Safety of Anti-Reelin Therapeutic Approaches for Chronic Inflammatory Diseases

Abstract

Reelin, a large extracellular glycoprotein, plays critical roles in neuronal development and synaptic plasticity in the central nervous system (CNS). Recent studies have revealed non-neuronal functions of plasma Reelin in inflammation by promoting endothelial-leukocyte adhesion through its canonical pathway in endothelial cells (via ApoER2 acting on NF-κB), as well as in vascular tone regulation and thrombosis. In this study, we have investigated the safety and efficacy of selectively depleting plasma Reelin as a potential therapeutic strategy for chronic inflammatory diseases. We found that Reelin expression remains stable throughout adulthood and that peripheral anti-Reelin antibody treatment with CR-50 efficiently depletes plasma Reelin without affecting its levels or functionality within the CNS. Notably, this approach preserves essential neuronal functions and synaptic plasticity. Furthermore, in mice induced with experimental autoimmune encephalomyelitis (EAE), selective modulation of endothelial responses by anti-Reelin antibodies reduces pathological leukocyte infiltration without completely abolishing diapedesis. Finally, long-term Reelin depletion under metabolic stress induced by a Western diet did not negatively impact the heart, kidney, or liver, suggesting a favorable safety profile. These findings underscore the promising role of peripheral anti-Reelin therapeutic strategies for autoimmune diseases and conditions where endothelial function is compromised, offering a novel approach that may avoid the immunosuppressive side effects associated with conventional anti-inflammatory therapies.

Keywords: ApoER2; NF-κB; Reelin; atherosclerosis; fibrosis; immune system; inflammation; leukocyte; multiple sclerosis; vascular system.

Figure 1

Reelin expression is stable between sex and during aging. (A) Reelin concentration was evaluated by ELISA in healthy patients (sex matched); n = 46 with 21 women and 25 men. (B) Reelin expression was evaluated by Western blot in WT mice at different ages.

Figure 2

Peripheral CR-50 treatment does not affect Reelin expression and function in the CNS. (A–C) Cx3cr1-GFP male mice were injected intraperitoneally with 100 µg of irrelevant IgG (n = 10) or CR-50 (n = 9) twice per week. To challenge the BBB and increase its permeability, EAE was induced by Myelin Oligodendrocyte Glycoprotein immunization using standard procedures [118], one week after the first antibody injection. (A) Reelin protein expression was evaluated in plasma by Western blot (n = 9–10). (B) One total brain hemisphere was lysed and Reelin protein expression was evaluated by immunoblotting (n = 5). Reelin expression was also evaluated by immunoblotting in the olfactory bulb and the hippocampus (n = 3). (C) Reelin protein expression in the spinal cord was evaluated by Western blot in both groups. (A–C) Adapted with permission from Calvier et al. Sci. Trans. Med., 2020 [53]. (D–F) WT mice were injected intraperitoneally with 100 µg of irrelevant IgG (n = 10) or CR-50 (n = 9). (D) LTP induced by theta-burst stimulation (TBS, 200 pulses in total) in the CA1 region of hippocampal slices from CR50 and control mono IgG-treated mice. Baselines were normalized to 1 by dividing each experiment by its 10 min average before the TBS paradigm. LTP was calculated as the average potentiation between 40 and 60 min after TBS. We did not find any significant difference between CR50 (1.396 ± 0.20, n = 11) and IgG-treated slices (1.390 ± 0.11, n = 14). (E) DAP5 is an NMDA receptor inhibitor. Block of NMDA receptors at rest disinhibits AMPA receptor insertion into the synapse and causes a rapid synaptic potentiation which stabilizes within 25–35 min. After a 20 min stable baseline, AP5 was applied to induce rapid synaptic scaling. There were no significant differences between CR50 (1.74 ± 0.21, n = 12) and IgG (1.49 ± 0.15, n = 7)-treated slices. (F) Input output curves are plotted as increasing stimulus intensities, which are normalized to fiber volley amplitudes vs. fEPSP slopes and best fitted with a line which showed no significant differences at various points.

Figure 3

Peripheral CR-50 treatment does not abolish basal diapedesis. Cx3cr1-GFP male mice were injected intraperitoneally with 100 µg of irrelevant IgG (n = 10) or CR-50 (n = 9) twice per week. To challenge the BBB and increase its permeability, EAE was induced by Myelin Oligodendrocyte Glycoprotein immunization using standard procedures [118], one week after the first antibody injection. Naïve mice are littermates with no antibody treatment or EAE induction. (A–C) Adhesion protein expression was evaluated by Western blot or immunohistochemistry in tissues, as indicated. (D) In the Cx3cr1-GFP-positive cell population, the total number of inflammatory cells (Cx3cr1-GFP-positive), monocytes (Cx3cr1-GFP-positive, Iba-1-negative; indicated by the arrows), and microglia (Cx3cr1-GFP and Iba1 double positive) were visualized by immunofluorescence (scale = 50 µm). (A–D) Figure adapted with permission from Calvier et al. Sci. Trans. Med., 2020 [53]. n ≥ 3; * p < 0.05 and ** p < 0.01.

Figure 4

Long-term Reelin depletion has no adverse effect on organ functions. Reelin conditional KO mice and WT littermates, both on LDLR KO background, were fed a Western diet for 4 months (for the 6-month time point) or for 10 months (for the 12-month time point). At the end of each time point, various organs including the heart, kidney, and liver were analyzed to find any adverse effect of prolonged Reelin depletion under the physiological stress imposed by a Western diet. Representative pictures are shown using either H&E or Masson’s trichrome staining.