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. 2019 Jun 19:10:1251.
doi: 10.3389/fimmu.2019.01251. eCollection 2019.

MAP7 and MUCL1 Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients

Juan Navarro-Barriuso  1   2 , María José Mansilla  1   2 , Bibiana Quirant-Sánchez  1   2 , Alicia Ardiaca-Martínez  1   2 , Aina Teniente-Serra  1   2 , Silvia Presas-Rodríguez  3   4 , Anja Ten Brinke  5 , Cristina Ramo-Tello  3 , Eva M Martínez-Cáceres  1   2
Affiliations

MAP7 and MUCL1 Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients

Juan Navarro-Barriuso et al. Front Immunol. .

Abstract

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both in vitro and in vivo. In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated CYP24A1, MAP7 and MUCL1 genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that MAP7 and MUCL1 genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that MAP7 and MUCL1 constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS.

Keywords: biomarkers; immune tolerance; multiple sclerosis; tolerogenic dendritic cells; vitamin D3.

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Figures

Figure 1
Figure 1
Expression of the selected genes as candidate biomarkers of vitD3-tolDC. (A) Expression of CA2, GZMB and SNORD30 in healthy donors by microarray (n = 5) and quantitative PCR (qPCR; n = 20, except CA2, in which n = 24) in immature dendritic cells (iDC), mature DC (mDC) and vitD3-tolDC. (B) Expression of CAMP, CLEC5A, CYP24A1, MAP7 and MUCL1 in healthy donors both by microarray (n = 5) and qPCR analysis (n = 24, except CYP24A1, in which n = 10) in iDC, mDC and vitD3-tolDC, and in multiple sclerosis (MS) patients by qPCR only (n = 10). (C) Expression of MAP7 and MUCL1 in vitD3-tolDC (n = 24) and in IL10-tolDC (n = 5) by qPCR. Data presented as the mean difference of expression (MeanDiff) or the decimal logarithm of fold change (logFC) expression for the microarray and qPCR results, respectively, in both cases normalized to mDC expression. Housekeeping genes GAPDH, TBP and CYPA were used as controls. One qPCR experiment was performed for each donor or patient, with triplicated measurements for each sample. Error bars corresponding to SEM. Dotted lines represent the logFC = 0.5 or −0.5 expression threshold. *p < 0.05. Friedman test with Dunn's correction, one-way ANOVA test with Geisser-Greenhouse correction or paired t-test.
Figure 2
Figure 2
Network of protein interactions between CYP24A1, MAP7 and MUCL1. The protein interactions network was built based in both our microarray results and previously reported data. Each node represents a different protein encoding gene. The color of the border of each node indicates the level of expression of each gene in vitD3-tolDC compared to immature dendritic cells (iDC) and the color of the body of each node indicates the expression of each gene compared to mature dendritic cells (mDC). The color scale indicates the level of mean difference expression (MeanDiff) of each gene according to our microarray study, from green (MeanDiff ≤ −1.35) to red (MeanDiff ≥ 1.35). Green and blue lines indicate protein interactions that are related to MAP7 and MUCL1 genes (respectively) in 3 or less parenthood levels, and the red lines indicate interactions that are shared by both genes in <6 parenthood levels. Genes have been grouped in functional clusters, as indicated by the colored bubbles. Arrows indicate those genes that were selected for further validation studies.
Figure 3
Figure 3
Immunocytochemistry study of MAP7 and MUCL1 protein expression in dendritic cells. (A) Relative levels of expression of microtubule-associated protein 7 (MAP7) in dendritic cells differentiated from healthy donor samples (n = 4) and MS patient samples (n = 3). (B) Relative levels of expression of mucin-like 1 (MUCL1) in dendritic cells differentiated from healthy donor samples (n = 4) and MS patient samples (n = 3). Results are calculated as percentage (%) of corrected total cell fluorescence (CTCF) in immature dendritic cells (iDC) and mature DC (mDC) compared to vitD3-tolDC. One single immunocytochemistry experiment was performed for each sample. Error bars corresponding to SEM. *p < 0.05. One-way ANOVA test with Geisser-Greenhouse correction or paired t-test. Representative pictures of the expression of (C) MAP7 and (D) MUCL1 in iDC, mDC and vitD3-tolDC from healthy donor and MS patient samples. α-tubulin staining is shown in green; either MAP7 or MUCL1 staining is shown in red; nuclei staining is shown in blue. The immunocytochemistry analysis was performed on a fluorescence microscope using a 63x objective. Immunocytochemistry primary staining was performed using mouse anti-human α-tubulin and either rabbit anti-human MAP7 or rabbit anti-human MUCL1 antibodies. Secondary stainings were performed using AlexaFluor (AF) 488 goat anti-mouse IgG and AF 594 goat anti-rabbit IgG antibodies. Nuclei staining was performed using DAPI.
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