Human urine-derived stem cells alleviate psoriasis by suppressing JAK2/STAT3 pathway-mediated macrophage polarization

Abstract

Background

Psoriasis is a chronic skin disease featured with aberrant keratinocyte proliferation, inflammatory cell infiltration, and immune dysregulated. Although the imbalance of M1/M2 macrophage polarization is implicated in its pathogenesis, the underlying mechanisms remain unclear. Mesenchymal stem cells exhibited potent immunomodulatory properties, representing a promising therapeutic approach for psoriasis. This study aimed to explore the role and the underlying mechanism of human urine-derived stem cells (hUSCs) in mouse psoriatic models.

Methods

hUSCs were isolated from urine of heath volunteer and cultured in serum-free medium, and characterized by multiple approaches such as morphological analysis, biological markers examination, differentiation potentials and tumorigenicity assay. Histological analysis, immunofluorescence staining, ELISA, flow cytometry, antibody array, western blot and qRT-PCR analysis were used to assess the therapeutic effects and the underlying mechanism of hUSCs in imiquimod (IMQ)-induced mouse psoriasis models and multiple cell models.

Results

hUSCs had the potential for self-renewal and multipotent differentiation with low immunogenicity and lacking tumorigenicity both in vitro and in vivo. Our results showed that hUSCs significantly alleviated IMQ-induced psoriasis via their paracrine, evidenced by improving morphologies, inhibiting the infiltration of macrophages, reducing the releases of the pro-inflammatory cytokines. Mechanistically, we revealed that the protective effects of hUSCs on psoriasis were involved in suppressing M1 and promoting M2 macrophage polarization, and inhibiting NETs formation through inhibiting JAK2/STAT3 pathway. Finally, we further demonstrated that hUSCs-derived TGF-β1 selectively inhibited the JAK2/STAT3 pathway-mediated the polarization of M1 and M2 macrophages to alleviate psoriasis in mouse and cellular models.

Conclusions

Our data demonstrated that hUSCs remarkably ameliorated psoriasis by suppressing M1 and promoting M2 macrophage polarization through they-derived TGF-β1 inhibiting the JAK2/STAT3 pathway. Our results have revealed the molecular mechanism of hUSCs in treating psoriasis, highlighting a safe and effective cellular treatment method for psoriasis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04870-w.

Background

Psoriasis is a prevalent, immune-mediated chronic inflammatory disease characterized by abnormal keratinocyte proliferation, immune cell infiltration and dysregulation of the cytokine network [1]. Although some advances have been made in the treatments of psoriasis such as topical therapies, phototherapy, and systemic biologics targeting TNF-α, IL-17, or IL-23, the challenges of the drug resistance, adverse effects and high relapse rates persist [2–4]. Moreover, a growing patients with severe psoriasis remains refractory to all currently available treatments [5], underscoring the urgent need for more advanced and potentially curative therapeutic approaches, including non-pharmacological interventions.

Extensive macrophages infiltration was observed in the skin lesions of psoriasis [6], and new evidence confirmed that the macrophage polarization played a critical role in the pathogenesis of psoriasis, in which divergent macrophage subtypes fulfilled distinct roles in disease progression [7]. Excessive M1 pro-inflammatory macrophages, which spur keratinocyte proliferation and neutrophil recruitment, and the reduced anti-inflammatory M2 macrophages were observed in psoriatic skin lesions [8, 9]. In addition, studies indicated that the activated neutrophils formed neutrophil extracellular traps (NETs), which stimulated keratinocyte proliferation and inflammatory responses, thereby exacerbating the severity of psoriasis [10]. It has been reported that M1 macrophages promote the formation of NETs, while M2 macrophages have an advantage in preventing and alleviating psoriasis [11, 12]. The JAK/STAT pathway plays a pivotal role in macrophage-mediated inflammatory response, whereby its activation not only promotes M1 pro-inflammatory polarization by IFN-γ and IL-12, but also mediates M2 anti-inflammatory polarization by IL-4, IL-13 and IL-6 [13]. It has been reported that cytokines such as IL-23, IL-17, and IL-6 activate members of the JAK family, triggering phosphorylation of STAT proteins and facilitating their nuclear translocation [14]. This molecular cascade subsequently modulated the transcription of pro-inflammatory cytokines, thereby exacerbating the inflammatory response in psoriasis pathogenesis. Nonetheless, the precise mechanism of macrophage polarization related to JAK/STAT signaling pathway in psoriasis is not fully elucidated. Therefore, further elucidation of the underlying mechanism should shed light on the pathogenesis of psoriasis, and targeting macrophage plasticity might be a promising therapeutic strategy.

Recently, studies indicated that mesenchymal stem cells (MSCs) with low complication risks have emerged as a vital therapeutic strategy in psoriasis [15, 16]. MSCs exhibit potent anti-inflammatory and immunomodulatory properties, demonstrating efficacy in autoimmune diseases through their paracrine effects [17, 18]. Human urine-derived stem cells (hUSCs) are a novel subpopulation of stem cells that have some advantages such as a non-invasive source, high self-renewal, proliferation, and multi-directional differentiation abilities, low or no immunogenicity and no tumorigenicity [19]. Studies have validated that hUSCs effectively treated many diseases such as dermatological [20, 21], urogenital [22, 23], and neurological disorders [24]. Notably, no investigations have yet explored the in-depth molecular mechanism of hUSCs in the treatment of psoriasis. This study aims to explore the therapeutic effects and the underlying molecular mechanisms of hUSCs in psoriasis mouse models, thereby providing experimental evidence for the treatment of psoriasis clinically.

