Antibody-mediated inhibition of TNFR1 attenuates disease in a mouse model of multiple sclerosis

Abstract

Tumour necrosis factor (TNF) is a proinflammatory cytokine that is known to regulate inflammation in a number of autoimmune diseases, including multiple sclerosis (MS). Although targeting of TNF in models of MS has been successful, the pathological role of TNF in MS remains unclear due to clinical trials where the non-selective inhibition of TNF resulted in exacerbated disease. Subsequent experiments have indicated that this may have resulted from the divergent effects of the two TNF receptors, TNFR1 and TNFR2. Here we show that the selective targeting of TNFR1 with an antagonistic antibody ameliorates symptoms of the most common animal model of MS, experimental autoimmune encephalomyelitis (EAE), when given following both a prophylactic and therapeutic treatment regime. Our results demonstrate that antagonistic TNFR1-specific antibodies may represent a therapeutic approach for the treatment of MS in the future.

Figure 1. EAE severity depends upon TNFR expression.

EAE was induced with MOG^35–55 in TNFR1^-/-, TNFR2^-/- and WT mice and the resulting disease was assessed daily using a score from 0 to 5, until 21 days after the onset of neurological symptoms. TNFR1^-/- mice had a much less severe disease course when compared to WT mice, whereas TNFR2^-/- mice showed more severe symptoms than WT mice (A). These observations were reflected in the significantly reduced cumulative EAE score in TNFR1^-/- mice, and the significantly increased cumulative score in TNFR2^-/- mice (B). Furthermore, TNFR1^-/- mice were more resistant to disease induction than both TNFR2^-/- and WT mice (C). * P<0.05, TNFR1^-/- n = 13; TNFR2^-/- n = 8; WT n = 14.

Figure 2. Histopathological analysis confirmed that EAE was less severe in TNFR1^-/- mice in comparison to both WT and TNFR2^-/- mice.

Spinal cords were analysed at day 21 of EAE for signs of inflammatory infiltration, demyelination and axonal degeneration. TNFR1^-/- mice had significantly less infiltration of both CD3-positive T cells (A) and Mac-3-positive activated microglia/macrophages (B) when compared to WT and TNFR2^-/- mice. Furthermore, TNFR1^-/- mice also had significantly less demyelination, as assessed by LFB staining (C) and axonal damage, as assessed by accumulation of β-APP-positive axonal profiles (D), when compared to both WT and TNFR2^-/- mice. Conversely, TNFR2^-/- had significantly increased numbers of β-APP positive damaged axons compared to WT mice.

Figure 3. Anti-TNFR1 inhibited acute TNF toxicity in vitro and in vivo.

L929 cells were incubated with recombinant mouse TNF in the presence or absence of 10 µg/ml anti-TNFR1 (A) or with anti-TNFR1 in the presence of 0.1 ng/ml mouse TNF (B) and cell survival was determined by crystal violet staining. C, D: Female C57BL/6 wild type mice were pretreated with PBS or anti-TNFR1 (10 mg/kg, i.p.). After 2 h, PBS or murine TNF were injected i.v. (1 mg/kg) and body temperature (C) as well as survival of animals (D) were determined.

Figure 4. Administration of anti-mouse TNFR1 on the day of immunization ameliorated EAE.

Anti-mouse TNFR1 was injected intra-peritoneally in C57BL/6 mice, on the day of disease induction, at a dosage of 100 µg (equivalent to 5 mg/kg). Mice were subsequently monitored on a daily basis until 21 days after the onset of clinical symptoms (EAE day 21). Antibody treatment resulted in a reduced EAE severity compared to mice receiving control IgG (A, B). Furthermore, mice injected with anti-TNFR1 also showed a significant delay in the onset of spinal cord symptoms in comparison to mice receiving control IgG (C). (A) Results from one representative experiment out of four shown (control IgG n = 4; anti TNFR1 n = 6), (B, C) results from four combined experiments (control IgG n = 16, anti-TNFR1 n = 19). * P<0.05, **P<0.01.

Figure 5. Anti-TNFR1 treatment on the day of immunization resulted in a significant reduction in demyelination and neuronal loss and a mild reduction in inflammatory infiltration.

Spinal cord histopathology was performed at day 21 of EAE, following treatment with anti-mouse TNFR1 on the day of immunization. Representative images are shown from control IgG treated mice, with an EAE score of 2.0 (B, E, H, and K) and from anti-TNFR1-treated animals, with an EAE score of 1.0 (C, F, I and L). The level of spinal cord demyelination was assessed using sections stained with LFB (A–C). Mice treated prophylactically with anti-TNFR1 had significantly reduced levels of demyelination compared to control-treated mice (A–C). Immunohistochemistry with an anti-CD3 antibody was used to detect T cells and showed a decrease, although not significant, in the number of T cells within the spinal cord of anti-TNFR1 treated mice, in comparison to control animals (D–F). Immunohistochemistry with an antibody to Mac-3 was used to detect activated microglia and macrophages and demonstrated a decrease in the number of positive cells in anti-TNFR1 treated mice, although again this was not significant (G–I). J-L: Immunohistochemistry with an anti-NeuN antibody was used to detect neuronal cell bodies, which were quantified within the spinal cord grey matter. Anti-TNFR1 treated mice had significantly elevated numbers of surviving neuronal cell bodies. Scale bars in B, C, E, F, H, I, K, L: 200 µm.

Figure 6. Anti-TNFR1 treatment reduced EAE severity in a therapeutic setting.

EAE was induced in C57BL/6 mice with MOG^35–55 and mice were assessed for neurological symptoms on a daily basis. 400 µg (equivalent to 20 mg/kg) anti-mouse TNFR1 was injected i.p. either once on the day of EAE onset, or twice; firstly on the day of EAE onset and again on day 4 of the disease. Control mice received two injections of control IgG on days 1 and 4 of EAE. Mice receiving two injections of anti-TNFR1 had significantly reduced neurological symptoms than mice receiving control IgG (A, B). Spinal cords were examined histopathologically at day 14 of EAE by LFB staining (C–E). Control IgG-treated mice (D, image from animal with an EAE score of 2.5) had more demyelination than mice treated with two injections of anti-TNFR1 (E, image from animal with an EAE score of 0.5), although this was not found to be statistically significant. Immunohistochemistry with an anti-CD3 antibody showed no difference in the number of T cells within the spinal cord of control IgG-treated animals (F, G, image from an animal with an EAE score of 2.5) and anti-TNFR1 treated mice (H, image from an animal with an EAE score of 2.0). Immunohistochemistry with an antibody to Mac-3 also demonstrated no difference in the number of activated microglia/macrophages within the spinal cord of control IgG-treated animals (I, J, image from an animal with an EAE score of 2.5) and anti-TNFR1 treated mice (K, image from an animal with an EAE score of 2.0). L–N: Immunohistochemistry with an antibody against NeuN showed a statistically non-significant trend towards neuroprotection in the anti-TNFR1-treated mice (M, image from a control IgG-treated animal with an EAE score of 1.5), (N, image from an anti-TNFR1-treated animal with an EAE score of 1.5). Scale bars in D, E, G, H, J, K, M, N: 200 µm. * P<0.05. Control IgG n = 9; anti-TNFR1: 1 injection n = 4; anti-TNFR1: 2 injections n = 8.