Understanding progressive CNS autoimmunity using transgenic mouse models

La sclérose en plaques (SP) est une maladie auto-immune chronique du système nerveux central (SNC) caractérisée par une neurodégénérescence et une invalidité croissante avec le temps. Au Canada, plus de 100 000 personnes sont atteintes de SP. Plusieurs facteurs peuvent contribuer à l’aggravation de la maladie, mais la cause exacte n’est toujours pas connue. Il est largement admis que les lymphocytes T et B franchissent la barrière hémato-encéphalique et invoquent une attaque inflammatoire contre la myéline du SNC. Le rôle des cellules T et B dans l'auto-immunité du SNC peut être étudié à l'aide d'un modèle animal appelé encéphalomyélite auto-immune expérimentale (EAE). Dans la première partie de la thèse, j'ai utilisé une souris transgénique sur un fond de diabète non obèse (DNO) appelé 1C6, dont les cellules T réagissent spécifiquement à un peptide de la myéline, la glycoprotéine de la myéline provenant des oligodendrocytes 35-55 (MOG[35-55]). Les cellules T CD4 de souris 1C6 mâles et femelles sont différenciées en cellules Th17 et sont transférées de manière adoptive à des souris DNOscid déficientes en lymphocytes. Les cellules 1C6 Th17 mâles sont devenues très pathogéniques par rapport aux femelles et ont induit une maladie évolutive sévère chez les receveurs. Les cellules Th17 des deux sexes présentaient une plasticité phénotypique telle que mesurée par leur expression de la cytokine Th1 classique IFN-©. Cependant, les Th17 mâles affichent une production accrue d’ IFN-© par les cellules Th17 mâles, ce qui est corrélé à la gravité de la maladie chez les souris receveuses. L'utilisation d'un modèle de génotype à quatre noyaux nous a permis de séparer l'effet des hormones sexuelles et des chromosomes sexuels dans l'EAE. Nous avons découvert qu'un gène de régulation immunitaire dans le chromosome X, appelé Jarid1c, s'est avéré être régulé négativement dans les cellules Th17 mâles ayant causé une plus grande sévérité, ainsi que dans les cellules T CD4+ provenant du sang périphérique d'hommes atteints de SP. Dans la deuxième partie de la thèse, j'ai utilisé une souris transgénique appelée IgH[MOG] sur un fond DNO dont les cellules B sont spécifiques de la protéine MOG. Lors de l'immunisation avec MOG[35-55], les souris IgH[MOG] ont présenté une EAE rapide et létale, qui est corrélée à l'inflammation et à la démyélinisation du système nerveux central de ces souris. Ceci est accompagné par l'infiltration de cellules B et de cellules T dans le système nerveux central. Chez les souris IgH[MOG], les cellules T CD4+ infiltrantes dans le SNC sont devenues très pro-inflammatoires, comme le montre leur production d'IL-17 et de GM-CSF dans le SNC. Par conséquent, nos données fournissent un aperçu des contributions des réponses des cellules T Th17, du sexe masculin et des cellules B dans l'auto-immunité chronique du SNC. À l’avenir, ces travaux pourraient nous permettre d’identifier des molécules et des voies pouvant être ciblées pour le traitement de la SP.Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) marked by neurodegeneration and accumulating disability over time. Over 100,000 people in Canada are affected by MS. Multiple factors could contribute to the worsening of the disease and yet the exact cause is still unknown. It is widely accepted that T and B lymphocytes cross the blood-brain barrier and invoke an inflammatory attack against CNS myelin. The role of T and B cells in CNS autoimmunity can be studied using an animal model called experimental autoimmune encephalomyelitis (EAE). In the first part of this thesis, I have used a transgenic mouse on a non-obese diabetic (NOD) background called 1C6 whose T cells possess specificity for a myelin-derived peptide, myelin oligodendrocyte glycoprotein 35-55 (MOG[35-55]). 1C6 CD4+ T cells from both male and female mice are differentiated into Th17 cells and are adoptively transferred into lymphocyte deficient NOD.Scid mice. Male 1C6 Th17 cells became highly pathogenic compared to the females and induced a severe progressive disease in the recipients. Th17 cells from both sexes exhibited phenotypic plasticity as measured by their expression of the classic Th1 cytokine IFN-©. However, male Th17 display increased production of IFN-© by male Th17 cells and this is correlated with disease severity in recipient mice. The use of four core genotype model has allowed us to segregate the effect of sex hormones and sex chromosomes in EAE. We uncovered an immune regulatory gene in the X chromosome called Jarid1c is found to be downregulated in both male Th17 cells that caused greater severity as well as in CD4+ T cells from the peripheral blood of men with MS. In the second part of the thesis, I utilized a transgenic mouse called IgH[MOG] on a NOD background whose B cells are specific for MOG protein. Upon immunization with MOG[35-55], IgH[MOG] mice displayed a rapid and lethal EAE, which is correlated to the inflammation and demyelination in the CNS of these mice. This is accompanied by the infiltration of B cells and T cells into the CNS. In IgH[MOG] mice, CNSinfiltrating CD4+ T cells became highly proinflammatory as measured by their production of IL-17 and GM-CSF in the CNS. Hence, our data provide insight into the contributions of Th17 T cell responses, male sex and B cells in chronic CNS autoimmunity. In the future, this work may permit us to identify targetable molecules and pathways for the treatment of MS