Methods

Isolation and culture of hUSCs

hUSCs were isolated and cultured with the established protocol [25]. Briefly, 100 mL of human urine was collected from each healthy male for total 4 individuals, and the urine was centrifuged at 400 g for 10 min, and the pellets were resuspended in 10 mL of phosphate buffered saline (PBS), followed by another centrifugation at 400 g for 10 min. The pellets were resuspended in 1 mL of serum-free medium containing 70% MCDB 153 medium (M7403, Sigma, USA), 20% MEM alpha medium (12571063, Gibco, USA) 2% human platelet lysate (PL-NH-500, SEXTON, USA), 1×Insulin-Transferrin-Selenium Ethanolamine (1× ITS-X) (51500056, Gibco), 1% Non-Essential Amino Acids (NEAA) (11140050, Gibco), 1% penicillin/streptomycin (15140-122, Gibco), 2 × 10^{− 9} mol/L 3,3′,5-Triiodo-L-thyronine (T2877, Sigma), hydrocortisone (H0888, Sigma), and 10 ng/mL epidermal growth factor (EGF) (AF-100-15, Peprotech, USA). The cells were then placed in a 5% CO₂ incubator for culture, and the hUSCs colonies usually appeared at about 7 days, designated passage 0 (P0). The cells were cultured and extended to a certain order of magnitude for the subsequent experiments. The morphologies of hUSCs under the culture conditions in serum-free medium (SFM) were monitored and compared with those of previous serum medium (SM) containing fetal bovine serum (FBS) (10099141 C, Gibco) [23] via an inverted microscope (Olympus, Japan). Serum medium contain Keratinocyte Serum-Free Medium (KSFM, 10785-012, Gibco), 22% High Glucose-Dulbecco’s Modified Eagle Medium (H-DMEM, 11965092, Gibco), 22% Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, 11330032, Gibco), 10% FBS, 1% insulin-transferrin-selenium (insulin-transferrin-Se, 41400045, Gibco), 100 U/mL penicillin and streptomycin, 10 mol/L cholera toxin (C8052, Sigma), adenine (A8626, Sigma), 2 × 10^–9 mol/L 3,3′,5-Triiodo-L-thyronine, hydrocortisone, and 10 ng/mL EGF.

Cell lines

Human immortalized keratinocytes (HaCaT) and mouse macrophages (RAW264.7) were cultured in H-DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin. Human Acute Promyelocytic Leukemia-60 (HL-60) was maintained in IMDM supplemented with 20% FBS and 1% Penicillin/Streptomycin. All cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Preparation and concentration of hUSCs-derived conditioned medium (hUSC-CM)

hUSCs from passages 3 to 5 were seeded in 10 cm dishes at 37 °C with 5% CO₂. Upon reaching 90% confluence, the medium was aspirated, and the cells were washed twice with PBS. Subsequently, 8 mL of basal medium was added to the cells, which were then cultured for 48 h. hUSC-CM was collected and centrifuged at 1500 rpm for 5 min to remove cell debris. The supernatant was transferred to a Millipore ultrafiltration tube (3 kDa, 15 mL) and concentrated 10-fold by centrifugation at 5000 × g for 60 min at 4 °C, prior to storage at −80 °C.

Flow cytometry analysis

Characterization of cultured hUSCs, classification of bone marrow cells, bone marrow-derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC) were analyzed using flow cytometry. Cells were washed and resuspended in PBS at a concentration of 1 × 10⁶ cells/mL, and then the cells were filtered through a 45 μm cell sieve. The corresponding antibodies were added and incubated in the dark at 4℃ for 30 min. Antibodies (most from BioLegend (USA) except for CD86 (BD Biosciences, USA)) include MSC markers (CD73-PE (344003), CD105-PE (323205)), and CD90-FITC (389803), hematopoietic cell markers (CD34-FITC (343503) and CD45-PerCP (982318)), major histocompatibility complex markers (HLA-DR-FITC (980402) and HLA-ABC-PE (311405) and costimulatory molecules (CD80-FITC (375405), CD86-PE (560957), and CD40-FITC (334305). Additional antibodies used for immune cell typing encompassed were as follows: F4/80-FITC (123107, Biolegend), CD86-PE (12–0862-81, eBioscience, USA), CD206-PE (12–2061-80, eBioscience) and CD11c (17–0114-81, Invitrogen, USA). RAW264.7 were incubated with 0.5 mL of DCFH-DA working solution (1:1000, CA1410, Solarbio, China) at 37 °C for 30 min under light-protected conditions. Data was acquired via flow cytometry for analysis (Beckman, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from hUSCs with Trizol reagent (15596026CN, Thermo Fisher, USA). The purity of the RNA was determined by calculating the absorbance ratio at 260 nm to 280 nm. The RNA was reverse-transcribed into cDNA using a cDNA synthesis kit (11141ES60, Yi Sheng, China) with 2 µg RNA as a templet. The primers of the targeting genes were presented in Supplementary Table 1. PCR was conducted using a thermal cycler (Thermo Hybaid, USA). The PCR products were analyzed on a 1.0% (m/v) agarose gel containing 0.5 µg/mL ethidium bromide for nucleic acid visualization under ultraviolet light. Human GAPDH acted as the internal control.

Immunofluorescence analysis

hUSCs, skin tissue sections and BMDM were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 and blocking with 5% bovine serum albumin (BSA). Subsequently, primary antibodies were incubated with the tissues/cells overnight at 4℃. The antibodies were used as follows: anti-SSEA4 (1:300, MC813, CST, USA), anti-Oct4 (1:200, D7O5Z, CST), anti-Nanog (1:200, D73G4, CST), anti-Ki67 (1:500, TW0001, Abmart, China), anti-Cytokeratin 18 (1:50, ab668, Abcam, USA), anti-MAB1281 (1:300, 32160702, Merck, Germany), anti-CD90 (1:500, ab307736, Abcam), anti-CD86 (1:100, NBP2-25208, NOVUS, USA), anti-CD206 (1:500, 18704-1-AP, Proteintech, USA), anti-IL-10 (1:50, 5750, PTMab, China), anti-iNOS (1:50, 6217, PTMab), anti-F4/80 (1:500, 28463-1-AP, Proteintech), anti-Ly6G (1:400, WLH4318, Wanleibio, China) and anti-Cit-H3 (1:200, 17939, Cayman, USA). The samples were subjected to a one-hour incubation in the dark at room temperature with HRP-conjugated goat anti-rabbit (1:500, 7074, CST) or anti-mouse (1:500, 8890, CST) IgG secondary antibody at a dilution of 1:1000 after washed with PBS. Cells were then washed with PBS and incubated with DAPI dye for 3 min at 37 °C. The cell nuclei were re-stained with anti-fluorescence quencher containing DAPI, observed by a laser confocal microscope (ZEISS, Germany) and photographed.

