Anti-inflammatory mechanisms and pharmacological actions of phycocyanobilin in a mouse model of experimental autoimmune encephalomyelitis: A therapeutic promise for multiple sclerosis

Abstract

Cytokines, demyelination and neuroaxonal degeneration in the central nervous system are pivotal elements implicated in the pathogenesis of multiple sclerosis (MS) and its nonclinical model of experimental autoimmune encephalomyelitis (EAE). Phycocyanobilin (PCB), a chromophore of the biliprotein C-Phycocyanin (C-PC) from Spirulina platensis, has antioxidant, immunoregulatory and anti-inflammatory effects in this disease, and it could complement the effect of other Disease Modifying Treatments (DMT), such as Interferon-β (IFN-β). Here, our main goal was to evaluate the potential PCB benefits and its mechanisms of action to counteract the chronic EAE in mice. MOG_35-55-induced EAE was implemented in C57BL/6 female mice. Clinical signs, pro-inflammatory cytokines levels by ELISA, qPCR in the brain and immunohistochemistry using precursor/mature oligodendrocytes cells antibodies in the spinal cord, were assessed. PCB enhanced the neurological condition, and waned the brain concentrations of IL-17A and IL-6, pro-inflammatory cytokines, in a dose-dependent manner. A down- or up-regulating activity of PCB at 1 mg/kg was identified in the brain on three (LINGO1, NOTCH1, and TNF-α), and five genes (MAL, CXCL12, MOG, OLIG1, and NKX2-2), respectively. Interestingly, a reduction of demyelination, active microglia/macrophages density, and axonal damage was detected along with an increase in oligodendrocyte precursor cells and mature oligodendrocytes, when assessed the spinal cords of EAE mice that took up PCB. The studies in vitro in rodent encephalitogenic T cells and in vivo in the EAE mouse model with the PCB/IFN-β combination, showed an enhanced positive effect of this combined therapy. Overall, these results demonstrate the anti-inflammatory activity and the protective properties of PCB on the myelin and support its use with IFN-β as an improved DMT combination for MS.

Keywords: antioxidants; experimental autoimmune encephalomyelitis; interferon-β; multiple sclerosis; phycocyanobilin; proinflammatory cytokines; remyelination.

Figure 1

Effect of PCB in a murine model of chronic progressive EAE. (A) Daily clinical assessment of the mice. (B) Clinical severity determined by the area under the curve (in arbitrary units). MOG_35–55 immunized mice received a daily i.p. injection of PBS (vehicle) or PCB at 0.1, 0.5 or 1 mg/kg, from day 0 until day 26 post-immunization. A numeric scale with the ascending graveness was used to score every day the clinical signs of the mice, ranging from 0 (no disease) to 5 (death). The naïve group (non-immunized animals) remained healthful during the entire study period (score 0). Data are expressed as the mean ± S.E.M (n=6-7 per group). The asterisk (*) is indicative of significant differences with vehicle-treated EAE mice (*p<0.05, ANOVA + Newman-Keuls tests).

Figure 2

Evaluation of the PCB effect on the brain expression of cytokines in EAE mice. MOG_35–55 immunized mice received a daily i.p. injection of PBS (vehicle) or PCB at 0.1, 0.5 or 1 mg/kg, from day 0 until day 26 post-immunization. At this moment, the protein levels of the cytokines (A) IL-17A, (B) IL-6, and (C) IL-10 were assessed by the respective ELISA commercial kits. Data are expressed as mean ± S.E.M. (n=6-7 per group). The ampersand (&) and asterisk (*) are indicative of significant differences with non-immunized naïve and vehicle-treated EAE mice, respectively (*p<0.05, **p<0.01, ***p<0.001; ^&&p <0.01, ^&&&p <0.001, Kruskal-Wallis + Dunn’s tests).

Figure 3

Effect of PCB on expression levels of demyelinating/remyelinating-related genes assessed by Real Time PCR in the brain of EAE mice. The panels show the mRNA levels of CXCL12, LINGO1, MAL, MOG, NKX2-2, NOTCH1, OLIG1 and TNF-α. MOG_35–55 immunized mice received a daily i.p. injection of PBS (vehicle) or PCB at 0.1, 0.5 or 1 mg/kg, from day 0 until day 26 post-immunization. Data are expressed as mean ± S.E.M. (n=4 per group). The ampersand (&) and asterisk (*) are indicative of significant differences with non-immunized naïve and vehicle-treated EAE mice, respectively (*p<0.05, ^&p<0.05, REST^© software).

