System-wide Analysis of the T Cell Response

SummaryThe T cell receptor (TCR) controls the cellular adaptive immune response to antigens, but our understanding of TCR repertoire diversity and response to challenge is still incomplete. For example, TCR clones shared by different individuals with minimal alteration to germline gene sequences (public clones) are detectable in all vertebrates, but their significance is unknown. Although small in size, the zebrafish TCR repertoire is controlled by processes similar to those operating in mammals. Thus, we studied the zebrafish TCR repertoire and its response to stimulation with self and foreign antigens. We found that cross-reactive public TCRs dominate the T cell response, endowing a limited TCR repertoire with the ability to cope with diverse antigenic challenges. These features of vertebrate public TCRs might provide a mechanism for the rapid generation of protective T cell immunity, allowing a short temporal window for the development of more specific private T cell responses

Adult neural stem cells in neuroinflammation

Adult neural stem cells (NSC) can be isolated from the subventricular zone (SVZ) of the lateral ventricles, the subgranular zone (SGZ) of the hippocampus and the central canal of the spinal cord. These cells are thought to have regenerative potential and pose important therapeutic possibilities in neuroinflammatory conditions such as Multiple Sclerosis (MS). The aim of this thesis was to investigate the function of these cells during neuroinflammation. To that end we employed both in vitro primary cultures of NSC and the experimental autoimmune encephalomyelitis (EAE) model. I. NSC generate neurons in demyelinated spinal cord lesions. In order to monitor NSC behaviour in EAE we labelled the endogenous NSC by injecting a lipophilic dye, DiI in the ventricle system of Dark Agouti rats. These rats were immunized to develop EAE and injected with BrdU to identify proliferating cells. We report that NSC proliferated and migrated to demyelinated lesions in the spinal cord of EAE diseased rats, where some of the cells started to express betaIIItubulin or NeuN. Ultimately, these NSC-derived neuronal-like cells could generate overshooting action potentials. II. Nitric oxide (NO) suppresses NSC-derived neurogenesis. In this study we used NSC cultures to analyse the effects of nitric oxide (NO) on NSC proliferation and differentiation. NO is produced within EAE and MS lesions and has been correlated with disease exacerbation in MS. We report that exposure of NSC to pathophysiological concentrations of NO diverted their differentiation potential from neurogenesis towards astrogliogenesis. Using immunocytochemistry we could demonstrate a lower percentage of betaIIItubulin-IR neurons but a higher percentage of O4-IR oligodendrocytes in NO-exposed cultures. The higher rate of gliogenesis in these cultures was also confirmed by western blotting for the astrocyte-specific protein GFAP and activated STAT1, a transcription factor involved in gliogenic differentiation. Moreover, the pro-neurogenic determinant neurogenin-2 was down-regulated subsequent to NO exposure, constituting a potential mechanism for the NO-mediated down-regulation of neurogenesis. III. High Mobility Group Box Protein 1 (HMGB1) expression correlates with inflammation in MS and EAE. In the third study we focused on the expression of the cytokine HMGB1 and its receptors RAGE, TLR-2 and TLR-4 in MS and EAE. Our interest in HMGB1 is related both to its cytokine function in inflammatory diseases and to its possible involvement in cell migration and differentiation. We detected cytoplasmic translocation of HMGB1, indicative of active release, in microglia and macrophages located in MS lesions. Moreover, the expression of HMGB1 and its receptors in immune cells isolated from the cerebrospinal fluid (CSF) of MS patients was significantly higher compared to cells from CSF of control patients. In EAE, an increased transcript level of HMGB1 correlated with higher disease severity. Finally, microglia cells could translocate HMGB1 to the cytoplasm, implying their ability to actively release this protein and indicating their potential contribution to inflammation. IV. TLR-2 and TLR-4 agonists induce TNFalpha release from NSC. TLR-2 and TLR-4 recognise bacterial moieties and can also ligate HMGB1. TLR activation in innate immune cells leads to release of inflammatory agents aimed at clearing invading pathogens. The connection between TLR and NSC originates from the Drosophila ortholog, Toll, which participates in neuronal patterning. Immunocytochemical investigations of primary NSC cultures revealed the presence of both TLR-2 and TLR-4 on these cells. Moreover, the expression of these receptors was differentially regulated by inflammatory conditions and cytokines. Agonist-induced TLR activation was not involved in differentiation or proliferation of NSC. Activation of these receptors prompted NSC to express the pro-inflammatory cytokine TNFalpha at both mRNA and protein levels. In conclusion, we demonstrated that inflammatory conditions can both promote and inhibit the ectodermal differentiation capacity of NSC, but also to yield them unexpected immune features