Protein translocation and retro-translocation across the endoplasmic reticulum are crucial to inflammatory effector CD4(+) T cell function

Effector CD4(+) T cells can be classified by the cytokines they secrete, with T helper 1 (Th1) cells generating interferon (IFN)gamma and Th17 cells secreting interleukin (IL)-17. Both Th1 and Th17 cells are strongly implicated in the initiation and chronicity of autoimmune diseases such as multiple sclerosis. The endoplasmic reticulum (ER) has been implicated as a potentially crucial site in regulating CD4(+) T cell function. Secretory and transmembrane proteins are shuttled into the ER via the Sec61 translocon, where they undergo appropriate folding; misfolded proteins are retro-translocated from the ER in a p97-dependent manner. Here, we provide evidence that both processes are crucial to the secretion of inflammatory cytokines from effector CD4(+) T cells. The pan-ER inhibitor eeeyarestatin-1 (ESI), which interferes with both Sec61 translocation and p97 retro-translocation, inhibited secretion of interferon (IFN)gamma, interleukin (IL)-2 and tumor necrosis factor (TNF)alpha from Th1 cells in a dose-dependent manner. Selective inhibition of Sec61 by Apratoxin A (ApraA) revealed that ER translocation is crucial for Th1 cytokine secretion, while inhibition of p97 by NMS-873 also inhibited Th1 function, albeit to a lesser degree. By contrast, none of ESI, ApraA or NMS-873 could significantly reduce IL-17 secretion from Th17 cells. ApraA, but not NMS-873, reduced phosphorylation ApraA had modest effects on activation of the Th17 transcription factor Stat3, while NMS-873 had no effect. Interestingly, NMS-873 was able to reduce disease severity in CD4(+) T cell-driven experimental autoimmune encephalomyelitis (EAE). Together, our data indicate that CD4(+) T cell function, and Th1 cell function in particular, is dependent on protein translocation and dislocation across the ER.Peer reviewe

A Scoping Review of Modifiable Risk Factors in Pediatric Onset Multiple Sclerosis: Building for the Future

Knowledge of the effect of modifiable lifestyle factors in the pediatric multiple sclerosis (MS) population is limited. We therefore conducted a scoping review, following the framework provided by Arksey and O’Malley. Four databases were searched for pediatric MS and modifiable lifestyle factors using index terms and keywords, from inception to May 2018. All quantitative and qualitative primary articles were included and limited to English and full text. Of the 7202 articles identified and screened, 25 full-text articles were relevant to our objective and were included. These articles focused on diet obesity, physical activity, and sleep. In cross-sectional analyses, these lifestyle factors were associated with increased risk of pediatric onset MS (POMS), and increased disease activity. Diet, particularly vitamin D and vegetable intake, was associated with reduced relapse rate. Obesity was linked to increased risk of POMS, and physical activity was associated with reduced relapse rate and sleep/rest fatigue. Thus, available studies of lifestyle related outcomes in pediatric MS suggest specific lifestyle related factors, including obesity, higher vitamin D levels, and higher physical activity may associate with lower disease burden in POMS. Studies reviewed are limited by their observational designs. Future studies with longitudinal and experimental designs may further clarify the role of modifiable lifestyle factors in this population

Interferon-β suppresses murine Th1 cell function in the absence of antigen-presenting cells.

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals

IFN-β suppresses Th1 cell proliferation.

<p>A. WT CD4⁺CD62L^hi T cells were labeled with CFSE and stimulated for 5 days with either soluble anti-CD3 plus irradiated splenocytes (APCs), or with plate-bound anti-CD3+anti-CD28, under Th1 or Th17 conditions, with the indicated concentrations of IFN-β. CFSE dilution was assessed by flow cytometry. Data representative of three experiments. B. WT or <i>IFNAR1-/-</i> CD4⁺CD62L^hi T cells were labeled with CFSE and stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions with the indicated concentrations of IFN-β for 5 days. CFSE dilution was assessed by flow cytometry. Data representative of two experiments. Gates represent the percentage of cells that underwent at least one division.</p

IFN-β regulates Stat1 and Stat4 expression on Th cells in the absence of APCs.

