Quantitative Proteome Profiling of CNS-Infiltrating Autoreactive CD4⁺ Cells Reveals Selective Changes during Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4⁺ lymphocytes into the CNS, where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labeling approach to investigate the proteome of CD4⁺ cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models: PLP_139–151-induced relapsing-remitting EAE and MOG_35–55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease, and remission phases of disease, and of these 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair, and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (annexin A1), which represent key events during EAE progression, were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4⁺ cell-driven processes that occur during the initiation of relapse and remission stages of disease

Clinical and histological outcome of MOG_35–55 EAE in <i>ngr1^−/−.</i>

<p>Data represent mean ± SEM. *p&lt;0.05, **p&lt;0.01. Wild Type (WT) and Nogo Receptor 1 deficient (<i>ngr1^−/−)</i> values are shown for the peak (18 days post-immunization, dpi) and the chronic (45dpi) stage of MOG peptide (MOG_35–55 )-EAE.</p

Clinical and histological outcome of rMOG EAE in <i>ngr1^−/−.</i>

<p>Data represent mean ± SEM. *p≤0.05. Wild Type (WT) and Nogo Receptor 1 deficient (<i>ngr1^−/−</i>) values are shown for the peak (18 days post-immunization, dpi) and the chronic (45dpi) stage of recombinant MOG-EAE.</p

Reduced severity of MOG₃₅_–55 peptide-EAE in <i>ngr1-/-</i> mice.

<p>(<b>A</b>) EAE was induced by immunization with MOG_35–55 peptide and animals were scored daily for disease clinical manifestations. <i>ngr1-/-</i> mice presented a less severe clinical disease than WT mice. Data were pooled from 2 independent experiments (n = 11-13; mean ± SEM). ***p&lt;0.05, two-way ANOVA. (<b>B</b>) Representative lumbar-thoracic spinal cord sections stained with hematoxylin-eosin for inflammation, luxol fast blue for demyelination and Bielschowsky silver impregnation for axonal damage. Histological examination was performed at 18 and 45 days post-immunization (dpi). Compared to WT controls, a trend towards reduced inflammation, demyelination and axonal damage could be observed in <i>ngr1-/-</i> spinal cords (magnification 20X, scale bar = 200 µm). (<b>C-D</b>) Flow cytometric analysis of spleen <b>(C)</b> and central nervous system (CNS) (<b>D</b>) mononuclear cells at 18 and 45 dpi. The proportion and number of immune cell populations analyzed did not differ significantly between <i>ngr-/-</i> and WT mice. Data represent mean ± SEM (n = 3-5).</p

Immune-phenotype and bone marrow (BM) progenitor status of naïve <i>ngr1-/-</i> and WT mice.

<p>(<b>A</b>) Comparative flow cytometric analysis of single cell suspension from spleen, BM, thymus, blood, lymph nodes and central nervous system (CNS) associated mononuclear cells. Proportion and total number of CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells, B220⁺ B cells, Gr-1⁺ granulocytes and F4/80 (Gr-1^loF4/80⁺) monocyte/macrophage are shown. Data represent mean ± SEM (n = 8-11). *p&lt;0.05 Mann-Whitney test. (<b>B</b>) Quantification of BM-derived colony number in naïve <i>ngr1-/-</i> and WT mice<b>.</b> After 8 days on methylcellulose culture, BM-derived progenitors from <i>ngr1-/-</i> animals were capable of producing granulocytes (G); macrophages (M) and mixed (GM) colonies at comparable numbers to WT mice. Bars represent mean colony number/plate ± SEM (n = 3).</p

Immune-phenotype of <i>ngr1-/-</i> mice during EAE induced with rMOG.

<p><i>ngr1-/-</i> and WT animals were immunized with rMOG and the mononuclear cells isolated at 18 (peak) and 45 (chronic) days post-immunization (dpi) were analyzed by flow cytometry. Proportion and total number of lymphocytes, granulocytes and monocyte/macrophages of bone marrow (BM; <b>A</b>); spleen, (<b>B</b>); lymph nodes, (<b>C</b>); thymus (<b>D</b>) and central nervous system (CNS; <b>E</b>) are shown. No significant differences were found between <i>ngr1-/-</i> and WT for all organs and time points examined. Data represent mean ± SEM (n = 6-11). (<b>F</b>) Further analysis of microglia (CD45^loCD11b⁺) and macrophages (CD45^hiCD11b⁺) in the CNS of <i>ngr1-/-</i> and WT mice was performed. <i>ngr1-/-</i> animals presented an increased proportion of microglial cells at 18 dpi and a decreased number of macrophages at 45 dpi (n = 3-4; *p&lt; 0.05 Mann-Whitney test).</p

Suppression of <i>in vitro</i> MOG-specific T-cell responses.

<p>(A) Fixed numbers of splenocytes from 2D2 mice stimulated with MOG_35–55 were cultured in the presence of varying numers of GS-N cells, 46C-NS cells or SVZ-NSCs (expressed as NSC: splenocyte ratio). Proliferative responses were measured by ³H-thymidine incorporation and expressed as the mean counts per minute (CPM). (B–G) Supernatants were collected from co-cultures after 48 hrs and the level of pro-inflammatory cytokines was quantified by cytometric bead array. Data represent the mean ± SEM (n = 4 mice). *P&lt;0.05, **P&lt;0.01, ∧P&lt;0.005, ^#P&lt;0.001 vs MOG_35–55 stimulated splenocytes unless otherwise indicated.</p

Peripheral immune response of <i>ngr1-/-</i> mice to rMOG.

<p>(<b>A</b>) <i>In vitro</i> proliferative response of <i>ngr1-/-</i> and WT splenocytes stimulated with rMOG or anti-CD3/CD28 at 18 and 45 days post immunization (dpi). <i>ngr1-/-</i> splenocytes showed an equivalent proliferative response to that of WT splenocytes. (<b>B</b>) Quantification of pro-inflammatory (INF-γ, TNFα, Interleukin (IL)-2, IL-17A and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines in supernatants derived from rMOG and anti-CD3/CD28 stimulated splenocytes cultures. <i>ngr1</i> deficiency had no impact on splenocyte cytokine production at either time points analyzed. Data represent mean ± SEM (n = 3-5). (<b>C</b>) Serum rMOG-specific IgG, IgG1, IgG2b and IgM antibody response as determined by ELISA. Data represent mean ± SEM (n = 2-6).</p

Differentiation potential of GS-N cells.

<p>(A–C) Immunofluorescence staining showed GS-N cells expressed nestin, red (A), A2B5, red (B) and βIII-Tubulin, red and MAP2, green (C). (D–E) Following neuronal or astrocytic differentiation, GS-N cells expressed MAP2, green and NeuN, red (D) or GFAP (E), respectively. (F–K) Electrophysiological assessment of GS-N cell-derived neurons. Neurons in high density (F) fired spontaneous action potentials (APs), which were blocked by tetrodotoxin (TTX) (G). The depolarization giving rise to the APs (H) was underpinned by an inward current (I). Sparsely growing neurons (J) did not display spontaneous activity but APs could be evoked by depolarising current steps (K).</p

Clinical and pathological outcome of EAE mice.

<p>Data are expressed as mean ± SEM.</p>*<p>p&lt;0.05,</p>**<p>p&lt;0.01 vs PBS.</p