Nogo-receptor 1 deficiency has no influence on immune cell repertoire or function during experimental autoimmune encephalomyelitis

Abstract

The potential role of Nogo-66 Receptor 1 (NgR1) on immune cell phenotypes and their activation during neuroinflammatory diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is unclear. To further understand the function of this receptor on haematopoietically-derived cells, phenotypic and functional analyses were performed using NgR1-deficient (ngr1-/-) animals. Flow cytometry-based phenotypic analyses performed on blood, spleen, thymus, lymph nodes, bone marrow and central nervous-system (CNS)-infiltrating blood cells revealed no immunological defects in naïve ngr1-/- animals versus wild-type littermate (WTLM) controls. EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or by MOG35-55 peptide, a B cell-independent model. We have demonstrated that in ngr1-/- mice injected with MOG35-55, a significant reduction in the severity of EAE correlated with reduced axonal damage present in the spinal cord when compared to their WTLM controls. However, despite a reduction in axonal damage observed in the CNS of ngr1-/- mice at the chronic stage of disease, no clinical differences could be attributed to a specific genotype when rMOG was used as the encephalitogen. Following MOG35-55-induction of EAE, we could not derive any major changes to the immune cell populations analyzed between ngr1-/- and WTLM mice. Collectively, these data demonstrate that NgR1 has little if any effects on the repertoire of immune cells, their activation and trafficking to the CNS.

Figure 1. Immune-phenotype and bone marrow (BM) progenitor status of naïve ngr1-/- and WT mice.

(A) 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<0.05 Mann-Whitney test. (B) Quantification of BM-derived colony number in naïve ngr1-/- and WT mice. After 8 days on methylcellulose culture, BM-derived progenitors from ngr1-/- 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).

Figure 2. Reduced severity of MOG_{35 –55} peptide-EAE in ngr1-/- mice.

(A) EAE was induced by immunization with MOG_35–55 peptide and animals were scored daily for disease clinical manifestations. ngr1-/- mice presented a less severe clinical disease than WT mice. Data were pooled from 2 independent experiments (n = 11-13; mean ± SEM). ***p<0.05, two-way ANOVA. (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 ngr1-/- spinal cords (magnification 20X, scale bar = 200 µm). (C-D) Flow cytometric analysis of spleen (C) and central nervous system (CNS) (D) mononuclear cells at 18 and 45 dpi. The proportion and number of immune cell populations analyzed did not differ significantly between ngr-/- and WT mice. Data represent mean ± SEM (n = 3-5).

Figure 3. Susceptibility of ngr1-/- mice to develop EAE provoked by rMOG.

(A) EAE was induced by immunization with the extracellular domain of mouse rMOG and animals were scored daily for disease clinical manifestations. There were no discernible variation in the severity of disease between ngr1-/- and WT mice. Data were pooled from 3 independent experiments (n = 19-22; mean ± SEM). (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). In contrast to WT controls, reduced axonal damage in ngr1-/- spinal cords could be observed at the chronic stage (45dpi) of EAE (magnification 20X, scale bar = 200 µm). (C) Amyloid precursor protein (APP)-positive, NF-200-positive axons (arrow) near DAPI-positive inflammatory infiltrates in optic nerves from rMOG-induced WT mice at 18 dpi (left, score 3). Reduced APP-immunopositive axons in optic nerves from rMOG-induced ngr1-/- mice (right, score 2.5). Scale bar  =  20 µm.

Figure 4. Immune-phenotype of ngr1-/- mice during EAE induced with rMOG.

ngr1-/- 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; A); spleen, (B); lymph nodes, (C); thymus (D) and central nervous system (CNS; E) are shown. No significant differences were found between ngr1-/- and WT for all organs and time points examined. Data represent mean ± SEM (n = 6-11). (F) Further analysis of microglia (CD45^loCD11b⁺) and macrophages (CD45^hiCD11b⁺) in the CNS of ngr1-/- and WT mice was performed. ngr1-/- animals presented an increased proportion of microglial cells at 18 dpi and a decreased number of macrophages at 45 dpi (n = 3-4; *p< 0.05 Mann-Whitney test).

Figure 5. Peripheral immune response of ngr1-/- mice to rMOG.

(A) In vitro proliferative response of ngr1-/- and WT splenocytes stimulated with rMOG or anti-CD3/CD28 at 18 and 45 days post immunization (dpi). ngr1-/- splenocytes showed an equivalent proliferative response to that of WT splenocytes. (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. ngr1 deficiency had no impact on splenocyte cytokine production at either time points analyzed. Data represent mean ± SEM (n = 3-5). (C) Serum rMOG-specific IgG, IgG1, IgG2b and IgM antibody response as determined by ELISA. Data represent mean ± SEM (n = 2-6).