Adult Neural Progenitor Cells Transplanted into Spinal Cord Injury Differentiate into Oligodendrocytes, Enhance Myelination, and Contribute to Recovery

Summary: Long-term survival and integration of neural progenitor cells (NPCs) transplanted following spinal cord injury (SCI) have been observed. However, questions concerning the differentiation choice, the mechanism of action, and the contribution of NPCs to functional recovery remains unanswered. Therefore, we investigated the differentiation of NPCs, global transcriptomal changes in transplanted NPCs, the effect of NPCs on neuroinflammation, and the causality between NPC transplantation and functional recovery. We found that NPCs transplanted following SCI differentiate mainly into oligodendrocytes and enhance myelination, upregulate genes related to synaptic signaling and mitochondrial activity, and downregulate genes related to cytokine production and immune system response. NPCs suppress the expression of pro-inflammatory cytokines/chemokines; moreover, NPC ablation confirm that NPCs were responsible for enhanced recovery in hindlimb locomotor function. Understanding the reaction of transplanted NPCs is important for exploiting their full potential. Existence of causality implies that NPCs are useful in the treatment of SCI. : In this article, Brundin and colleagues show that NPCs transplanted following SCI differentiated mainly into oligodendrocytes and enhanced myelination, upregulated genes related to synaptic signaling and mitochondrial activity, suppressed pro-inflammation, and were responsible for enhanced recovery in hindlimb function. Understanding the reaction of transplanted NPCs is important for exploiting their full potential. Existence of causality implies that NPCs are useful in the treatment of SCI. Keywords: spinal cord injury, neural progenitor cells, global transcriptomal changes, neuroinflammation, oligodendrocyte, myelination, regeneration, hindlimb locomotor function, transplantatio

Neurogenesis in the adult spinal cord in an experimental model of multiple sclerosis

Multiple sclerosis is an inflammatory disease of the central nervous system characterized by inflammation, demyelination, axonal degeneration and accumulation of neurological disability. Previously, we demonstrated that stem cells constitute a possible endogenous source for remyelination. We now addressed the question of whether neurogenesis can occur in neuroinflammatory lesions. We demonstrated that, in experimental autoimmune encephalomyelitis, induced in rats 1,1'-dioctadecyl-6,6'-di(4sulphopentyl)-3,3,3',3'tetramethylindocarbocyani n(DiI)-labelled ependymal cells not only proliferated but descendants migrated to the area of neuroinflammation and differentiated into cells expressing the neuronal markers beta-III-tubulin and NeuN. Furthermore, these cells were immunoreactive for bromodeoxyuridine and PCNA, markers for cells undergoing cell proliferation. Using the whole-cell patch-clamp technique on freshly isolated 1, DiI-labelled cells from spinal cord lesions we demonstrated the ability of these cells to fire overshooting action potentials similar to those of immature neurones. We thus provide the first evidence for the initiation of neurogenesis in neuroinflammatory lesions in the adult spinal cord

DNA methylation changes in glial cells of the normal-appearing white matter in Multiple Sclerosis patients

Multiple Sclerosis (MS), the leading cause of non-traumatic neurological disability in young adults, is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). Due to the poor accessibility to the target organ, CNS-confined processes underpinning the later progressive form of MS remain elusive thereby limiting treatment options. We aimed to examine DNA methylation, a stable epigenetic mark of genome activity, in glial cells to capture relevant molecular changes underlying MS neuropathology. We profiled DNA methylation in nuclei of non-neuronal cells, isolated from 38 post-mortem normal-appearing white matter (NAWM) specimens of MS patients (n = 8) in comparison to white matter of control individuals (n = 14), using Infinium MethylationEPIC BeadChip. We identified 1,226 significant (genome-wide adjusted P-value < 0.05) differentially methylated positions (DMPs) between MS patients and controls. Functional annotation of the altered DMP-genes uncovered alterations of processes related to cellular motility, cytoskeleton dynamics, metabolic processes, synaptic support, neuroinflammation and signaling, such as Wnt and TGF-β pathways. A fraction of the affected genes displayed transcriptional differences in the brain of MS patients, as reported by publically available transcriptomic data. Cell type-restricted annotation of DMP-genes attributed alterations of cytoskeleton rearrangement and extracellular matrix remodelling to all glial cell types, while some processes, including ion transport, Wnt/TGF-β signaling and immune processes were more specifically linked to oligodendrocytes, astrocytes and microglial cells, respectively. Our findings strongly suggest that NAWM glial cells are highly altered, even in the absence of lesional insult, collectively exhibiting a multicellular reaction in response to diffuse inflammation

