Type I IFNs in Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis

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Type I IFNs and Lymphocytic Choriomeningitis Virus (LCMV) Infection

LCMV is a member of the Arenaviridae family of viruses and contains a bi-segmented single-stranded ambisense RNA genome. The genome contains two genes on each of the two segments, termed L (7.2 kb) and S (3.4 kb), separated by a unique hairpin structure. The L segment contains the viral RNA dependent RNA polymerase (L) and a small RING-finger protein (Z) which acts a matrix protein linking the viral nucleoprotein and membrane proteins and is the major protein involved in viral budding (151-153). The S segment contains the nucleoprotein (NP) and glycoprotein polyprotein (GP) that becomes cleaved post-translationally into SSP (signal peptide), GP1 and GP2 (154-156). Following cleavage, the GP complex is responsible for the initial binding of virions to cells and affecting their entry into cells.

LCMV, as well as several other arenavirus family members, uses the ubiquitously

expressed cell surface molecule α-dystroglycan for viral entry (157, 158). Differences in binding affinity of various strains of LCMV for α-dystroglycan has been correlated with changes in tissue tropism and disease outcome, high affinity binding leads to higher levels of replication, increased infection of antigen-presenting cells, and overall higher levels of immunosupression. Less pathogenic strains of LCMV are more likely to use alternative receptors for cell entry, but these receptors remain less well characterized (159, 160).
LCMV is a natural pathogen in mice that has been used to elucidate many aspects of T cell immunity (161). LCMV is particularly useful as a model system because infection can lead to acute or chronic infections depending upon host and virus factors (159, 162). Peripheral inoculation of adult mice with the acutely replicating strain LCMV Armstrong (ARM) induces a potent anti-viral CD8 T cell response that is capable of clearing virus within 7-10 days. Infection with LCMV strain Clone 13 (C13), which is a genetic variant of the Armstrong (ARM) strain, leads to persistently high viremia for up to three months with some tissues never clearing virus (163-165). A study showed that type I IFN signals were required for CD8+ expansion in response to lymphocytic choriomeningitis virus (LCMV). The absence of type I IFN lead to the blockade CD8+ T cells during LCMV (96).

Immune Responses to Acute LCMV infection

The LCMV Armstrong (ARM) response is associated with a robust CD8+ T cell response that is composed of three distinct stages: naive, effector and memory. Following acute ARM infection, CD8+ T cell responses are directed at the immunodominant LCMV peptides NP396-404, GP33-41, and to a lesser extent GP276-286 in the context of MHC Class I H-2Db (166). CD8+ T cells reactive to these epitopes expand rapidly and reach a peak of expansion by day 8. These effector cells extravasate to peripheral tissues and control the infection, often by killing infected cells and producing effector cytokines Following this expansion and viral clearance >90% of these cells undergo apoptosis leaving a small population from which a stable pool of memory CD8+ T cells is generated (167, 168).
During the expansion phase of LCMV ARM infection, CD8+ T cells express effector proteins and begin to increase expression of the genes for IFN-γ, TNF-α, IL-2, perforin and granzyme B (166, 169, 170). T cell contraction following expansion is believed to limit the immunopathology that results from excessive cytolysis and cytokine production, and also resets the immune system following infection to become more able to respond to new infections (167). The memory CD8+ T cells that persist following acute infection are characterized by the ability to survive long-term in the absence of antigen, proliferate in response to homeostatic signals, and to rapidly divide and produce effector cytokines upon re-challenge (171).
Although CD8+ T cells are essential for the clearance of LCMV ARM, there is also a robust CD4+ T cells response that is dispensable for the CD8+ cells response. Virus specific-CD4+ T cells expand early during infection, though in significantly lower numbers and with slower kinetics than CD8+ T cells and reach a peak expansion 1-2 days after CD8+ T cells (172, 173). The CD4+ T cell responses are primarily Th1 in nature, producing IFN-γ and IL-2 (174, 175). Experiments in CD4+ deficient mice demonstrated that CD4+ T cells were dispensable to the clearance of acute infections, but in the absence of CD4+ T cell help, CD8+ T cells became functionally impaired and failed to maintain a stable memory population (176-178). The activation of CD4+ T cells in response to LCMV has been shown to have a much higher dependence on signaling through co-stimulatory molecules such as CD28, CD154, and OX40 (179, 180).

Immune Responses to Chronic LCMV infection

LCMV clone 13 (C13) was isolated from mice persistently infected with LCMV Armstrong virus (181). It has 2 silent mutations and 2 amino acid changes from the parental ARM strain. Infection of naïve mice with a high dose of the C13 strain of LCMV results in a protracted viremia that lasts approximately 2-3 months, with virus persisting in select tissues such as the kidney, brain and salivary glands (165). C13 infection does not lead to a robust CD8+ and CD4+ T cell responses capable of clearing virus and protecting against future infection. During chronic LCMV infection, LCMV-specific CD8+ T cells also lose of their lytic capacity and the production of cytokines such as IL-2, TNF-α, and IFN-γ (165). The expansion of CD8+ T cells is diminished in C13 infected mice and the hierarchy of immunodominance is altered (182).
Although CD8+ T cells specific for NP396-404 are the immunodominant population during ARM infection, this CD8+ T cell population becomes nearly undetectable following C13 infection (165, 183, 184). Responses to GP epitopes are less affected and normally subdominant, but CD8+ clones specific for GP276-286 become immunodominant during C13 infection (183). It is widely accepted that chronic antigen exposure leads to diminished function and eventual deletion of cells that most recognize the most frequent viral-peptide: MHC Class I complexes (185-187). Because NP is the most abundantly expressed viral antigen, NP396-404-reactive clones are more likely to be stimulated and eventually exhausted, while clones recognizing the less abundant GP will be present and remain functional for longer periods of time (167, 176).
Recent studies analyzed the molecular mechanisms of T cell exhaustion during chronic infection. Expression of the inhibitory receptor, programmed death-1 (PD-1), is the hallmark of CD8+ T cell exhaustion. Blocking the PD-1/PD-L1 signaling pathway restores effector T cell function and leads to resolution of chronic LCMV infection (188). Further analysis of gene expression in exhausted T cells led to the identification of several other inhibitory molecules that mediate T cell exhaustion, including LAG-3, Tim-3 and 2B4 (189-191). These data revealed a complex pattern of regulation of CD8+ T cells by an array of co-expressed inhibitory receptors during chronic viral infection.

