice used in allergy studies,10.Matheu V Navikas V Issazadeh-Navikas S Susceptibility of B10.RIII mouse strain to develop inflammatory allergic pulmonary disease: a murine asthma model.Allergol Immunol Clin. 2001; 16: 282-290Google Scholar calcitriol is able to increase the TH2 response, measured as specific IgE production, as we and others have suggested before.11.Matheu V Mondoc E Back O Issazadeh S Vitamin D enhances allergic response in mice.Scand J Immunol. 2001; 54: 50Google Scholar, 12.Wjst M Dold S Genes, factor X, and allergens: what causes allergic diseases?.Allergy. 1999; 54: 757-759Crossref PubMed Scopus (99) Google Scholar Furthermore, Overbergh et al have recently reported exacerbation of anaphylactic symptoms with higher levels of specific IgE in hen egg white lysozyme-immunized NOD mice after treatment with calcitriol.Whether dietary vitamin D,7.Cantorna M.T Munsick C Bemiss C Mahon B.D 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease.J Nutr. 2000; 130: 2648-2652Crossref PubMed Scopus (438) Google Scholar which can be converted to the active metabolite calcitriol in vivo, contributes to the development of allergy11.Matheu V Mondoc E Back O Issazadeh S Vitamin D enhances allergic response in mice.Scand J Immunol. 2001; 54: 50Google Scholar could be addressed in properly controlled studies in human beings if nutritionists, epidemiologists, and immunologists come together. Then we will know if there is an association between allergy and vitamin D supplementation in our patients. To the Editor: We are grateful to Mr Vieth1.Vieth R Misuse of terminology to imply that 1,25-dihydroxy-vitamin D is a nutrient: there is no evidence for an association between vitamin D and allergy.J Allergy Clin Immunol. 2004; 113: 790-791Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar for paying attention to our recent article published in The Journal of Allergy and Clinical Immunology.2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Vitamin is a general term for a number of unrelated organic substances that occur in many foods in small amounts and that are necessary in trace amounts for the normal metabolic function of the body. “Vitamin D” is any one of several fat-soluble compounds, known commonly as calciferol. The recommendations of International Union of Pure and Applied Chemistry and International Union of Biochemistry and Molecular Biology, based on H. F. DeLuca's and other workers' proposal, say that the term vitamin D should be used as a general term to describe all steroids that exhibit qualitatively the biological activity of calciol.3.IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) Nomenclature of vitamin D. Recommendations 1981.Mol Cell Biochem. 1982; 49: 177-181PubMed Google Scholar Definitely, many compounds can be called vitamin D, and some of them4.Endres B Kato S DeLuca H.F Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo.Biochemistry. 2000; 39: 2123-2129Crossref PubMed Scopus (29) Google Scholar with activity in vitamin D receptor, but either 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2-vitamin D3) or 1,25-dihydroxycholecalciferol or calcitriol had been a more exact term in our paper.2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Discussing the effects of vitamin D on allergic inflammation in mice,2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar Mr Vieth is claiming that it was not a nutritional study. We totally agree. The paper is a study of pharmacologic effects of 1α,25(OH)2-vitamin D3 (1,25-dihydroxycholecalciferol or calcitriol), an active metabolite of vitamin D, on the immune system in mice in short-term experiments.2.Matheu V Back O Mondoc E Issazadeh-Navikas S Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease.J Allergy Clin Immunol. 2003; 112: 585-592Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar This was not hidden, as Mr Vieth claims. Our aim was to investigate if it is possible to interfere with the TH1/TH2 balance through administration of an active vitamin D–metabolite.5.Overbergh L Decallonne B Waer M Rutgeerts O Valckx D Casteels K.M et al.1-Alpha,25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524-543).Diabetes. 2000; 49: 1301-1307Crossref PubMed Scopus (160) Google Scholar Calcitriol, given as subcutaneous injections every second day, was used6.Lemire J.M Adams J.S Sakai R Jordan S.C 1-Alpha,25dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells.J Clin Invest. 1984; 74: 657-661Crossref PubMed Scopus (388) Google Scholar, 7.Cantorna M.T Munsick C Bemiss C Mahon B.D 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease.J Nutr. 2000; 130: 2648-2652Crossref PubMed Scopus (438) Google Scholar to control the blood levels in all mice. Furthermore, the dose 100 ng of calcitriol is taken from the mouse immunology literature, there given either as intraperitoneal injections or as special diets.7.Cantorna M.T Munsick C Bemiss C Mahon B.D 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease.J Nutr. 2000; 130: 2648-2652Crossref PubMed Scopus (438) Google Scholar, 8.Overbergh L Decallonne B Branisteanu D.D Valckx D Kasran A Bouillon R et al.Acute shock induced by antigen vaccination in NOD mice.Diabetes. 2003; 52: 335-341Crossref PubMed Scopus (16) Google Scholar The possibility that vitamin D status is an environmental factor affecting the prevalence of autoimmune diseases has been published elsewhere.9.Cantorna M.T Vitamin D and autoimmunity: is vitamin D status an environmental factor affecting autoimmune disease prevalence?.Proc Soc Exp Biol Med. 2000; 223: 230-233Crossref PubMed Scopus (205) Google Scholar The other side of the coin might be that vitamin D or analogues could contribute to the development of allergic diseases. Mr Vieth claims that our study provides no association between allergy and vitamin D. We think we have clearly demonstrated that in C57BL/10 mice, which are not a common strain of mice used in allergy studies,10.Matheu V Navikas V Issazadeh-Navikas S Susceptibility of B10.RIII mouse strain to develop inflammatory allergic pulmonary disease: a murine asthma model.Allergol Immunol Clin. 2001; 16: 282-290Google Scholar calcitriol is able to increase the TH2 response, measured as specific IgE production, as we and others have suggested before.11.Matheu V Mondoc E Back O Issazadeh S Vitamin D enhances allergic response in mice.Scand J Immunol. 2001; 54: 50Google Scholar, 12.Wjst M Dold S Genes, factor X, and allergens: what causes allergic diseases?.Allergy. 1999; 54: 757-759Crossref PubMed Scopus (99) Google Scholar Furthermore, Overbergh et al have recently reported exacerbation of anaphylactic symptoms with higher levels of specific IgE in hen egg white lysozyme-immunized NOD mice after treatment with calcitriol. Whether dietary vitamin D,7.Cantorna M.T Munsick C Bemiss C Mahon B.D 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease.J Nutr. 2000; 130: 2648-2652Crossref PubMed Scopus (438) Google Scholar which can be converted to the active metabolite calcitriol in vivo, contributes to the development of allergy11.Matheu V Mondoc E Back O Issazadeh S Vitamin D enhances allergic response in mice.Scand J Immunol. 2001; 54: 50Google Scholar could be addressed in properly controlled studies in human beings if nutritionists, epidemiologists, and immunologists come together. Then we will know if there is an association between allergy and vitamin D supplementation in our patients.