Differentiation analysis of hUSCs

The potentials of hUSCs differentiating into adipocytes and osteocytes were determined according to manufacturer’s instructions (HUXMX-90031/HUXXC-90021, OriCell^®, Cyagen, China). hUSCs were plated in a 6-well plate with a density of 1.5 × 10⁵ cells per well. Adipocyte differentiation was induced using alternating adipogenic induction media A and B, with Oil Red O staining for lipid droplet detection after 30 days. Osteoblast differentiation was induced with osteogenic medium, followed by Alizarin Red staining for calcium salt detection after 30 days. Differentiation outcomes were confirmed via inverted microscope imaging.

Soft agar assay for tumorigenicity

The bottom layer of the six-well plate was coated with 1.2% soft agar, while hUSCs or HepG2 cells with 1 × 10³/well were distributed in the upper layer of 0.6% soft agar. The plates were then incubated at 37 °C in a 5% CO₂ atmosphere for 30 days. HepG2 served as a control, and colonies were observed and imaged using an inverted microscope.

Tumorigenicity test in vivo

A total of 1 × 10⁶ cells of hUSCs or breast cancer cells (4T1, CBP60352, Nanjing Cobioer Biosciences Co., Ltd.) were suspended in PBS (100 µL), and administered into the breast pads of NOD/SCID mice at 8-week-old that had been anesthetized with tribromoethanol. Tumor development was monitored by measuring the size of tumor every three days and rigorously tracked over the course of 20 weeks. Mice injected with 4T1 cells were humanely killed when tumor formation approached 2000 mm³. Tumor dimensions were quantified daily using vernier calipers.

Animals

Balb/c mice (male, 6–8 weeks old, 18–22 g) were purchased from GemPharmatech Co., Ltd and maintained with free access to pellet food and water in plastic cages and kept on a 12 h light/dark cycle in specific pathogen-free facilities. All animal procedures were conducted in compliance with the approval of the Animal Care and Use Committee of Nanchang University. The work has been reported in line with the ARRIVE guidelines 2.0.

Comprehensive safety profiling of hUSCs in vivo

hUSCs (1.5 × 10⁶ cells) were injected into mice (n = 6/group) daily intravenously (i.v.) or subcutaneously (s.c.) for 7 consecutive days before euthanasia. Major organs (skin, heart, liver, spleen, lung and kidney) were harvested for histopathological analysis. Hematological profiling was performed with blood which was collected via mouse eyeball.

Induction and treatment of psoriasis mouse models

A total of 32 mice were randomly divided into 4 groups (n = 8/group), designated as normal group (Normal), imiquimod model group (IMQ), tail vein injection hUSCs group (hUSCs (i.v.)) and subcutaneous injection hUSCs group (hUSCs (s.c.)). Mice received a daily topical dose of 62.5 mg of commercially available IMQ cream (5%; Sichuan Mingxin Pharmaceutical Co., Ltd, Chengdu, China) on the shaved back for 7 consecutive days. Following the induction of symptoms, with the exception of the Normal and IMQ groups, hUSCs transplantation was conducted on days 2, 4, and 6 post-symptom induction. Each mouse received 1.5 × 10⁶ cells per injection in a volume of 200 µL. Disease severity was evaluated using the applicable Psoriasis Area and Severity Index (PASI) for human clinical settings, which included scoring erythema, scaling, and skin thickening on a scale of 0 to 4 [17]. The total PASI score was calculated by summing the scores for the three clinical parameters. Caliper-based measurements were used to assess the thickness of the dorsal skin. Ultimately, the mice were anesthetized via intraperitoneal injection of 1.25% tribromoethanol (0.2 mL/10 g, Lab Anim Tech, China) and subsequently euthanized by cervical dislocation. The skin, serum and bone marrow cells were collected for further experiments.

Histopathology analysis

Skin tissues were collected, fixed, and immersed in 4% paraformaldehyde for 24 h. Subsequently, the tissues were dehydrated, embedded, sectioned into 4 μm slices, and stained with hematoxylin and eosin (H&E).

Routine analysis of blood

On day 7 post-modeling, blood was collected via mouse eyeball enucleation. A 45-µL blood sample was collected from each mouse into an EDTA-2Na-coated tube (5 µL) and mixed thoroughly. Hematological parameters, including white blood cells (WBC), lymphocytes (Lymph), mid cells (Mid), granulocytes (Gran), red blood cell (RBC), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), red blood cell distribution width-coefficient of variation (RDW-CV) and red blood cell distribution width-standard deviation (RDW-SD) were analyzed using an Auto Hematology Analyzer (MAXCOM, China).

Enzyme-linked immunosorbent assay (ELISA)

Blood was left at room temperature for 3 h and the samples underwent centrifugation at 1000 × g for 20 min at 4℃ to facilitate serum separation. The supernatant (serum) was collected for further analysis. The serum levels of IL-17 A (E-MSEL-M0006), TNF-α (E-MSEL-M0002) and IL-10 (E-MSEL-M0031) were detected by ELISA kits (Elabscience, USA) following the instructions of the manufacturer.