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Role of Tregs in LCMV infection

After an infection, the balance between Tconv and Treg is critical. Pathogen-specific CD4+ and CD8+ T cells rapidly expand after infection and, in most cases, clear the infection. Treg may play a role during acute infection, but their importance is still unclear (211). However, during chronic infection, Treg can play critical roles by limiting excessive immune activation and tissue damage, while at the same time facilitating pathogen persistence and maintenance of immunity (212). We have previously shown that expansion of Treg following chronic LCMV (C13) infection was most prominent amongst a subset of Treg expressing a particular TCR Vβ subunit(s) (Vβ5 in C57BL/6 mice and Vβ5 and 12 in BALB/c mice) (213). Furthermore, our study demonstrated that Treg expansion in chronic LCMV infection was not directly mediated by LCMV, but was secondary to induction of Mammary tumor virus (Mtv)-superantigens (Sag) encoded in the mouse genome (eSag). A recent study has claimed that type I IFNs directly inhibit co-stimulation–dependent Treg cell activation and proliferation in vivo during acute infection with LCMV. In studies using mixed bone marrow chimeras between WT mice and IFNAR KO mice, loss of the IFNAR specifically in Treg cells resulted in functional impairment of virus-specific CD8+ and CD4+ T cells and inefficient viral clearance. The study claims that inhibition of Treg cells by IFNs is necessary for the generation of optimal antiviral T cell responses during acute LCMV infection (150).

Type I IFNs in Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE)

Given the diverse effects of IFNα/β in the innate and adaptive immune system, it is not surprising that these cytokines play a role in several autoimmune diseases. Psoriasis and systemic lupus erythematosus are improved by the inhibition of Type I IFNs (214, 215), while arthritis and multiple sclerosis benefit from the administration of Type I IFNs (216). Although the associations between Type I IFN and these diseases are established, the mechanisms responsible for the differential effects of IFN have not yet been elucidated.

Treg respond to Type I IFN in vitro and in vivo

The ability of both CD4+ and CD8+ T effector (Teff) cells to respond to Type I IFN is well established (286, 287), but the role of Type I IFN in Treg function remains poorly understood. While all cells express the IFNAR and can potentially respond to type I IFNs, we initially assessed the ability of Treg to directly respond to Type I IFN ex vivo by measuring the phosphorylation of STAT1 following a 15-min stimulation with recombinant IFN-α2 or IFN-β. The response of Treg was similar to CD4+ and CD8+ Teff over a range of cytokine concentrations (Fig. 5A).
We then addressed a potential role for Type I IFN signaling in Treg function by assessing the relative ability of different T cell subpopulations to respond to Type I IFN in vivo. Injection of mice with the synthetic double-stranded RNA homolog poly (I:C) has been shown to induce large amounts of Type I IFN in a Toll-like receptor-3-dependent manner (288). Previous studies have shown that stimulation of lymphocytes with Type I IFN rapidly induces the expression of CD69 (104, 289). We initially measured CD69 expression on various T lymphocyte subpopulations 24h following poly (I:C) injection as a readout for the response to Type I IFN. CD4+, CD8+ and CD4+Foxp3+ T cells uniformly expressed high levels of CD69 (Fig. 5B) following treatment of the mice with poly (I:C). As a negative control, CD69 up-regulation was not observed in any cell population following treatment of IFNAR KO mice with poly (I:C) (data not shown). IFN-α was detected in the serum at 24h following poly (I:C) injection, but not 72h after injection indicating that the secretion of IFN-α was transient with this dose of poly (I:C) (Fig. 5C)

Table of contents :

ACKNOWLEDGEMENTS
ABREVIATIONS
RESUME EN FRANÇAIS
ABSTRACT
LIST OF FIGURES
I. INTRODUCTION
A. Type I interferons
Type I IFN production
Type IFN signaling
B. Effects of Type I IFNs on Cells of the Immune System
Effects of type I IFNs on CD4+ T cells
Effects of type I IFN on effector and memory CD8+ T cells
C. Effects of Type I IFNs on T Regulatory T Cells (Treg)
D. Type I IFNs and Lymphocytic Choriomeningitis Virus (LCMV) Infection
Immune Responses to Acute LCMV infection
Immune Responses to Chronic LCMV infection
The Role of type I IFN During LCMV infection
Role of Tregs in LCMV infection
E. Type I IFNs in Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis
(EAE)
Multiple Sclerosis (MS)
Tregs in MS
Experimental Autoimmune Encephalomyelitis (EAE)
Tregs in EAE
Role of Type I IFNs in MS/EAE
II. MATERIALS AND METHODS
III. EXPERIMENTAL RESULTS
IV. FIGURES
V. DISCUSSION
REFERENCES

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