Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage ...processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential T cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca
2+ concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.
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► Th17 cells establish immune-neuronal synapses and induce neuronal cell death in vitro ► Th17 cells directly contact neurons irrespective of their CNS-antigen specificity ► Th17 cell-neuronal interaction leads to severe neuronal dysfunction ► Th17 cell-mediated Ca
2+ elevation is partially reversible by blocking excitotoxicity
Abstract
Background
T helper (Th) 17 cells are a highly plastic subset of T cells, which in the context of neuroinflammation, are able to acquire pathogenic features originally attributed to Th1 ...cells (resulting in so called ex-Th17 cells). Thus, a strict separation between the two T cell subsets in the context of experimental autoimmune encephalomyelitis (EAE) is difficult. High variability in culture and EAE induction protocols contributed to previous conflicting results concerning the differential contribution of Th1 and Th17 cells in EAE. Here, we systematically evaluate the role of different T cell differentiation and transfer protocols for EAE disease development and investigate the functional dynamics of encephalitogenic T cells directly within the inflamed central nervous system (CNS) tissue.
Methods
We compiled the currently used EAE induction protocols reported in literature and investigated the influence of the different Th1 and Th17 differentiation protocols as well as EAE induction protocols on the EAE disease course. Moreover, we assessed the cytokine profile and functional dynamics of both encephalitogenic Th1 and Th17 cells in the inflamed CNS using flow cytometry and intravital two-photon laser scanning microscopy. Lastly, we used astrocyte culture and adoptive transfer EAE to evaluate the impact of Th1 and Th17 cells on astrocyte adhesion molecule expression in vitro and in vivo.
Results
We show that EAE courses are highly dependent on in vitro differentiation and transfer protocols. Moreover, using genetically encoded reporter mice (B6.IL17A-EGFP.acRFP x 2d2/2d2.RFP), we show that the motility of interferon (IFN)γ-producing ex-Th17 cells more closely resembles Th1 cells than Th17 cells in transfer EAE. Mechanistically, IFNγ-producing Th1 cells selectively induce the expression of cellular adhesion molecules I-CAM1 while Th1 as well as ex-Th17 induce V-CAM1 on astrocytes.
Conclusions
The behavior of ex-Th17 cells in EAE lesions in vivo resembles Th1 rather than Th17 cells, underlining that their change in cytokine production is associated with functional phenotype alterations of these cells.
Extracellular matrix (ECM) proteins secreted by blood-brain barrier (BBB) endothelial cells (ECs) are implicated in cell trafficking. We discovered that the expression of ECM epidermal growth ...factor-like protein 7 (EGFL7) is increased in the CNS vasculature of patients with multiple sclerosis (MS), and in mice with experimental autoimmune encephalomyelitis (EAE). Perivascular CD4 T lymphocytes colocalize with ECM-bound EGFL7 in MS lesions. Human and mouse activated T cells upregulate EGFL7 ligand αvβ3 integrin and can adhere to EGFL7 through integrin αvβ3. EGFL7-knockout (KO) mice show earlier onset of EAE and increased brain and spinal cord parenchymal infiltration of T lymphocytes. Importantly, EC-restricted EGFL7-KO is associated with a similar EAE worsening. Finally, treatment with recombinant EGFL7 improves EAE, reduces MCAM expression, and tightens the BBB in mouse. Our data demonstrate that EGFL7 can limit CNS immune infiltration and may represent a novel therapeutic avenue in MS.
Background Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence ...points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System xc - or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system xc -, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT-/-) mice and irradiated mice reconstituted in xCT-/- bone marrow (BM), to their proper wild type (xCT+/+) controls. Results xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT+/+ mice, xCT-/- mice were equally susceptible to EAE, whereas mice transplanted with xCT-/- BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system xc - on immune cells invading the CNS participates to EAE. Since a total loss of system xc - had no net beneficial effects, these results have important implications for targeting system xc - for treatment of MS.