Quantitative reverse transcription‑polymerase chain reaction (qRT‑PCR)

Mouse skin tissue (15–20 mg) was mechanically disrupted in TRIzol reagent with grinding beads (G0201, Servicebio, China) using a homogenizer (1800 rpm, 60 s × 8). HaCaT, BMDM, RAW264.7 and neutrophil-like cells were seeded in a six-well plate and lysed with 500 µL of TRIzol reagent per well for total RNA extraction, and then the samples were collected and transferred to nuclease-free EP tube. After centrifugation (14,000 rpm, 15 min), chloroform substitute was added to the supernatant, mixed, and incubated on ice. After a second centrifugation (14,000 rpm, 15 min), isopropanol was added to precipitate RNA, which was washed with chilled 75% ethanol. The RNA pellet was resuspended in DEPC-treated water, and its concentration and purity were quantified. Reverse transcription was performed using 2 µg of RNA, followed by qPCR on an ABI-ViiA7 instrument with a two-step amplification protocol to assess the expression of IL-17 A, TNF-α, IL-6, CXCL15, Arg-1, CD206, iNOS, TGF-β, IL-8, IL-10, IL-1β, Sprr2b, Rptn, Cstdc6, Lce3b, Lce3f and CD11b. The qPCR primers were provided in Supplementary Tables 2, and the expression levels of the target genes were standardized using the housekeeping gene GAPDH as a reference.

Isolation and culture of BMDM and BMDC

Balb/c mice at 6–8 weeks were euthanized and their bone marrow cells were isolated according to an established method [17]. In brief, mouse bilateral femurs and tibias were aseptically harvested and flushed to prepare single-cell suspensions. Cells were resuspended in H-DMEM or RPMI 1640 with 10% FBS, adjusted to 1 × 10⁶/mL and plated in 6-well plates. M-CSF (20 ng/mL) or GM-CSF (20 ng/mL) + IL-4 (20 ng/mL) were added into the cells for 5–7 days, and the medium was renewed every 3 days for generating BMDM or BMDC. The introduction of BMDM and BMDC maturation were exposed to 1 µg/mL LPS for 24 h.

Differentiation of neutrophil-like cells and the induction of NETs

The neutrophil-like cells differentiated from HL60 cells were cultured in a CO₂ incubator for 5 days in the presence of 1.25% DMSO. Neutrophilic-like cells were exposed to phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 4 h to trigger NETs formation.

Fluorescence assay

HaCaT, BMDM and RAW264.7 cells were inoculated into 12-well plates. After the cell fusion reached 90%, the cells were incubated with hUSCs (hUSCs: target cells = 1:5) [26, 27] or hUSC-CM by adding LPS (1 µg/mL) or TNF-α (20 ng/mL) for 24 h. DCFH-DA working solution (1:1000, CA1410, Solarbio), JC-1 (1:1000, M8650, Solarbio) and Lipid Peroxidation Probe working solution, BDP 581/591 C11 (1:1000, L267, Dojindo, Japan) were prepared according to the manufacturer instructions. The cells were cultured in 0.5 mL working solution in a 37 °C incubator for 20 min avoiding exposure to light. Then, the cells were purged thrice with serum-free cell culture medium or JC-1 staining buffer or HBSS to completely remove fluorescent dye that has not entered the cells respectively. The fluorescence was then observed under an inverted fluorescence microscope (Olympus, Japan).

Western blot analysis

Total protein lysates were prepared through standard homogenization protocols. Briefly, skin tissue, BMDM, RAW264.7 and neutrophil-like cells were homogenized in lysis buffer (R0010, Solarbio) to extract total proteins, with skin tissue requiring additional grinding with grinding beads (G0103, Servicebio, China), and then centrifuged at 15,000 rpm for 25 min. Protein concentrations were determined using the Bicinchoninic Acid (BCA) method (23227, Thermo Fisher Scientific, USA). The proteins were loaded on 10%−12% denaturing SDS-PAGE gels for electrophoresis (The protein ladder: 26616, Thermo Fisher Scientific), followed by electrotransferring onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated with primary antibodies at 4℃ overnight, whereas β-actin was used as a loading control. Then, the membranes were probed with secondary antibodies (G21234/G21040, Invitrogen, USA) at room temperature for 1 h. Protein bands were visualized and quantified using Super Signal West Pico or Femto chemiluminescent detection system (Pierce). The following primary were employed in this study: anti-p-JAK2 (1:1000, WL02997, Wanleibio), anti-JAK2 (1:1000, WL02188, Wanleibio), anti-p-STAT3 (1:2000, T56566, Abmart), anti-STAT3 (1:2000, T55292, Abmart), anti-MPO (1:4000, 66177-1, Proteintech), anti-Cit-H3 (1:200, 17919, Cayman) and anti-β-actin (1:5000, AC004, ABclonal, China).

Antibody array analysis

The expression levels of cytokines in hUSC-CM were quantified in triplicate using a Human Cytokine Array (RayBiotech, Guangzhou, China) following the manufacturer’s instructions.

Statistical analysis

Results were expressed as mean ± SEM. Student’s t-test was used for analysis between two groups. One-way analysis of variance (ANOVA) was performed to compare data from three or more groups. Differences of P-value of < 0.05 were considered statistically significant. All statistical analyses were conducted using GraphPad Prism 10.

Results

Identification and characterization of hUSCs

Human urine-derived hUSCs formed 3 to 6 large cell colonies with a cobblestone-like morphology after they were cultured for approximately 12 days. Compared with serum medium (SM), hUSCs cultured in a cost-effective serum-free medium (SFM) still maintained robust proliferation and morphology (Fig. 1A). The results showed that hUSCs cultured in SFM highly expressed mesenchymal stem cell markers (CD73, CD105, CD90, CD29), embryonic stem cell markers (Oct4, Nanog, SSEA4), and human leukocyte antigen (HLA-ABC), did not express hematopoietic stem cell markers (CD34, CD45, CD133), HLA-DR, and co-stimulatory molecules (CD40, CD80, and CD86) (Fig. 1B–D), indicating that hUSCs cultured with SFM possessed the similar features such as the robust stemness and low immunogenicity compared with SM. Differentiation experiments showed that hUSCs exhibited the multipotent differentiation potentials, as evidenced by having differentiated into adipocytes (stained with Oil-Red-O) and osteoblasts (stained with Alizarin-Red) (Fig. 1E). In vitro tumorigenicity assay showed that numerous colonies were formed in soft agar 30 days after inoculation of HepG2 cells, whereas no colonies were observed in the hUSCs group (Fig. 1F). Additionally, in vivo studies revealed that none of the NOD/SCID mice inoculated with hUSCs developed tumors over a 20-week period, while control mice injected with 4T1 cells exhibited tumor formation within 7–9 weeks (Fig. 1G). Collectively, these results indicated that hUSCs had the potential for self-renewal and multipotent differentiation with low immunogenicity, and no tumor formation was observed both in vitro and in vivo.

