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. 2017 Oct 16:8:1335.
doi: 10.3389/fimmu.2017.01335. eCollection 2017.

Divergent Immunomodulation Capacity of Individual Myelin Peptides-Components of Liposomal Therapeutic against Multiple Sclerosis

Vilena V Ivanova  1 , Svetlana F Khaiboullina  1   2 , Marina O Gomzikova  1 , Ekaterina V Martynova  1 , André M Ferreira  1 , Ekaterina E Garanina  1 , Damir I Sakhapov  1 , Yakov A Lomakin  1   3 , Timur I Khaibullin  4 , Evgenii V Granatov  4 , Farit A Khabirov  4 , Albert A Rizvanov  1 , Alexander Gabibov  1   3   5 , Alexey Belogurov Jr  1   3   5
Affiliations

Divergent Immunomodulation Capacity of Individual Myelin Peptides-Components of Liposomal Therapeutic against Multiple Sclerosis

Vilena V Ivanova et al. Front Immunol. .

Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination and consequent neuron injury. Although the pathogenesis of MS is largely unknown, a breach in immune self-tolerance to myelin followed by development of autoreactive encephalitogenic T cells is suggested to play the central role. The myelin basic protein (MBP) is believed to be one of the main targets for autoreactive lymphocytes. Recently, immunodominant MBP peptides encapsulated into the mannosylated liposomes, referred as Xemys, were shown to suppress development of experimental autoimmune encephalomyelitis, a rodent model of MS, and furthermore passed the initial stage of clinical trials. Here, we investigated the role of individual polypeptide components [MBP peptides 46-62 (GH17), 124-139 (GK16), and 147-170 (QR24)] of this liposomal peptide therapeutic in cytokine release and activation of immune cells from MS patients and healthy donors. The overall effects were assessed using peripheral blood mononuclear cells (PBMCs), whereas alterations in antigen-presenting capacities were studied utilizing plasmacytoid dendritic cells (pDCs). Among three MBP-immunodominant peptides, QR24 and GK16 activated leukocytes, while GH17 was characterized by an immunosuppressive effect. Peptides QR24 and GK16 upregulated CD4 over CD8 T cells and induced proliferation of CD25+ cells, whereas GH17 decreased the CD4/CD8 T cell ratio and had limited effects on CD25+ T cells. Accordingly, components of liposomal peptide therapeutic differed in upregulation of cytokines upon addition to PBMCs and pDCs. Peptide QR24 was evidently more effective in upregulation of pro-inflammatory cytokines, whereas GH17 significantly increased production of IL-10 through treated cells. Altogether, these data suggest a complexity of action of the liposomal peptide therapeutic that does not seem to involve simple helper T cells (Th)-shift but rather the rebalancing of the immune system.

Keywords: T helper cells; T regulatory cells; cytokines/chemokines; dendritic cells; liposomal peptide therapeutic; multiple sclerosis; myelin basic protein; treatment.

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Figures

Figure 1
Figure 1
Expression of the cell surface markers by peripheral blood mononuclear cells (PBMCs) exposed to the individual myelin basic protein (MBP) peptides comprising the liposomal peptide therapeutic. The PBMCs from the healthy donors (HDs) and multiple sclerosis (MS) patients were incubated with 10 µg mL−1 of MBP peptides GH17 (blue bars), GK16 (green bars), QR24 (red bars), or PBS (black bars) for 24 h. Percentages of human leukocyte antigen (HLA-DR)+ (A), CD80+ (B), CD123+ (C), and CD25+ (E) cells were further determined by flow cytometry. Data are presented as a percentage of positive cells within the CD45+ PBMC population. The percentage of CD25+ cells was estimated in the CD4+ population. CD4/CD8 ratio (D) was estimated as an absolute count of CD4 cells divided by the absolute count of CD8 cells in each sample. Asterisk denotes statistically significant difference with a control. NS, non-significant.
Figure 2
Figure 2
Cytokine production by peripheral blood mononuclear cells (PBMCs) treated by individual myelin basic protein (MBP) peptides comprising the liposomal peptide therapeutic. The PBMCs from the healthy donors (HDs, left panels) and multiple sclerosis (MS) patients (right panels) were incubated with 10 µg mL−1 of MBP peptides GH17 (blue curves), GK16 (green curves), QR24 (red curves), or PBS (black curves). Levels of cytokines [from top to bottom, interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF), interleukin (IL)-6, IL-8, and IL-10] in the supernatants of the PBMCs were determined at indicated time points (0.5, 1, 3, 6, 12, and 24 h).
Figure 3
Figure 3
Level of mRNA-encoding tumor necrosis factor (TNF) in peripheral blood mononuclear cells (PBMCs) in response to incubation with individual myelin basic protein (MBP) peptides comprising the liposomal peptide therapeutic. The PBMCs from the healthy donors (HD) and multiple sclerosis (MS) patients were incubated with 10 µg mL−1 of MBP peptides GH17 (blue bars), GK16 (green bars), QR24 (red bars), or PBS (black bars) for 12 h. Further levels of mRNA-encoding TNF were measured by qPCR. All measurements were performed in triplicates, and values were normalized to corresponding values of 18S ribosomal RNA.
Figure 4
Figure 4
Expression of the cell surface markers by plasmacytoid dendritic cells (pDCs) exposed to the individual myelin basic protein (MBP) peptides comprising the liposomal peptide therapeutic. The pDCs from the healthy donors (HD) and multiple sclerosis (MS) patients were incubated with 5 µg mL−1 of MBP peptides GH17 (blue bars), GK16 (green bars), QR24 (red bars), or PBS (black bars) for 24 h. Percentages of human leukocyte antigen (HLA-DR)+ (A), CD80+ (B), and CD123+ (C) cells were further determined by flow cytometry. Data are presented as a percentage of positive cells within the CD45+ PBMC population. Asterisk denotes a statistically significant difference with the control. NS, non-significant.
Figure 5
Figure 5
Cytokine production by plasmacytoid dendritic cells (pDCs) treated by individual myelin basic protein (MBP) peptides comprising the liposomal peptide therapeutic. The pDCs from the healthy donors (HDs, left panels) and multiple sclerosis (MS) patients (right panels) were incubated with 5 µg mL−1 of MBP peptides GH17 (blue curves), GK16 (green curves), QR24 (red curves), or PBS (black curves). Levels of cytokines [from top to bottom, IFN-α, IFN-γ, tumor necrosis factor (TNF), IL-6, IL-8, and IL-10] in the supernatants from the peripheral blood mononuclear cells were determined at indicated time points (0.5, 1, 3, 6, 12, and 24 h).
Figure 6
Figure 6
Fine tuning of the immune system by individual peptide components of the liposomal peptide therapeutic. Liposome-encapsulated myelin peptides GH17, GK16, and QR24 are proposed to be mainly trapped by dendritic cells through CD206 receptors due to the exposed mannose residues on the surface of liposomes. It seems that GH17 has a limited effect on leukocyte activation, rebalances T cells to being CD8+, and induces production of anti-inflammatory cytokine IL-10 that may significantly contribute to the amelioration of the first disease attack. By contrast, QR24 and GK16, which are effective mainly in preventing a second wave of exacerbation, induce the release of pro-inflammatory cytokines, shifting the CD4/CD8 ratio to CD4 T cells and promoting proliferation of CD4+CD25+ lymphocytes, which are important for maintaining further immune tolerance.
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