Fig 1: Flow cytometric analysis of EVs from PBMC supernatants before (CTR, ✕) and after stimulation with 10 µg/mL LPS, 50 ng/mL PMA, 500 ng/mL ionomycin (LPS, ○). DiO+ small particles (SP) of stimulated PBMC were analyzed by flow cytometry after EV isolation using Invitrogen Total Exosome Isolation reagent. (A) Small particles were gated for single events according to DiO-H to DiO-A ratio. Single events were first gated for CD9+ SP, followed by gating for DiO-stained SP, excluding high-SSC DiO-dye crystals. Henceforth, DiO+CD9+ SP were called extracellular vesicles (EVs). EVs were cross-checked with size reference beads. (B) DiO+ stained single events were investigated for their CD9 and CD63 expression. CD9 RFI calculated from CD9 MFI of sample in relation to isotype control. (C) DiO+CD9+ EVs were investigated for CD83 expression. RFI calculated from CD83 MFI of sample in relation to isotype control. Data were analyzed by paired t-test and are depicted as mean (n = 6). *p < 0.05; **p < 0.01. RFI, Relative Immunofluorescence.
Fig 2: CD19+ cells were isolated by magnetic separation and stimulated with 10 µg/mL LPS, 50 ng/mL PMA, 500 ng/mL ionomycin (○) in the presence of pregnancy-related factors. E2, estradiol (2 ng/mL), P4: progesterone (30 ng/mL), TGFβ, TGF-β1 (2 ng/mL), hCG (10 IU/mL) Dexa, dexamethasone (40 ng/mL). The expression of CD83 was assessed by qPCR (A) and the secretion of MMP-7 was analyzed by ELISA (B). Data were analyzed by paired ANOVA with Dunnett post-test and are depicted as mean (n = 6). *p < 0.05; **p < 0.01; ****p < 0.0001.
Fig 3: Low sCD83 levels correlate with a severe disease course in MOGAD and NMOSD patients. Correlation between sCD83 levels and the volume of the brain and VEP in MOGAD and NMOSD patients. (a) There is a significant correlation between total brain volume and sCD83 level in the sera of MOGAD patients (r = 0.66, p = 0.0004) (b) There is no significant correlation between total brain volume and sCD83 level in the sera of NMOSD patients (r = -0.28, p = 0.10) (c) There is no significant correlation between VEP and sCD83 level in the sera of MOGAD patients (r=-0.26, p=0.35). (d) There is a significant correlation between VEP and sCD83 level in the sera of NMOSD patients (r = -0.50, p = 0.008) MOGAD, myelin oligodendrocyte glycoprotein antibody-associated disease; NMOSD, Neuromyelitis optica spectrum disorders; sCD83, soluble CD83; VEP, Visual Evoked Potentials.
Fig 4: sCD83 levels can predict a relapsing MOGAD disease course. (a) sCD83 expression of monophasic (n=14) versus relapsing (n=12) MOGAD patients (1.36 ± 1.03 RQ vs 0.62 ± 0.60 RQ, p=0.04). (b) sCD83 levels in the sera of monophasic (n=21) and relapsing (n=27) MOGAD patients. Monophasic MOGAD patients had significantly higher sCD83 levels compared to relapsing MOGAD patients (10694.80 ± 9846.70 pg/mL vs 3049.57 ± 4621.34 pg/mL, p=0.0009) (c) For sCD83 in the sera, a value of 706.85 pg/mL yielded the highest ROC-AUC of 0.811 (with a sensitivity of 59.26% and specificity of 95.24% (d) sCD83 levels in the CSF of monophasic (n=10) and relapsing (n=13) MOGAD patients. No significant difference was found between monophasic and relapsing MOGAD patients (287.60 ± 299.80 pg/mL vs 202.30 ± 194.90 pg/mL, p = 0.42). MOGAD: myelin oligodendrocyte glycoprotein antibody-associated disease; sCD83: soluble CD83; ROC: receiver operating characteristic; AUC: area under the curve. * p<0.05 ** p<0.01 *** p<0.001 **** p<0.0001.
Fig 5: NMOSD patients have low sCD83 levels. (a) sCD83 expression in PBMCs of HCs (n=40), MOGAD (n=37), MS (n=17) and NMOSD patients (n=20). The expression level of NMOSD patients was significantly lower than that of MOGAD patients (0.44 ± 0.22 RQ vs 1.16 ± 0.92 RQ, p = 0.02), and HCs (0.44 ± 0.22 RQ vs 1.07 ± 0.91 RQ, p = 0.05) (b) sCD83/mCD83 expression ratio in PBMCs of HCs (n=40), MOGAD (n=37), MS (n=17) and NMOSD patients (n=20). The sCD83/mCD83 ratio of NMOSD patients was significantly lower than that of MOGAD patients (0.71 ± 0.16 vs 1.02 ± 0.39, p = 0.04), MS patients (0.71 ± 0.16 vs 1.16 ± 0.45, p=0.006) and HCs (0.71 ± 0.16 vs 1.04 ± 0.48, p = 0.02) (c) sCD83 levels in the sera of HCs (n=64), MOGAD (n=64), MS (n=47) and NMOSD (n=56) patients. NMOSD patients had significantly lower sCD83 levels compared to HCs (1129.34 ± 2073.49 pg/mL vs 10076.75 ± 10366.37 pg/mL, p<0.0001) and MOGAD (1129.34 ± 2073.49 pg/mL vs 7019.97 ± 9225.68 pg/mL, p=0.0008) patients. MS patients had significantly lower sCD83 concentration compared to HCs (5777.94 ± 8319.23 pg/mL vs 10076.49 ± 10366.21 pg/mL, p = 0.04) and higher sCD83 concentration compared to NMOSD patients (5777.94 ± 8319.23 pg/mL vs 1129.34 ± 2073.49 pg/mL, p = 0.03). (d) sCD83 levels in the CSF of ONNIDs (n=15), MOGAD (n=37), MS (n=23) and NMOSD (n=26) patients. NMOSD patients had significantly lower sCD83 levels compared to MOGAD patients (215.20 ± 202.80 pg/mL vs 44.77 ± 47.84 pg/mL, p=0.02) and ONNIDs (311.30 ± 411.80 pg/mL vs 44.77 ± 47.84 pg/mL, p=0.002). HC, healthy control; MOGAD, myelin oligodendrocyte glycoprotein antibody-associated disease; NMOSD, neuromyelitis optica spectrum disorders; MS, multiple sclerosis; ONNIDs, other non-inflammatory neurological disorders; sCD83, soluble CD83; PBMCs, Peripheral blood mononuclear cells. * p<0.05 ** p<0.01 *** p<0.001 **** p<0.0001.
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