Fig. 1.

Characterization of human urine stem cells (hUSCs). A Morphologies of hUSCs were determined by microscope under the serum medium (SM) and serum-free medium (SFM) (scale bar = 100 μm). B The surface markers including CD73, CD105, CD90, CD34, CD45, HLA-DR, HLA-ABC, CD40, CD80 and CD86 of hUSCs (SM and SFM) were determined by flow cytometry. C The mRNA expression of the surface markers including CD133, CD34, CD29, CD105, Oct4 and Nanog was examined by qRT-PCR analysis. D The embryonic stem cell markers including Oct4, SSEA-4 and Nanog of hUSCs were identified by immunofluorescence localization studies (scale bar = 100 μm). E The multilineage differentiation potentials of hUSCs were determined by adipogenesis (Oil-Red-O staining) and osteogenesis (Alizarin-Red staining). F The tumorigenicities of hUSCs and HepG2 cells were assessed in soft agar culture in vitro. G The tumorigenicities were determined by injection of hUSCs (1 × 10⁶ cells in 100 µL) or 4T1 tumor cells (1 × 10⁶ cells in 100 µL) in NOD-SCID mice

hUSCs alleviated IMQ-induced psoriasis in mice

Topical application of IMQ induces both localized and systemic inflammation, leading to a significant increase in epidermal thickness [28]. To evaluate the impact of hUSCs transplantation on IMQ-induced psoriasis, we first conducted a safety assessment of hUSCs in mice. Following a 7-day treatment of hUSCs, H&E staining revealed negligible toxicity in the skin, heart, liver, lungs, spleen, and kidneys (Supplementary Fig. 1 A), and the blood routine data also showed that there were no alterations in various blood cells (Supplementary Fig. 1B), suggesting that hUSCs-based cell therapy might offer a safe therapeutic approach for psoriasis. After the appearance of symptoms in mice treated with IMQ, the mice were transplanted with hUSCs by tail vein and subcutaneous injection three times (Fig. 2A). A typical symptom of psoriasis was induced by IMQ administration for 7 consecutive days in mice, evidenced by the emergence of erythematous plaques, with the skin surface being covered by numerous silvery scales and a significant thickening of the affected skin (Fig. 2B). In comparison, the results from H&E staining and PASI scoring showed that both administration of hUSCs in i.v or s.c. significantly alleviated IMQ-induced psoriasis, seen as a significant reduction in epidermal hyperplasia (Fig. 2C–E). In addition, intravenous injection of hUSCs yielded better therapeutic outcomes. Furthermore, hUSCs remarkably inhibited IMQ-induced increases in Ki67 and cytokeratin 18-positive cells which represent the enhanced proliferation of keratinocytes (Fig. 2F). In-depth analysis of the transcriptome data from GSE248626 revealed that the genes related to immune inflammation, keratinocyte differentiation and barrier function were significantly upregulated in mouse skin tissues in the IMQ group compared with normal group (Supplementary Fig. 2 A). Moreover, hUSCs markedly suppressed the expression of the genes related to keratinocyte differentiation and barrier function (Supplementary Fig. 2B).

Fig. 2.

hUSCs mitigated IMQ-induced mouse psoriasis models. A Schematic illustration of IMQ-induced mouse psoriasis models and hUSCs administration. B Representative macroscopic appearance of dorsal skin lesions on day 7 post-treatment of hUSCs. C Histopathological alterations were analyzed by H&E staining for skin sections (scale bar = 20 μm). D The lesions of the dorsal skin were assessed by PASI scores. E Measurement of murine epidermal thickness. F The co-localization analyses of Ki67 proliferation marker (red) and Cytokeratin staining (green) were detected by immunofluorescent staining in cutaneous tissues (scale bar = 20 μm). G The co-localization analyses of MAB1281 (green) and CD90 (red) were detected by immunofluorescent staining in cutaneous tissue (scale bars = 50 μm (10×) or 20 μm (40×)). H The peripheral blood leukocytes including WBC, Lymph, Mid and Gran were quantitated by the routine blood examination. I Serum IL-17 A, TNF-α and IL-10 were quantified by ELISA. J The relative mRNA expression of IL-17 A, TNF-α, IL-6 and CXCL15 was determined by qRT‑PCR in murine skin tissues. Data are shown as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus IMQ, n = 3–6

Additionally, by trancing the co-staining of human-specific MAB1281 (green fluorescence) and mesenchymal stem cell marker CD90 (red fluorescence), we observed that hUSCs could home to the lesional skin without differentiation, indicating that their therapeutic effects were achieved through their paracrine effects (Fig. 2G). Furthermore, hematological results showed that hUSCs remarkably suppressed IMQ-induced increases in WBC, Gran, Mid and Lymph in blood (Fig. 2H). ELISA analysis also revealed that hUSCs markedly reduced IMQ-induced the elevations of the pro-inflammatory factors such as IL-17 A, and TNF-α, and enhanced the production of the anti-inflammatory cytokine IL-10 in serum (Fig. 2I). Moreover, qRT-PCR analysis further confirmed that hUSCs significantly inhibited IMQ-induced the increased mRNA expression of IL-17 A, TNF-α, IL-6, and CXCL15 in skin tissues (Fig. 2J). Collectively, these results demonstrated that hUSCs effectively alleviated IMQ-induced psoriasis by suppressing pro-inflammatory cytokines and promoting anti-inflammatory factors in mouse psoriasis models.

hUSCs restrained psoriasis progression by inhibiting JAK2/STAT3-mediated M2-to-M1 macrophage polarization

