Disclosing the Novel Protective Mechanisms of Ocrelizumab in Multiple Sclerosis: The Role of PKC Beta and Its Down-Stream Targets

Abstract

Ocrelizumab (OCR) is a humanized anti-CD20 monoclonal antibody approved for both Relapsing and Primary Progressive forms of Multiple Sclerosis (MS) treatment. OCR is postulated to act via rapid B cell depletion; however, by analogy with other anti-CD20 agents, additional effects can be envisaged, such as on Protein Kinase C (PKC). Hence, this work aims to explore novel potential mechanisms of action of OCR in peripheral blood mononuclear cells from MS patients before and after 12 months of OCR treatment. We first assessed, up-stream, PKCβII and subsequently explored two down-stream pathways: hypoxia-inducible factor 1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF), and human antigen R (HuR)/manganese-dependent superoxide dismutase (MnSOD) and heat shock proteins 70 (HSP70). At baseline, higher levels of PKCβII, HIF-1α, and VEGF were found in MS patients compared to healthy controls (HC); interestingly, the overexpression of this inflammatory cascade was counteracted by OCR treatment. Conversely, at baseline, the content of HuR, MnSOD, and HSP70 was significantly lower in MS patients compared to HC, while OCR administration induced the up-regulation of these neuroprotective pathways. These results enable us to disclose the dual positive action of OCR: anti-inflammatory and neuroprotective. Therefore, in addition to B cell depletion, the effect of OCR on these molecular cascades can contribute to counteracting disease progression.

Keywords: HIF-1α; HSP70; HuR; MnSOD; VEGF; multiple sclerosis; ocrelizumab; protein kinase C.

Figure 1

PKCβII, HIF-1α, and VEGF basal protein expression in PBMCs from HC and MS patients. Upper side: cropped representative western blotting images; lower side: boxplots of PKCβII (A), HIF-1α (B), and VEGF (C) protein expression in PBMCs from HC (n = 17) and MS patients (T0, n = 17), and from MS patients in relation to baseline EDSS scores (D). The results are expressed as the mean grey level ratios × 10³ (mean ± S.E.M.) of the PKCβII (A), HIF-1α (B), and VEGF (C,D) immunoreactivities measured by western blotting and normalized on ponceau signals. Data were analyzed by the Mann–Whitney test; * p < 0.05. HC = healthy controls; T0 = MS patients before OCR (ocrelizumab) therapy; EDSS: Expanded Disability Status Scale.

Figure 2

Effect of ocrelizumab on the PKCβII/HIF-1α/VEGF cascade. Upper side: cropped representative western blotting images; lower side: boxplots of PKCβII (A), HIF-1α (B), and VEGF (C) protein expression in PBMCs at baseline (T0) and after 12 months of OCR treatment (T12, n = 17). The results are expressed as the mean grey level ratios × 10³ (mean ± S.E.M.) of the PKCβII (A), HIF-1α (B), and VEGF (C) immunoreactivities measured by western blotting and normalized on ponceau signals. Data were analyzed by the Wilcoxon signed-rank test; * p < 0.05, *** p < 0.001. T0 = MS patients before OCR (ocrelizumab) therapy, T12 = MS patients after 12 months of OCR treatment.

Figure 3

Effect of ocrelizumab on the HuR, MnSOD, and HSP70 neuroprotective cascades in MS patients. Upper side: cropped representative western blotting images; lower side: boxplots of HuR (A,D), MnSOD (B,E), and HSP70 (C,F) protein expression in PBMCs from HC (n = 17) and MS patients (T0: n = 17 and T12: n = 17). The results are expressed as the mean grey level ratios × 10³ (mean ± S.E.M.) of the HuR, MnSOD, and HSP70 immunoreactivities measured by western blotting and normalized on ponceau signals. Data were analyzed by the Mann–Whitney test for independent samples (HC-T0) and the Wilcoxon signed-rank test for paired samples (T0 and T12); * p < 0.05, ** p < 0.01, *** p < 0.001. HC = healthy controls; T0 = MS patients before OCR (ocrelizumab) therapy; T12 = MS patients after 12 months of OCR treatment.

Figure 4

Comparison of protein expression between healthy controls and MS patients after 12 months of ocrelizumab treatment. Boxplots of PKCβII (A), HIF-1α (B), VEGF (C), HuR (D), MnSOD (E), and HSP70 (F) protein expression in PBMCs from HC (n = 17) and MS patients (T12, n = 17). The results are expressed as the mean grey level ratios × 10³ (mean ± S.E.M.) of the PKCβII, HIF-1α, VEGF, HuR, MnSOD, and HSP70 immunoreactivities measured by western blotting and normalized on ponceau signals. Data were analyzed by the Mann–Whitney test for independent samples (HC-T12); ns = not significant. HC = healthy controls; T12 = MS patients after 12 months of ocrelizumab (OCR) treatment.

Figure 5

Synthetic representation of the two proposed novel mechanisms of action of ocrelizumab: anti-inflammatory and neuroprotective. See text for details.