miR‐31 regulates energy metabolism and is suppressed in T cells from patients with Sjögren's syndrome

Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a Tcell-specific reduction of miR-31-5p levels, both after intramuscular injection of IFN and in patients with Sjogren's syndrome (SjS). To interrogate the role of miR-31-51p in Tcells we transfected human CD4(+) Tcells with a miR-31-5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR-31-5p. Furthermore, treatment with IFN- also increased the basal levels of human CD4(+) T-cell metabolism. In all, our results suggest that reduced levels of miR-31-5p in Tcells of SjS patients support autoimmune T-cell responses during chronic type I IFN exposure

CCL11 and CCR3 protein levels in the inguinal lymph nodes.

<p>(A) Western blot analysis of CCL11 and its main receptor CCR3 in inguinal lymph nodes on day 7 p.i. Protein levels of both targets were increased in the congenic animals compared to the controls (data normalized to β- actin). CCL11, CCR3 and β- actin shown in original representative blots and graphically; three blots performed for each target within two separate EAE experiments. (B) Co-staining with antibodies directed against CCL11 and other immune cell types on paraffin cross sections of inguinal lymph nodes performed on day 7 p.i. in the congenic <i>Eae18b</i> strain. CCL11 co-localized with the macrophage/microglia markers ED1 and Iba-1 (first two images from the left, upper row). CCL11 was not expressed by CD4⁺ and CD8⁺ T cells (last two images, upper row). CCL11 was also not detected in MHC class II⁺ (Ox6⁺) and CD11b⁺ populations, neither in Ox62⁺ dendritic, nor in CD45RA⁺ B cells (second row, from left to right). (C) The main CCL11 receptor CCR3 was detected on W3/13⁺ T lymphocytes and ED1⁺ macrophages, but not on CD45RA⁺ B cells (five congenic and five control rats analyzed, two whole inguinal lymph nodes per each animal).</p

<i>Eae18b</i> regulates intrathecal production of CCL11 and influences the BBB integrity.

<p>(A) During the whole disease course CCL11 protein levels were increased in the cerebrospinal fluid (CSF), in the congenics (Eae18b) compared to controls (DA). Experiments repeated twice, three to five animals per group analyzed. (B) mRNA levels of <i>Ccl11</i> measured in the late disease time point (day 23 p.i.) showed upregulation in the congenic spinal cord. Error bars represent SEM, and (*) indicates p-value <0.05, (**) indicates p-value <0.01, (***) indicates p-value <0.001 calculated by Student’s t-test in (A) and by Mann-Whitney non-parametrical test in (B). (C) Representative images of occludin, a marker for the BBB tight junction components; more abundant in the congenic spinal cord (cross sections of seven congenic and eight control animals analyzed on day 12 p.i.). (D) Representative image of CCR3, the main receptor for CCL11, expressed on ependymal cells of the spinal cord canalis centralis, including a detail in high magnification (160×). Five congenic and four control animals analyzed on day 15 p.i.</p

<i>Eae18b</i> congenic animals significantly upregulate <i>Ccl11</i> mRNA.

<p>qPCR was used to measure mRNA expression of <i>Ccl2, Ccl7, Ccl11, Ccl12</i> and <i>Ccl1</i> in draining lymph nodes (A) and spinal cord tissue (B) on day 7 and 12 p.i. <i>Ccl11</i> mRNA was significantly upregulated in the congenic strain in both lymph node and spinal cord tissue at both time-points. Five to eight animals per strain were used at each time-point and experiments were repeated three times. Error bars represent SEM, statistics were calculated using Mann-Whitney non-parametric test, (*) indicates p-value <0.05, (**) indicates p-value <0.01.</p