To explore the underlying mechanism of hUSCs alleviating psoriasis, the effects of hUSCs on the maturation of macrophages and dendritic cells were examined. The results showed that hUSCs markedly reduced the ratio of F4/80⁺CD86⁺ and CD11c⁺CD86⁺ cells in mouse bone marrow without altering F4/80⁺CD206⁺ populations (Fig. 3A and Supplementary Fig. 3A, B), suggesting that hUSCs might exert immunomodulatory effects by preferentially regulating myeloid cell differentiation. In addition, the immunofluorescent data showed that hUSCs significantly downregulated the expression of CD86 and iNOS, and upregulated CD206 and IL-10 levels in lesional skin. This shift in the macrophage population provides direct evidence that hUSCs ameliorate the local immune microenvironment in tissues by suppressing M1 and promoting M2 polarization, thereby reducing inflammation and promoting tissue repair (Fig. 3B–E). Furthermore, our qPCR analysis further confirmed that hUSCs upregulated the expression of M2 macrophage markers such as CD206, Arg-1, and TGF-β, and downregulated the expression of M1 macrophage marker iNOS (Fig. 3F). Moreover, the western blot results showed that hUSCs significantly attenuated phosphorylation of JAK2 and STAT3 (Fig. 3G, H), indicating that the effect of hUSCs on macrophage polarization was involved in suppressing JAK2/STAT3 signaling pathway in macrophages.

Fig. 3.

hUSCs inhibited M1 macrophage and promoted M2 macrophage polarization through suppressing the JAK2/STAT3 signaling axis. A The F4/80⁺CD86⁺ cells were quantitatively analyzed by flow cytometry in bone marrow cells. B–E The expression of CD86 (B), CD206 (C), IL-10 (D) and iNOS (E) was determined by immunofluorescence staining in murine skin tissues (scale bar = 20 μm). F The relative mRNA expression of CD206, TGF-β, iNOS and Arg-1 was determined by qRT‑PCR in murine skin tissues. G, H The expression of p-JAK2, JAK2, p-STAT3 and STAT3 was detected by western blot assay in skin tissues. Data are shown as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus IMQ, n = 3–6

hUSCs and hUSC-CM ameliorated the TNF-α/LPS-induced inflammatory response and oxidative stress in HaCaT cells

Our animal studies have demonstrated that hUSCs exerted therapeutic effects primarily through paracrine. To further explore their mechanisms, the conditioned medium of hUSCs (hUSC-CM, 10 ×) was prepared to evaluate their role in psoriasis using HaCaT cells (Fig. 4A). Fluorescence analysis revealed that both hUSCs and hUSC-CM significantly inhibited TNF-a and LPS-induced the elevations of reactive oxygen species (ROS) (Fig. 4B, C) and the expression of the inflammatory cytokines such as TNF-α, IL-6, and IL-8 in HaCaT cells (Fig. 4D). Furthermore, hUSC-CM exhibited superior therapeutic potential by prominently upregulating anti-inflammatory TGF-β expression (Fig. 4D). Moreover, hUSC-CM not only suppressed IL-17 A and IL-8 expression, but also enhanced the expression of TGF-β in LPS-induced models (Fig. 4E). Taken together, these results indicated that hUSCs/hUSC-CM possessed the antioxidant and anti-inflammatory effects in HaCaT cells.

Fig. 4.

hUSCs and hUSC-CM revealed the comparable efficacy in suppressing LPS/TNF-α-triggered inflammatory response and oxidative damage. A Schematic grouping of LPS or TNF-α- induced HaCaT cells. HaCaT cells were co-treated with LPS (1 µg/mL) or TNF-α (20 ng/mL) and either hUSCs or hUSC-CM (10×) for 24 h. B, C TNF-α (B) or LPS (C)-induced ROS generations were determined by DCFH-DA in HaCaT cells under fluorescence microscope (scale bar = 100–300 μm), respectively. D, E The mRNA expression of TNF-α, IL-6, IL-8, TGF-β and IL-17 A was determined with qRT-PCR in HaCaT cells induced by TNF-α (D) or LPS (E). Data are shown as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus TNF-α or LPS, n = 3

hUSCs and hUSC-CM suppressed macrophage M1 polarization and promoted M2 polarization via inhibiting JAK2/STAT3 signaling pathway in vitro

To systematically investigate the immunomodulating properties of hUSCs and hUSC-CM in vitro, LPS was employed to induce the activation of BMDM, RAW264.7 and BMDC (Fig. 5A). Immunofluorescence and flow cytometry validated the lineage-specific markers: F4/80⁺ for BMDM and CD11c⁺ for BMDC, confirming successful in vitro differentiation (Supplementary Fig, 4 A, B). It has been reported that reducing intracellular ROS levels modulated macrophage polarization [29], whereas LPS stimulated BMDM and RAW264.7 to elevate oxidative stress. The results showed that hUSCs and hUSC-CM drastically lowered ROS level, reducing oxidative damage in BMDM (Fig. 5B). In addition, the results showed that both hUSCs and hUSC-CM prevented LPS-induced mitochondrial membrane potential loss (Supplementary Fig. 5 A) and suppressed lipid peroxidation (Supplementary Fig. 5B), suggesting that hUSCs might maintain mitochondrial function and protect membrane integrity. Furthermore, our flow cytometry results revealed that hUSCs and hUSC-CM markedly downregulated CD86 and upregulated CD206 in LPS-induced BMDM, respectively and BMDC (Fig. 5C and Supplementary Fig. 4 C). qRT-PCR and immunofluorescent results showed that hUSCs or hUSC-CM remarkably reduced LPS-induced the increases in iNOS, IL-6 and TNF-α expression, and augmented the expression of CD206 and Arg-1 in BMDM (Fig. 5D, E). ELISA results also showed that hUSCs or hUSC-CM decreased TNF-α and elevated IL-10 in the supernatant of LPS-induced BMDM (Fig. 5F). Moreover, our western blot results showed that hUSCs and hUSC-CM markedly inhibited JAK2/STAT3 pathways (Fig. 5G), whereas WP1066, a selective inhibitor of JAK2 and STAT3, remarkably reversed LPS-mediated macrophage polarization (Fig. 5H). Moreover, similar results were also observed in RAW264.7 (Supplementary Fig. 6). Taken together, our in vitro results demonstrated that hUSCs and hUSC-CM orchestrate macrophage polarization dynamics via selective suppression of the JAK2/STAT3 signaling axis.