Abstract BACKGROUND: Bevacizumab (BEV) plus chemotherapy has shown high response rates in recurrent glioblastoma (GBM) and patients who achieve response have an improved overall survival as well as quality of life. Recent retrospective analysis of the randomized phase III trial, AVAglio, indicate that patients with the proneural GBM subtype have a survival benefit when treated with BEV in combination with standard treatment. However, no validated biomarkers able to predict BEV response have been identified and the biology reflecting a clinical BEV response is poorly understood. The primary objective of this study was to evaluate the predictive and prognostic value of GBM subtypes in recurrent GBM patients treated with BEV therapy. The secondary objective was to identify biomarkers able to predict response to BEV therapy in recurrent GBM patients. METHODS: A total of 90 recurrent GBM patients treated with BEV combination treatment according to a previously published treatment protocol were included. Inclusion criteria: BEV plus irinotecan treatment in the period between May 2005-2011; available GBM tissue (according to WHO); response evaluable (RANO). RNA from tumor tissue was analyzed by the NanoString platform covering 800 genes. Raw data was assigned to molecular subtypes for each of the samples using the PAMR classifier model, previously trained on the AVAglio dataset. In order to identify novel candidate biomarkers able to predict response, differentially expressed genes (fold-change difference &amp;gt; 1.5) between patients responding versus progressing on BEV were identified using a t-test. Biomarkers significantly (P&amp;lt;0.05) associated with response in multivariate logistic regression analysis adjusted for recently validated clinical prognostic factors were selected for the final predictive model. RESULTS: Molecular subtypes were not associated with response or overall survival. However, two novel independent predictive biomarkers (gene1 down-regulated and gene2 up-regulated in responders) of BEV response and overall survival were identified. Results will be presented. Citation Format: Thomas Urup, Signe Regner Michaelsen, Lars Rønn Olsen, Anders Toft, Ib Jarle Christensen, Kirsten Grunnet, Helle Broholm, Michael Kosteljanetz, Shohreh Issazadeh-Navikas, Hans Skovgaard Poulsen, Ulriik Lassen. Predictive biomarkers for bevacizumab response in recurrent glioblastoma patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A25.

