Table 1.
List antibodies used in this study.
Fig 1.
Characteristics and multilineage differentiation potential of T-MSCs.
(A) Morphology of cultured T-MSCs observed under light microscopy (original magnification, 200×). (B) Flow cytometric analysis of surface marker expression in T-MSCs. Histograms show absence of CD11b, CD34, and CD45 (negative markers) and expression of CD73, CD90, and CD105 (positive markers). Red areas represent isotype controls; green areas represent specific antibody staining. (C) Multilineage differentiation potential of T-MSCs. Left: adipogenic differentiation visualized by Oil Red O staining of lipid droplets. Center: osteogenic differentiation shown by Alizarin Red S staining of calcium deposits. Right: chondrogenic differentiation demonstrated by Alcian Blue staining of proteoglycans (original magnification, 200×).
Fig 2.
Neutrophil-like differentiation of HL-60 cells.
(A) Morphological changes in HL-60 cells during differentiation. Cells were treated with 0.8% dimethylformamide (DMF) and observed at Day 0 (undifferentiated) and Day 4 (differentiated). Images were captured using an Olympus DP28 microscope (original magnification, 200×). (B) Giemsa staining of differentiated HL-60 cells. Cells were collected by cytospin centrifugation, fixed in methanol, and stained with Giemsa solution. Images were captured using an Olympus DP28 microscope (original magnification, 200×). Differentiated HL-60 cells are indicated by arrows. Scale bar = 10 µm. (C) Flow cytometric analysis of neutrophil surface markers. Expression of CD11b, CD35, and CD71 was compared between Day 0 (undifferentiated) and Day 4 (differentiated) HL-60 cells.
Fig 3.
Effects of T-MSCs in the DNCB-induced skin inflammation model.
(A) Experimental scheme showing the timeline of DNCB application and T-MSC injection in the mouse model of skin inflammation. DNCB or vehicle was applied on days 1, 2, 3, 8, and 9. T-MSCs or PBS were administered intravenously on day 2. Mice were sacrificed on day 10 for analysis. (B) Body weight changes over 10 days following DNCB treatment and T-MSC injection. The graph shows body weight as a percentage of baseline (day 0) for each group (n = 4 per group). (C) Representative photographs of dorsal skin from each group taken at sacrifice on day 10. (D) Macroscopic images of lymph nodes (LN) and spleens (SP) harvested from each group. (E) Quantification of LN length across groups. DNCB treatment significantly increased LN length compared with vehicle (n = 2 per group). (F) Quantification of SP length across groups. Data were analyzed using two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (* P < 0.05 and ** P < 0.01). Vehicle (V), vehicle + T-MSCs (V + T), DNCB (D), and DNCB + T-MSCs (D + T).
Fig 4.
Histological analysis of inflammatory features by H&E staining.
(A) Representative H&E-stained dorsal skin sections from each group (Vehicle, Vehicle + T-MSCs, DNCB, and DNCB + T-MSCs) at 100 × magnification. Scale bar = 100 µm. (B) H&E-stained LN sections from each group at 4 × magnification. Scale bar = 50 µm. (C) H&E-stained spleen (SP) sections from each group at 4 × magnification. Scale bar = 50 µm. (D) Quantification of epidermal thickness across groups. Twenty measurements per group (5 per slide, 4 slides; n = 20) were included. (E) Quantification of LN medullary sinus area as a percentage of total LN area. Three slides per group (n = 3) were analyzed. (F) Quantification of splenic follicle area as a percentage of total SP area. Eight slides per group (n = 8) were analyzed. Images were analyzed with ImageJ, and results were analyzed by two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (* P < 0.05, ** P < 0.01, ***P < 0.001 and ****P < 0.0001). ns: not significant. Vehicle (V), vehicle + T-MSCs (V + T), DNCB (D), and DNCB + T-MSCs (D + T).
Fig 5.
Countined.
Fig 6.
Immunofluorescence analysis of cutaneous lymphocyte antigen (CLA).
(A) Representative immunofluorescence images of skin sections from each group (Vehicle, Vehicle + T-MSCs, DNCB, and DNCB + T-MSCs) stained for CLA (red) and DAPI (blue). (B) Quantification of CLA+ cells per field of view across groups. Data were analyzed using two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (** P < 0.01 and ****P < 0.0001).
Fig 7.
Continued.
Fig 8.
Myeloperoxidase (MPO) expression and neutrophil infiltration.
(A) Representative immunohistochemistry images of skin sections from each experimental group stained for MPO. Scale bar = 100 µm. (B) Quantification of MPO expression in skin sections across groups. Data were analyzed using two-way ANOVA (**** indicates P < 0.0001). (C) Representative flow cytometry plots showing neutrophil populations (CD11b⁺Ly-6G⁺) in skin samples from each group. (D) Quantification of neutrophil infiltration in skin samples across groups. Data were analyzed using two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (*** P < 0.001 and **** P < 0.0001).
Fig 9.
(A) Representative images of differentiated HL-60 (dHL-60) cells used as neutrophil-like cells for in vitro experiments. (B) Quantification of NETosis in dHL-60 cells under various treatment conditions: untreated, DNCB-treated, co-cultured with T-MSCs, or co-cultured with T-MSCs and DNCB. Data were analyzed using two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (*P < 0.05 and **P < 0.01). (C) Immunoblot analysis of citrullinated histone H3 (CitH3) and β-actin in dHL-60 cells treated with PMA, T-MSCs, PAD4 inhibitor (GSK484) and NADPH oxidase inhibitor (DPI) for NETosis quantification. (D) Densitometric quantification of CitH3 expression normalized to β-actin. Bars represent mean ± SD of three independent experiments. Data were analyzed using two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (* P < 0.05). ns: not significant.
Fig 10.
Transwell migration assay of Jurkat cells.
(A) Experimental design of the Transwell migration assay. Jurkat cells (human T cell leukemia line) were seeded in the upper chamber either alone, co-cultured with dHL-60 cells (neutrophil-like cells), or co-cultured with DNCB-treated dHL-60 cells. T-MSCs were placed in the lower chamber. Two pore sizes (5 µm and 8 µm) were tested. (B) Flow cytometry-based quantification of Jurkat migration under different conditions. Groups included Jurkat alone, Jurkat + dHL-60, Jurkat + DNCB-treated dHL-60, and each of these with or without T-MSCs. Migrated cells were quantified by flow cytometry for both pore sizes. (C) Quantification of Jurkat migration using 5 µm pore Transwells. Bar graphs show the percentage of migrated cells. (D) Quantification of Jurkat migration using 8 µm pore Transwells. Data were analyzed using two-way ANOVA, followed by Tukey’s post hoc test for multiple comparison (a: P < 0.001 for Jurkat vs. Jurkat + T-MSCs, b: P < 0.001 for dHL-60 + Jurkat vs. dHL-60 + Jurkat + T-MSCs, c: P < 0.001 for dHL-60 + DNCB + Jurkat vs. dHL-60 + DNCB + Jurkat + T-MSCs, d: P < 0.001 for Jurkat + T-MSCs vs. dHL-60 + Jurkat + T-MSCs, and e: P < 0.001 for Jurkat + T-MSCs vs. dHL-60 + DNCB + Jurkat + T-MSCs).