Fig. 5.

hUSCs and hUSC-CM orchestrated BMDM polarization via inhibiting JAK2/STAT3 signaling pathway. A Schematic grouping of LPS-indued macrophages, in which BMDM were treated with LPS (1 µg/mL) and either hUSCs or hUSC-CM (10×) for 24 h. B The ROS levels labeled with DCFH-DA in BMDM were analyzed under fluorescence microscope (scale bar = 100 μm). C Relative fluorescent intensities of F4/80⁺CD86⁺ and F4/80⁺CD206⁺ were assessed by flow cytometry. D The relative mRNA expression of iNOS, IL-6, TNF-α, CD206 and Arg-1 was determined by qRT-PCR in LPS-induced BMDM. E The CD206 expression was evaluated by Immunofluorescence microscopy in LPS-stimulated BMDM (scale bar = 100 μm). F The releases of TNF-α and IL-10 were determined by ELISA with the supernatant of LPS-stimulated BMDM. G The expression of p-JAK2, JAK2, p-STAT3 and STAT3 was detected by western blot in BMDM. H The relative mRNA expression of iNOS, IL-6, TNF-α, CD206 and Arg-1 was determined by qRT‑PCR with BMDM treated with LPS (1 µg/mL) and either WP1066 (5 µM) or hUSC-CM (10×) for 24 h. Data are shown as means ± SEM, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus LPS, n = 3

hUSCs suppressed the formation of NETs through driving M2 macrophage polarization

Given the elevated NETs deposition in psoriatic lesions [30], we assessed NETs formation via immunofluorescence and western blot. Our data showed that hUSCs downregulated neutrophils markers Ly6G and NETs-associated markers such as Cit-H3 and MPO, suggesting that hUSCs might suppress the formation of NETs (Fig. 6A, B). Since ROS generation is indispensable for NETs formation [31], we first established an in vitro NETs induction model (Fig. 6C, D). Subsequent analyses identified that both hUSCs and hUSC-CM effectively attenuated ROS burst in neutrophil-like cells (Fig. 6E). Building upon the observation that hUSC-CM upregulated the expression of M2 phenotypic markers such as CD206 and Arg-1 in BMDM, we exposed BMDM to hUSC-CM and then co-cultured them with neutrophil-like cells (Fig. 6F, G). Strikingly, hUSC-CM intervention led to markedly downregulating the expression of MPO and Cit-H3 (Fig. 6H), indicating that hUSCs mediated immunomodulation by reprogramming macrophage toward a NETs-suppressive phenotype.

Fig. 6.

hUSCs-mediated M2 polarization reduced NETs formation. A The co-localization of Ly6G and CitH3 was determined by immunofluorescent analysis in cutaneous tissues. B The expression of MPO and Cit-H3 was detected by western blot analysis in skin tissues, ****p < 0.0001 versus IMQ. C Schematic illustration of neutrophilic-like cells and NETs induction. D The expression of CD11b was determined by qRT-PCR in neutrophilic-like cells, ***p < 0.001 versus Normal. E The DHE levels were analyzed by fluorescence microscope in neutrophilic-like cells treated with PMA (100 ng/mL) and either hUSCs or hUSC-CM (10×) for 4 h (scale bar = 100 μm). F The expression of CD206 and Arg-1 was determined by qRT-PCR in BMDM pretreated with hUSC-CM for 6 h, *p < 0.05 and ****p < 0.0001 versus Normal. G Schematic diagram of co-culture of BMDM pretreated with hUSC-CM and neutrophil-like cells. H The expression of MPO and Cit-H3 was detected by western blot analysis in BMDM cultured with neutrophilic-like cells, *p < 0.05 and **p < 0.01 versus PMA. Data are shown as means ± SEM, n = 3

TGF-β1 derived from hUSC-CM orchestrated LPS-stimulated macrophage polarization through suppression of JAK2/STAT3 signaling cascade

To identify the critical components in hUSC-CM, an antibody array for cytokines was analyzed. Aligning with our aforementioned results, KEGG enrichment analysis revealed that the active factors were mainly enriched in the JAK-STAT pathway (Fig. 7A), and the antibody array analysis showed that there was a high expression of TGF- β1 in hUSC-CM (Fig. 7B, C, Supplementary Table 3). It has been reported that TGF-β1 modulated the polarization of macrophage and the JAK/STAT signaling pathway [32, 33], suggesting that hUSCs-mediated macrophage polarization might be associated with TGF-β1-suppressing JAK/STAT signaling pathway in macrophages. Our western blot results showed that hUSC-CM significantly inhibited LPS-induced the elevations of p-JAK2 and p-STAT3 expression, and TGF-β1 also exhibited a comparable inhibitory effect, whereas LY2109761, an TGF-β receptor inhibitor, weakened the inhibitory effects of hUSC-CM in BMDM (Fig. 7D, E, H and I) or RAW264.7 (Fig. 7F, G, J and K). In addition, the qRT-PCR results showed that hUSC-CM and TGF-β1 remarkably reduced LPS-induced increases in iNOS, TNF-α, IL-6, and CXCL15 expression, and promoted the expression of CD206, Arg-1 and IL-10, whereas LY2109761 reversed these effects of hUSC-CM in BMDM (Supplementary Fig. 7A-D) or RAW264.7 (Supplementary Fig. 7E-H). These results indicated that hUSC-CM-derived TGF-β1 suppressed M1 macrophage polarization and facilitated the shift towards M2 polarization by inhibiting JAK2/STAT3 pathway.

