image_1_Distinct Gene Profiles of Bone Marrow-Derived Macrophages and Microglia During Neurotropic Coronavirus-Induced Demyelination.TIF

<p>Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and axonal loss. Demyelinating lesions are associated with infiltrating T lymphocytes, bone marrow-derived macrophages (BMDM), and activated resident microglia. Tissue damage is thought to be mediated by T cell produced cytokines and chemokines, which activate microglia and/or BMDM to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. However, the relative contributions of BMDM and microglia to demyelinating pathology are unclear, as their identification in MS tissue is difficult due to similar morphology and indistinguishable surface markers when activated. The CD4 T cell-induced autoimmune murine model of MS, experimental autoimmune encephalitis (EAE), in which BMDM are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with BMDM and microglia, respectively. Using a murine model of demyelination induced by a gliatropic coronavirus, in which BMDM are redundant for demyelination, we herein characterize gene expression profiles of BMDM versus microglia associated with demyelination. While gene expression in CNS infiltrating BMDM was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mRNA upregulation coinciding with peak demyelination after viral control. This delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. Furthermore, while BMDM exhibited a mixed phenotype of M1 and M2 markers, microglia repressed the vast majority of M2-markers. Overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas BMDM retained their gene expression profile independent of the changing environment. As demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for MS pathogenesis.</p

Additional file 1: of Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation

TNF blockade reduces astrocyte and myeloid cell reactivity during progressive EAE in GFAPγR1Δ mice. Longitudinal spinal cord sections from WT and GFAPγR1Δ mice treated with isotype control or anti-TNF mAb were stained for astrocyte (GFAP) or myeloid cell (Iba-1) reactivity at d19 and d30 as indicated. Data represents three to four separate fields per mouse with two to three mice per group. (TIF 1843 kb

Additional file 3: of Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation

OPC maturation and recruitment is not impaired in non-lesioned areas during progressive EAE in GFAPγR1Δ mice. Longitudinal spinal cord sections from WT and GFAPγR1Δ mice, treated with isotype control or anti-TNF mAb, were stained for OLG and OPC during acute (d19) and chronic (d30) EAE. A. Quantification of differentiated myelinating OLG (CC1+Olig2+) per square millimeter non-lesioned area. D. Quantification of OPCs (CC1−Olig2+) per square millimeter non-lesioned area. Data represent the mean ± SEM of five to seven separate fields per mouse with two to three mice per group from two independent experiments. P values were determined by Wilcoxon rank sum-test. GFAPγR1Δ in all panels represents GFAPγR1Δ mice treated with isotype control mAb. (TIF 153 kb

Additional file 2: of Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation

TNF neutralization limits OLG and T cell apoptosis during progressive EAE in GFAPγR1Δ mice. Longitudinal spinal cord sections from WT and GFAPγR1Δ mice, treated with isotype control or anti-TNF mAb, were stained for apoptosis during acute (d19) and chronic (d30) EAE. A. Quantification of apoptotic mature OLG (TUNEL+ CC1+ Olig2−) per square millimeter non-lesioned area. C. Quantification of apoptotic T cells (TUNEL+ CD3+) per square millimeter lesion area. Data represent mean ± SEM of two to three separate fields per mouse with two to three mice per group from two independent experiments. P values were determined by Student’s t test. GFAPγR1Δ in all panels represents GFAPγR1Δ mice treated with isotype control mAb. (TIF 133 kb

IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability

<div><p>Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.</p> </div

IFN-γ signaling to astrocytes does not influence brain inflammation.

<p>(<b>A</b>) CD45^hi bone marrow derived inflammatory cells and CD4⁺ T cells in brains (day 16 p.i.) analyzed by flow cytometry following isolation by Percoll gradients. R1 gate = CD45^hi inflammatory cells; R2 gate = CD4⁺ T cells within R1. Representative of 4 separate experiments at days 14 – 16 p.i. (<b>B</b>) IFN-γ and IL-17 secreting MOG-specific CD4⁺ T cells during acute EAE. Brain derived cells enriched by Percoll gradients at day 16 p.i. stimulated with MOG^35–55 peptide for 4 hrs in the presence of GolgiStop. p>0.05 comparing the frequency of MOG-specific CD4⁺ T cells secreting IFN-γ; p<0.05 comparing IL-17 in wt and GFAPγR1Δ mice. CNS cells pooled from 4 – 5 mice per experiment. Data are representative of 3 – 4 separate experiments at days 14 – 16 p.i. (<b>C</b>) Foxp3⁺ regulatory T cells within CD4⁺ T cells enriched from brain at day 16 p.i. CNS cells pooled from 4 – 5 mice per experiment. p<0.05 comparing wt and GFAPγR1Δ mice. Data are representative of 3 separate experiments at days 14 – 16 p.i.</p

Astrocytes regulate IL-1, CCL5 and TNF mRNA during acute EAE.

<p>Relative gene expression in spinal cord from GFAPγR1Δ (average clinical score = 3.9) and wt mice (average clinical score = 3.1) during acute disease (day 18 p.i.) determined by qRT-PCR. Representative of 2 experiments (n = 3 – 4 mice/group) analyzed in triplicate. *p<0.05 comparing wt and GFAPγR1Δ mice.</p

IFN-γ signaling regulates astrocyte activation within grey matter during chronic EAE.

<p>Cross section of spinal cord grey matter from wt (upper panel) and GFAPγR1Δ mice (lower panel) during chronic disease (day 35 p.i.). Grey matter regions shown are distinct from areas of demyelination. Data are representative of 3 separate experiments with 3 – 4 individuals per experiment and 6 cross sections per spinal cord and 2 separate experiments in which the spinal cords were sectioned longitudinally.</p

EAE in GFAPγR1Δ tg mice.

a<p>Peak disease score determined at day 18 post immunization.</p>b<p>Mortality determined at day 50 post immunization.</p>*<p>p≤0.05.</p

Sustained inflammation and increased pro-inflammatory genes during chronic EAE.

<p>(<b>A</b>) CD45^hi inflammatory cells and MHC class II expression on CD45^low microglia from spinal cord during chronic disease (day 42 p.i.). R1 gates depict total inflammatory cells. The R2 gate depicts relative MHC Class II expression. Representative of 4 similar experiments. (<b>B</b>) Relative frequencies of MOG specific IFN-γ and IL-17 CD4⁺ T cells derived from spinal cord during chronic disease (day 42 p.i.). Pools of 5 – 7 mice per experiment. Representative of 2 separate experiments. (<b>C</b>) Relative gene expression in spinal cord mRNA from GFAPγR1Δ (average clinical score = 3.9) and wt mice (average clinical score = 0.9) during chronic disease (day 43 p.i.) determined by qRT-PCR. Representative of 2 experiments (n = 3 – 4 mice/group) analyzed in triplicate. *p<0.05 comparing wt and GFAPγR1Δ mice.</p