Production of IL-27 in multiple sclerosis lesions by astrocytes and myeloid cells: Modulation of local immune responses.

The mechanisms whereby human glial cells modulate local immune responses are not fully understood. Interleukin-27 (IL-27), a pleiotropic cytokine, has been shown to dampen the severity of experimental autoimmune encephalomyelitis, but it is still unresolved whether IL-27 plays a role in the human disease multiple sclerosis (MS). IL-27 contribution to local modulation of immune responses in the brain of MS patients was investigated. The expression of IL-27 subunits (EBI3 and p28) and its cognate receptor IL-27R (the gp130 and TCCR chains) was elevated within post-mortem MS brain lesions compared with normal control brains. Moreover, astrocytes (GFAP(+) cells) as well as microglia and macrophages (Iba1(+) cells) were important sources of IL-27. Brain-infiltrating CD4 and CD8 T lymphocytes expressed the IL-27R specific chain (TCCR) implying that these cells could respond to local IL-27 sources. In primary cultures of human astrocytes inflammatory cytokines increased IL-27 production, whereas myeloid cell inflammatory M1 polarization and inflammatory cytokines enhanced IL-27 expression in microglia and macrophages. Astrocytes in postmortem tissues and in vitro expressed IL-27R. Moreover, IL-27 triggered the phosphorylation of the transcription regulator STAT1, but not STAT3 in human astrocytes; indeed IL-27 up-regulated MHC class I expression on astrocytes in a STAT1-dependent manner. These findings demonstrated that IL-27 and its receptor were elevated in MS lesions and that local IL-27 can modulate immune properties of astrocytes and infiltrating immune cells. Thus, therapeutic strategies targeting IL-27 may influence not only peripheral but also local inflammatory responses within the brain of MS patients.

Interleukin-15 enhances proinflammatory T-cell responses in patients with MS and EAE.

OBJECTIVE: We posit that interleukin-15 (IL-15) is a relevant contributor to MS pathobiology as this cytokine is elevated in the CNS and periphery of patients with MS. We aim to investigate (1) the impact of IL-15 on T lymphocytes from patients with MS and (2) the in vivo role of IL-15 using the experimental autoimmune encephalomyelitis (EAE) mouse model. METHODS: We compared the impact of IL-15 on T lymphocytes obtained from untreated patients with MS (relapsing-remitting, secondary progressive, and primary progressive) to cells from age/sex-matched healthy controls (HCs) using multiparametric flow cytometry and in vitro assays. We tested the effects of peripheral IL-15 administration after EAE disease onset in C57BL/6 mice. RESULTS: IL-15 triggered STAT5 signaling in an elevated proportion of T cells from patients with MS compared with HCs. This cytokine also enhanced the production of key proinflammatory cytokines (interferon γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-17, and tumor necrosis factor) by T cells from both MS and controls, but these effects were more robust for the production of IL-17 and GM-CSF in T-cell subsets from patients with MS. At the peak of EAE disease, the proportion of CD4+ and CD8+ T cells expressing CD122+, the key signaling IL-15 receptor chain, was enriched in the CNS compared with the spleen. Finally, peripheral administration of IL-15 into EAE mice after disease onset significantly aggravated clinical scores and increased the number of inflammatory CNS-infiltrating T cells long term after stopping IL-15 administration. CONCLUSIONS: Our results underscore that IL-15 contributes to the amplification of T-cell inflammatory properties after disease onset in both MS and EAE.

NKG2D and Its Ligand MULT1 Contribute to Disease Progression in a Mouse Model of Multiple Sclerosis.

NKG2D is an activating receptor expressed on the surface of immune cells including subsets of T lymphocytes. NKG2D binds multiple ligands (NKG2DL) whose expression are differentially triggered in a cell type and stress specific manner. The NKG2D-NKG2DL interaction has been involved in autoimmune disorders but its role in animal models of multiple sclerosis (MS) remains incompletely resolved. Here we show that NKG2D and its ligand MULT1 contribute to the pathobiology of experimental autoimmune encephalomyelitis (EAE). MULT1 protein levels are increased in the central nervous system (CNS) at EAE disease peak; soluble MULT1 is elevated in the cerebrospinal fluid of both active and passive EAE. We establish that such soluble MULT1 enhances effector functions (e.g., IFNγ production) of activated CD8 T lymphocytes from wild type but not from NKG2D-deficient (Klrk1-/-) mice in vitro. The adoptive transfer of activated T lymphocytes from wild type donors induced a significantly reduced EAE disease in Klrk1-/- compared to wild type (Klrk1+/+) recipients. Characterization of T lymphocytes infiltrating the CNS of recipient mice shows that donor (CD45.1) rather than endogenous (CD45.2) CD4 T cells are the main producers of key cytokines (IFNγ, GM-CSF). In contrast, infiltrating CD8 T lymphocytes include mainly endogenous (CD45.2) cells exhibiting effector properties (NKG2D, granzyme B and IFNγ). Our data support the notion that endogenous CD8 T cells contribute to passive EAE pathobiology in a NKG2D-dependent manner. Collectively, our results point to the deleterious role of NKG2D and its MULT1 in the pathobiology of a MS mouse model.

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry.

BACKGROUND: Flow cytometry is an efficient and powerful technique to characterize and quantify numerous cells. However, the strengths of this technique have not been widely harnessed in neurosciences due to the critical step of CNS tissue preparation into a single cell suspension. Previous reports assessed either neural cells or infiltrating leukocytes but simultaneous detection has not been extensively implemented. We optimized CNS tissue preparation for flow cytometry analysis. NEW METHOD: We subjected CNS tissue from individual adult mice to different digestion protocols and Percoll™ methods. We quantified and characterized by flow cytometry neural cells (neurons, oligodendrocytes, microglia) and leukocytes (macrophages, T lymphocytes). RESULTS: The one step Percoll™ method significantly increased cell yield compared to the gradient Percoll™ method. The collagenase D+DNase I digestion led to the maximal cell number recovery while preserving cell marker (O4, NeuN, CD45, CD11b, CD3, CD4, CD8) integrity compared to papain, trypsin digestion, and no digestion. The combination of collagenase D+DNase I digestion and one step Percoll™ method was optimal for the recovery and analysis of cells from the CNS of naïve and experimental autoimmune encephalomyelitis (multiple sclerosis model) mice. COMPARISON WITH EXISTING METHOD(S): Although flow cytometry does not reveal CNS localization, this technique allows concurrent quantification of multiple parameters. In contrast to other protocols, our novel method simultaneously analyzes neural and immune cells in individual mice in healthy and pathological conditions. CONCLUSIONS: We strongly believe that the field of neurosciences will benefit from an optimal use of flow cytometry to elucidate physiological and pathological processes.