The proneurotrophin receptor sortilin is a protein with dual functions, being involved in intracellular protein transport, as well as cellular signal transduction. The relevance of the receptor for ...various neuronal disorders, such as dementia, seizures, and brain injury, is well established. In contrast, little is known about the role of sortilin in immune cells and inflammatory diseases. The aim of our study was to elucidate the distribution of sortilin in different immune cell types in mice and humans and to analyze its function in autoimmune CNS inflammation. Sortilin was expressed most profoundly in murine and human macrophages and dendritic cells and to a much lesser extent in B and T cells. In dendritic cells, sortilin had an impact on Ag processing. Accordingly, sortilin was highly expressed by infiltrated perivascular myeloid cells, mainly in vessel cuffs, in the CNS of patients suffering from multiple sclerosis, the most common inflammatory autoimmune disease of the CNS. Yet, sortilin gene-targeted mice (Sort1(-/-)) and chimeras deficient in sortilin in the immune system were as susceptible as wild-type littermates to T cell-dependent experimental autoimmune encephalomyelitis. Considering our results and recent data from other investigators, we conclude that the proneurotrophin receptor sortilin plays a role in innate, rather than in adaptive, immune processes and, thus, not in autoimmune neuroinflammation.
The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a ...3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed ...central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.
We applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.
By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.
The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.
Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system, which is thought to be triggered by environmental factors in genetically susceptible individuals ...leading to activation of autoreactive T lymphocytes. Large multi-centre genome-wide association studies have identified multiple genetic risk loci in multiple sclerosis. In this study, we investigated T cell transcriptomic changes in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We correlated these findings with the multiple sclerosis risk genes postulated by the most recent Immunochip analysis and found that multiple sclerosis susceptibility genes were significantly regulated in experimental autoimmune encephalomyelitis. Our data indicate that nine distinct genes associated with multiple sclerosis risk, Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7 and Thada, can be confirmed to be differentially regulated in pathogenic CD4(+) T cells. During the effector phase within the inflamed CNS, CD4(+) T cells undergo comprehensive transformation and we identified key transcription factors and signalling networks involved in this process. The transformation was linked to metabolic changes with the involvement of liver X receptor/retinoid X receptor signalling and cholesterol biosynthesis, which might control the T cell effector function in the central nervous system. Thus, our study confirms the involvement of multiple sclerosis risk genes in the pathophysiology of the animal model and sheds light on additional disease-relevant inflammatory networks.
Abstract
We established humanized mouse models by transplantation of human hematopoietic (stem) cells to immunodeficient mice. Engraftment results in the development of a functional human immune ...system. By co-transplantation of human patient-derived xenografts (PDX) we successfully generated a fully human tumor-immune-cell (TIC) model in mice. These humanized TIC-PDX models for melanoma and lung cancer were further characterized by treatment with immune therapy drugs like the CTLA4 inhibitor ipilimumab (Ipi) and the PD-1 inhibitor nivolumab (Nivo). In our current studies we monitored the effects of the human immune cells and evaluated concepts for combination therapies i.e. with chemotherapy or radiation. We further established new human TIC-PDX models from other tumor entities like lymphoma, pancreatic or breast cancer.
Hematopoietic stem cell engraftment was monitored by regular FACS analysis for human immune cells in the blood. PD-L1 expression on tumors as a target for immunotherapy was determined by FACS and immunohistochemistry. PD-L1 positive and negative PDX from 10 different entities were transplanted on humanized mice and treated with Nivo or Ipi alone or in combination therapy with radiation. Blood and tumor samples were analysed by FACS and immunohistochemistry for immune cell infiltration and activation.
The transplanted stem cells showed engraftment in immunodeficient mice with proliferation and differentiation and established a functional human immune system with T-cells, B-cells, NK-cells, monocytes, and dendritic cells. So far, we have transplanted more than 40 different PDX from 10 different tumor entities on humanized mice. Most of investigated PDX (>70%) successfully engrafted on humanized mice and showed no difference in tumor growth compared to growth on non-humanized mice. However, for a few PDX we observed a delayed tumor growth or a complete rejection. These results suggest an immune reaction of the engrafted human immune cells against the PDX. The tumor engraftment or rejection in the humanized mice seems to be independent from the tumor entity. To evaluate whether engraftment of the PDX in mice with a human immune system is in part enabled by checkpoint mediated immune-tolerance, we have treated over 30 PDX models with Ipi or Nivo alone or in combination. Significant tumor growth delay, observed in most models, accompanied by an increased number of T-cells in the blood and in the tumor, has functionally validated our immune-PDX model. Response to the checkpoint inhibitors seems to correlate with innate immune responsiveness and PD-L1 expression and can be further increased by combination with radiotherapy.
Our humanized immune-PDX models enable appropriate preclinical translational research in tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy.
Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Magdalena Paterka, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. Humanized immune-PDX mouse models for 10 different tumor entities in translational immuno-oncology research abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1145.
Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we ...have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.