I is a self-destructive process that can lead to irreversible chronic tissue destruction. The defective generation or function of Tregulatory/Treg cells contributes to chronic autoimmune inflammation. We report the first identification of FoxA1 as a novel transcription factor in T-cells that upon ectopic expression conveys suppressive properties in a new Treg population, hereby called FoxA1Tregs. FoxA1 bound to the pdl1 promoter, inducing PD-L1, which was essential for FoxA1+Tregs to kill activated T-cells. FoxA1+Tregs had a distinct transcription profile. They express CD4, CD47and PD-L1hi. IFN-β induces FoxA1+Tregs requiring IFNAR signaling; consequently Ifnb–/– and Ifnar–/– mice were defective in FoxA1+Tregs. Adoptive transfer of stable FoxA1+Tregs inhibited experimental autoimmune encephalomyelitis mediated by functional FoxA1 and PDL1. In patients with relapsing-remitting multiple sclerosis, response to IFN-β-treatment was associated with expansion of suppressive FoxA1+Tregs. FoxA1 is a lineage-specification factor with a specialized role in supporting differentiation and the suppressive function of FoxA1+Treg cells.

Expression of Th1 and Th2 cytokines in the blood plasma of WT and IFN‐βKO mice during the first week post‐surgery. a-d Plasma IFN-γ, IL-2, IL-4, IL-10, IL-12, mKC, and TNF-α protein concentrations (pg/mL), respectively. We present individual data points and the mean (blue dash). (PDF 93 kb)

Shape-memory polymers (SMPs), as a class of stimuli-responsive materials, can remember a temporary shape and return to the original shape exposed to an external stimulus, including heat, humidity, light, and electrical and magnetic fields. Electrospun shape-memory polymer fibers (ESMPFs), generally categorized as nano/microfibers with different morphologies and structures, are fabricated via electrospinning. ESMPFs, as promising materials, have potential applications in tissue engineering, drug release, smart cloth, etc. because of their large surface areas, porous structures, and shape-changing properties. In this chapter, we mainly present various SMPs and their composites as well as electrospinning used to fabricate ESMPFs. Moreover, different ESMPF structures, including nonwoven, core-shell, oriented, and particle-filled or -reinforced composites, achieved by adjusting processing parameters, are discussed. Furthermore, actuation methods covering traditional and remote-controlling approaches are also highlighted. Finally, potential applications in many different fields are elaborated along with potential challenges and perspectives.

The publisher regrets that the author listing was published incorrectly in the above article. The correct author listing is given above. GABA, a natural immunomodulator of T lymphocytesJournal of NeuroimmunologyVol. 205Issues 1-2Previewγ-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could be activated by the low (nM–1 µM), physiological concentrations of GABA present around neurons in the brain. The cells expressed the α1, α4, β2, β3, γ1 and δ GABAA channel subunits and formed functional, extrasynaptic-like GABA channels that were activated by 1 µM GABA. Full-Text PDF