Fig 2: EphA2 Is Required for Maximal Fungal Killing of Serum-Opsonized Yeast-Phase C. albicans(A) Killing of serum-opsonized yeast-phase C. albicans by peripheral blood (PB) and bone marrow (BM) neutrophils isolated from wild-type and EphA2-/- mice. The percentage of organisms killed was determined by colony counting. Results are means ± SD from 4 experiments, each performed in triplicate. ****p < 0.0001 (Mann-Whitney test).(B) Killing of unopsonized C. albicans yeast by neutrophils from the indicate mouse strains. NS, not significant.(C) Neutrophil killing of opsonized C. albicans hyphae.(D) Killing of serum-opsonized yeast-phase C. albicans by bone marrow-derived macrophages.(E) Killing of opsonized yeast C. albicans by a mixed wild-type and EphA2-/- population. Results are means ± SD from 3 independent experiments, each performed in duplicate. ***p < 0.001; ****p < 0.0001 (Mann-Whitney test corrected for multiple comparisons).(F) Phagocytosis of opsonized C. albicans yeast by wild-type and EphA2-/- neutrophils. NS, not significant. Data were normalized to the phagocytosis of wild-type neutrophils.(G) Neutrophil killing of serum-opsonized S. aureus.(H) Body weight of wild-type and EphA2-/- mice after oral infection with C. albicans. Results are median ± interquartile range of 5 mice per strain in a single experiment. *p < 0.05 (Mann-Whitney test for each day).(I) Oral fungal burden of wild-type and EphA2-/- mice after 4 days of OPC. Results are median ± interquartile range of five mice per group in a single experiment. **p < 0.01 (Mann-Whitney test). The y axis is set at the limit of detection (20 CFUs per gram of tissue).(J) Fc?R signaling is required for killing of serum-opsonized C. albicans yeast. The neutrophil Fc?Rs, CD16, CD32, and CD64 were blocked using specific Abs, after which the killing of serum-opsonized yeast-phase C. albicans was determined by colony counting. Results are means ± SD from 4 experiments in triplicate. ****p < 0.0001, NS, not significant (Mann-Whitney test).(K) Fc?R signaling is required for yeast phagocytosis. The neutrophil Fc?Rs, CD16, CD32, and CD64 were blocked using specific Abs, after which phagocytosis of opsonized C. albicans yeast by wild-type was determined. Results are means ± SD from 3 independent experiments, each performed in duplicate. **p < 0.01 (Mann- Whitney test).(L) Oral fungal burden of wild-type and FcyRI-/- mice after 2 days of OPC. Results are median ± interquartile range of five mice per group in a single experiment. **p < 0.01 (Mann-Whitney test). The y axis is set at the limit of detection.See also Figure S4.

Fig 3: MSCs-IT inhibit CCL2 production by macrophages and fibroblasts.A Immunofluorescence staining of CCL2 (red) in skin sections from MSCs-IT treated or untreated mice on day 30. Scale bars, 100 μm. B Skin sections were stained with anti-Vimentin (green) and anti-CCL2 (red) antibodies to visualize the colocalization of fibroblasts and CCL2 in BLM-treated mice on day 30. White dashed arrows indicate the double-positive cells. Scale bars, 35 μm. C Skin sections were stained with anti-CD64 (green) and anti-CCL2 (red) antibodies to visualize the colocalization of macrophages and CCL2 in BLM-treated mice on day 30. White dashed arrows indicate the double-positive cells. Scale bars, 35 μm. D MSCs-IT were stimulated with 10 ng/mL IFN-γ and 10 ng/mL TNF-α in the inserts of transwells for 24 h, and then the inserts bearing MSCs-IT were placed on the top of multiwell plates adhering fibroblasts or macrophages in the presence of 10 ng/mL BLM. Then, the expression of CCL2 in fibroblasts or macrophages was examined at 24 h. E The expression of Ccl2 mRNA in fibroblasts was examined by qPCR. n = 4. F The expression of Ccl2 mRNA in macrophages was examined by qPCR. n = 4. G Fibroblasts were stained with anti-CCL2 (green) antibody to visualize the expression of CCL2. Scale bars, 100 μm. n = 3. H Macrophages were stained with anti-CCL2 (green) antibody to visualize the expression of CCL2. Scale bars, 100 μm. n = 3. Data were shown as means ± SEM. Data are representative of three experiments with similar results. *P < 0.05; ***P < 0.001.

Fig 4: MSCs-IT ameliorate SSc by acting on macrophages.A, B Mice were i.c. injected with BLM or PBS daily for 4 weeks to induce SSc and then euthanized and examined on day 30. A The infiltration of macrophages in the skin was assessed by immunofluorescence staining of CD64 (green). Scale bars, 100 µm. B The colocalization of CD64 (red) and TGF-ß1 (green) in the skin of SSc mice was determined by immunofluorescence staining. Scale bars, 100 µm. C Experimental design of the BLM-induced SSc mouse model, Clodronate liposome (Clo)/Control liposome (Ctrl) treatment, and MSC-IT treatment. Mice were euthanized and examined on day 30. n = 4–5 mice for each group. D 100 µL Clo liposomes (5 mg/mL) or 100 µL of Ctrl liposomes was administered to normal mice, and the depletion efficiency of Clo liposome treatment on macrophages (CD11b+CD64+) in the skin was analyzed by flow cytometry on day 3. n = 3 mice for each group. E H&E staining of the skin sections. The distance between the two yellow lines was assessed. Scale bars, 200 µm. F Sirius Red staining of the skin sections. The distance between two black dotted lines was assessed. Scale bars, 200 µm. G Immunofluorescence staining of a-SMA (red) in the skin sections. The percentage of a-SMA+ area was assessed. Scale bars, 200 µm. Data were shown as means ± SEM. Data are representative of two or three experiments with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.

Fig 5: Myeloid cells play a key role in the pathogenesis of SSc.A–D Mice were i.c. injected with BLM or PBS daily for 4 weeks. MSCs-IT (2 × 105) were i.v. injected into SSc mice on days 7, 14, and 21. Mice were sacrificed and subjected to flow cytometry analysis on day 30. n = 5–7 mice for each group. A Flow cytometry analysis of CD45+ immune cells in the skin. B Flow cytometry analysis of CD11b+CD64+ macrophages in the skin. C Flow cytometry analysis of CD11b+Ly6G+ neutrophils in the skin. D Flow cytometry analysis of CD3+ T cells and CD19+ B cells in the skin. E, F Rag 2-/- mice and WT mice were i.c. injected with BLM or PBS daily for 4 weeks. Mice were sacrificed and examined on day 30. n = 4–6 mice for each group. E H&E staining of skin sections from Rag 2-/- mice and WT mice. The distance between the two yellow lines was assessed. Scale bars, 200 µm. F Sirius Red staining of skin sections from Rag 2-/- mice and WT mice. The distance between two black dotted lines was assessed. Scale bars, 200 µm. G Experimental design of the BLM-induced SSc model of CD11b-DTR mice and DT treatment. Mice were sacrificed and examined on day 30. n = 5–6 mice for each group. H Depletion efficiency of DT treatment on CD11b+ myeloid cells in the skin of CD11b-DTR mice was analyzed by flow cytometry on day 30. I H&E staining of skin sections from CD11b-DTR mice. The distance between the two yellow lines was assessed. Scale bars, 200 µm. J Sirius Red staining of skin sections from CD11b-DTR mice. The distance between two black dotted lines was assessed. Scale bars, 200 µm. Data were shown as means ± SEM. Data are representative of two or three experiments with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.