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

Sustained demyelination, axonal loss and astrocyte activation during chronic disease.

<p>Cross section of spinal cord from wt (upper panels) and GFAPγR1Δ mice (lower panels) during chronic disease (day 35 p.i.). Inflammation (HE), demyelination (LFB), axonal damage and loss following visualization with mAb SMI31 and SMI32 (SMI), GFAP expression and hypertrophy of astrocytes (GFAP) in wt and GFAPγR1Δ mice. Data are representative of 3 separate experiments with 3 – 4 individuals per experiment and 6 cross sections per spinal cord.</p

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 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

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

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

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

Astrocyte regulation of spinal cord demyelination and axonal loss during acute EAE.

<p>Cross section of spinal cord from wt (upper panels) and GFAPγR1Δ mice (lower panels) during acute disease (day 16 p.i.). Inflammation (HE), demyelination (LFB), axonal damage and loss (SMI), GFAP expression and hypertrophy of astrocytes in wt and GFAPγR1Δ mice. Data are representative of 3 separate experiments with 3 – 4 individuals per experiment and 6 cross sections per cord.</p

IFN-γ dependent secretion of IL-27 by astrocytes.

<p>IL-27 secretion by astrocytes derived from wt and GFAPγR1Δ tg mice. Cultures were treated with 100 ng/ml rIFN-γ for 48 hr. IL-27 concentrations determined by ELISA. Representative of 3 experiments. *p<0.05 comparing wt and GFAPγR1Δ mice.</p