<p>CD4⁺CD62L^hiCD25^- T cells were sorted from the spleens and lymph nodes of C57BL6/J mice. They were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 or Th17 conditions, with the indicated concentrations of IFN-β, for 48 hours, and cell lysates were generated. A. Expression of Stat1 and pStat1(Y701) were assessed by Western blot. Representative of two experiments. B. Expression of Stat4 and pStat4(Y693) were assessed by Western blot. Representative of three experiments. GAPDH, loading control.</p

IFN-β suppresses the encephalitogenic potential of Th1 cells.

<p>CD4⁺CD62L^hi T cells were isolated from female 2D2 mouse spleens and lymph nodes, and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, in the presence or absence of 100 U mL^-1 of IFN-β, for 5 days. They were then transferred to female, 6-week old, <i>Rag1-/-</i> mice (5x10⁶ cells/mouse). Recipient mice were monitored for clinical signs of EAE. n = 5 Th1, n = 4 Th1+IFN-β. Right graph, linear regression curves of the disease courses. The slopes are significantly different between the disease courses (p<0.0006). B. Brains and spinal cords were isolated from mice in (A) at d32 and single mononuclear cell suspensions were obtained. The frequency of CNS-infiltrating CD4⁺ cells was assessed by flow cytometry. * p<0.05, two-tailed Student’s <i>t</i> test.</p

IFN-β suppresses cytokine secretion from Th1 cells.

<p>A. CD4⁺CD62L^hi T cells were sorted from the spleens and lymph nodes of C57BL6/J mice and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, with the indicated concentrations of IFN-β, for 5 days. Cells were restimulated for 48 hours under Th1 conditions with the same concentration of IFN-β as at the initial stimulation. A. Generation of IFN-γ and IL-2 was assessed by intracellular cytokine staining and flow cytometry. Representative of three experiments. B. Expression of Tim-3 and PD-1 were assessed by flow cytometry. Representative of three experiments. C. Expression of T-bet was assessed by flow cytometry. Representative data from one of three mice assessed individually. Solid line with open histogram, Th1; dashed line with open histogram, Th1 + 10 U mL^-1 IFN-β; dotted line with open histogram, Th1 + 100 U mL^-1 IFN-β; shaded histogram, FMO control. Data in all panels gated on CD4⁺ cells.</p

Th1 cells express the type I interferon receptor.

<p>A. Naïve (CD62L^hi) and effector (CD62L^lo) CD4⁺ and CD8⁺ C57BL/6J T cells were assessed for surface expression of Ifnar1 by flow cytometry. Open histogram, Ifnar1; shaded histogram, fluorescence minus one (FMO) control. B. Th1 and Th17 cells were cultured and assessed for surface expression of Ifnar1 after 5 days. Solid line with open histogram, Th1; Dotted line with open histogram, Th17; shaded histogram, FMO control. Data representative of one of three experiments.</p

Peripheral adaptive immunity of the triple transgenic mouse model of Alzheimer’s disease

Abstract Background Immunologic abnormalities have been described in peripheral blood and central nervous system of patients suffering from Alzheimer’s disease (AD), yet their role in the pathogenesis still remains poorly defined. Aim and methods We used the triple transgenic mouse model (3xTg-AD) to reproduce Aβ (amyloid plaques) and tau (neurofibrillary tangles) neuropathologies. We analyzed important features of the adaptive immune system in serum, primary (bone marrow) as well as secondary (spleen) lymphoid organs of 12-month-old 3xTg-AD mice using flow cytometry and ELISPOT. We further investigated serum cytokines of 9- and 13-month-old 3xTg-AD mice using multiplex ELISA. Results were compared to age-matched non-transgenic controls (NTg). Results In the bone marrow of 12-month-old 3xTg-AD mice, we detected decreased proportions of short-term reconstituting hematopoietic stem cells (0.58-fold, P = 0.0116), while lymphocyte, granulocyte, and monocyte populations remained unchanged. Our results also point to increased activation of both B and T lymphocytes. Indeed, we report elevated levels of plasma cells in bone marrow (1.3-fold, P = 0.0405) along with a 5.4-fold rise in serum IgG concentration (P < 0.0001) in 3xTg-AD animals. Furthermore, higher levels of interleukin (IL)-2 were detected in serum of 9- and 13-month-old 3xTg-AD mice (P = 0.0018). Along with increased concentrations of IL-17 (P = 0.0115) and granulocyte-macrophage colony-stimulating factor (P = 0.0085), these data support helper T lymphocyte activation with Th17 polarization. Conclusion Collectively, these results suggest that the 3xTg-AD model mimics modifications of the adaptive immunity changes previously observed in human AD patients and underscore the activation of both valuable and harmful pathways of immunity in AD