Figure 4

Histological and immunohistochemical assessment of the PCB effects on myelination and inflammation markers in spinal cords of EAE mice. The representative cross-sectional images and the respective morphometric evaluation per variable are shown in each panel. (A) Demyelination (LFB/PAS). (B) Macrophages/activated microglia (Mac-3). (C) T cells (CD3). MOG_35–55 immunized mice received a daily i.p. injection of PBS (vehicle) or PCB at 0.1, 0.5 or 1 mg/kg, from day 0 until day 26 post-immunization. The areas marked with black pencil indicate demyelination. The percentage of demyelination and the cell densities (number of positive marked cells per mm²) were calculated by dividing with total area of the white matter in transversal sections. Data are expressed as mean ± SEM (n=4-7 per group). The asterisks indicate statistically significant differences vs. EAE + vehicle group (**p<0.01, ***p<0.001, Kruskal-Wallis + Dunn’s tests for LFB/PAS and Mac-3, ANOVA + Newman-Keuls tests for CD3). Bar: 50 μm.

Figure 5

Immunohistochemical assessment of the PCB effects on oligodendrocyte and neuronal markers in spinal cords of EAE mice. The representative cross-sectional images and the respective morphometric evaluation per variable are shown in each panel. (A) Oligodendrocytes precursor cells (Olig2). (B) Mature oligodendrocytes (TPPP/p25). (C) Acute axonal damage (APP). MOG_35–55 immunized mice received a daily i.p. injection of PBS (vehicle) or PCB at 0.1, 0.5 or 1 mg/kg, from day 0 until day 26 post-immunization. The cell densities (number of positive marked cells per mm²) were calculated by dividing with total area of the white matter in transversal sections. Data are expressed as mean ± SEM (n=4-7 per group). The ampersand (&) and asterisk (*) are indicative of significant differences with non-immunized naïve and vehicle-treated EAE mice, respectively (*p<0.05, **p<0.01, ***p<0.001; ^&p<0.05, ^&&&p<0.001, ANOVA + Newman-Keuls tests). Bar: 50 μm.

Figure 6

Effect of the treatment with the combination PCB/IFN-β and each compound individually on the clinical progression of EAE mice. (A) Daily clinical assessment of the mice. (B) Clinical severity determined by the area under the curve (in arbitrary units). MOG_35–55 immunized mice were treated either with oral PCB at 1 mg/kg daily (once a day), with subcutaneous IFN-β at 5000 U every other day, or with their combination, initiating at day 0 (prophylactic schedule), or after the occurrence of the first disease symptoms at day 14 post-induction (late therapeutic schedule), lasting until day 24 post-immunization. A numeric scale with the ascending graveness was used to score every day the clinical signs of the mice, ranging from 0 (no disease) to 5 (death). The naïve group (non-immunized animals) remained healthful during the entire study period (score 0). Data are expressed as the mean ± S.E.M. (n=9-10 per group). The ampersand (&) and asterisk (*) are indicative of significant differences with IFN-β-treated or vehicle-treated EAE mice, respectively (**p<0.01, ***p<0.001, ^&p<0.05, ^&&p<0.01, ANOVA + Newman-Keuls tests).

Figure 7

Effect of the treatment with the combination PCB/IFN-β and each compound individually on the cerebral expression of cytokines (protein levels) and of demyelinating/remyelinating-related genes (mRNA levels), as well as on the Treg levels in spleen of EAE mice. Panels show (A) the cytokines levels IL-17A, IL-6 and IL-10 from brain as assessed by ELISA, (B) Treg cells from mice spleen analyzed in the CD4⁺ CD25^high gate and expressed as Foxp3 Mean Fluorescence Intensity (MFI), (C) mRNA levels of LINGO1 and MAL genes as assessed by quantitative Real Time PCR from the mice brain. MOG_35–55 immunized mice were treated either with oral PCB at 1 mg/kg daily (once a day), with subcutaneous IFN-β at 5000 U every other day, or with their combination, initiating at day 0 (prophylactic schedule), or after the occurrence of the first disease symptoms at day 14 post-induction (late therapeutic schedule), lasting until day 24 post-immunization. Data are expressed as mean ± S.E.M. (n=4-7 per group). The ampersand (&) and asterisk (*) are indicative of significant differences in comparison to non-immunized naïve and to vehicle-treated EAE mice, respectively. (*p<0.05, **p<0.01, ^&p<0.05, ^&&p<0.01, ^&&&p <0.001, Kruskal-Wallis + Dunn’s tests when compared with EAE + vehicle group for IL-17A and IL-16, or according to ANOVA + Newman-Keuls tests for IL-10 and MFI of Fopx3).