Fig. 7.

hUSCs-derived TGF-β1 shifted the polarization of macrophages by inhibiting JAK2/STAT3 pathway in LPS-activated macrophages. A The expression of the differential genes was analyzed by KEGG, showing that most of them were enriched in the JAK/STAT signaling pathway. B The expression of the related cytokines was showed by the Heat map in hUSC-CM. C The expression of cytokines was determined by antibody array in hUSC-CM. D-G The expression of p-JAK2, JAK2, p-STAT3 and STAT3 was determined by western blot in BMDM (D, E) and RAW264.7 (F, G) treated with LPS (1 µg/mL) and either hUSC-CM (10×) or TGF-β1 (10 ng/mL) for 24 h. H-K The expression of p-JAK2, JAK2, p-STAT3 and STAT3 was determined by western blot in BMDM (H, I) and RAW264.7 (J, K) treated with LPS (1 µg/mL) and either hUSC-CM (10×) or LY2109761 (10 µM) + hUSC-CM (10×) for 24 h. Data are shown as means ± SEM, ****p < 0.0001 versus LPS, n = 3

Discussion

As the etiology and specific molecular mechanisms of psoriasis have not been fully elucidated, an increasing number of patients with severe psoriasis remain refractory to currently available treatments [34]. This highlights the urgent need for more advanced and potential therapeutic approaches. Recent studies indicated that MSCs derived from umbilical cord [15], adipose tissue [35], mouse bone marrow [17] and other sources significantly improved psoriasiform skin lesions. However, the clinical application of stem cells necessitates stringent requirements regarding safety, immunogenicity and ethical standards. In alignment with the prevailing trend toward defined culture systems that enhance batch-to-batch consistency and eliminate clinical risks of animal serum, this study expanded hUSCs in SFM [36, 37]. The resulting SFM-cultured hUSCs exhibited robust stemness, comparable proliferation and characteristics to serum-cultured cells, as well as practical advantages including simple preparation, abundant source, low cost, low immunogenicity, and no detected tumorigenicity. According to emerging evidence, hUSCs have reparative capacity across multiple pathological conditions, for instance, premature ovarian failure [23], diabetes and its complications [38], renal ischemia-reperfusion injury [39], articular cartilage repair [40] and diabetic wound healing [41]. Here, we presented novel evidence that hUSCs mitigated IMQ-induced psoriasiform dermatitis via paracrine activity (hUSC-CM), with mechanistic insights showing their lesional homing capacity coupled with dual suppression of epidermal hyperproliferation and cutaneous inflammation. Importantly, intravenous administration proved superior to subcutaneous injection, as it enables systemic dissemination of hUSCs to immune organs [23, 42] and distant skin lesions, resulting in broader immunomodulation and more effective suppression of both systemic inflammation and local pro-inflammatory signals, thus offering a more effective strategy for systemic psoriasis.

Existing studies have conclusively established macrophages as pivotal regulators in psoriasis pathogenesis leveraging their remarkable plasticity to orchestrate disease progression [43, 44]. The levels of the circulating monocytes were observed in psoriasis patients with displaying distinct M1-skewed polarization [45]. Integrated human and murine analyses revealed that there were the enriched M1-polarized macrophages (CD68⁺iNOS⁺) in psoriatic plaques, paralleling heightened Th1/Th17 cytokine production (IL-1α, IL-6, IL-23, TNF-α) [3, 46]. Noteworthily, these cells exhibited disease-specific functional differentiation, in which under IL-23 stimulation, certain subsets were able to co-produce TNF-α and IL-17 A/IL-22 [47]. Paradoxically, CD163⁺ M2-like populations also expand in skin lesions, concurrently secreting anti-inflammatory mediators and pathogenic IL-23/IL-12p40 [48], suggesting context-dependent functional plasticity. The depletion of these cell populations following TNF-α inhibitor therapy further confirmed that macrophages served as the central hub in the integration of the psoriasis inflammatory network [49, 50]. Additionally, neutrophils and NETs were accumulated in the blood and skin lesion, correlating with disease severity of the psoriatic patients [30, 51]. NETs and macrophages formed a complex of the inflammatory network [10]. On the one hand, M1 macrophages secreted cathepsin C, activating neutrophil p38/MAPK pathways and ROS production, thus inducing NETosis [11]. On the other hand, NETs-released components like LL37 activated macrophage TLR receptors, prompting the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, and IL-23, which intensified the inflammatory response [10]. Our results proved that hUSCs effectively decreased the proportion of F4/80⁺CD86⁺ cells in the bone marrow of psoriatic mice, while modulating macrophage polarization, as shown by significant downregulation of M1 macrophage markers (CD86, iNOS, TNF-α and IL-6) and upregulation of M2 macrophage phenotype markers (CD206, IL-10 and TGF-β) in skin lesions, BMDM, and RAW264.7 cells. In addition, we observed that hUSC-CM inhibited the formation of NETs by promoting the M2 polarization of macrophages. These results indicate that hUSCs mediated macrophage reprogramming serves as a key therapeutic approach for alleviating psoriatic inflammation.

Accumulating studies revealed that JAK-STAT pathway critically governs macrophage polarization, as evidenced by both foundational studies [52–54] and clinical observations in psoriasis at where JAK/STAT3 modulated cytokine expression in serum and skin lesions [55]. Compelling evidence demonstrated that aberrant activation of the JAK/STAT pathway drives macrophage polarization toward the pro-inflammatory M1 phenotype, which secretes copious inflammatory cytokines (TNF-α, IL-6, IL-1β), exacerbating psoriatic inflammation [52, 56]. Notably, JAK/STAT signaling concurrently amplifies the IL-23/Th17 axis, upregulating the key pathogenic cytokines such as IL-17/IL-22 to psoriasis pathogenesis [56]. Pharmacological inhibition of this pathway not only reduces M1 macrophage abundance but also promotes M2 polarization, thereby attenuating inflammatory cascades [57]. This mechanistic rationale underpins the clinical efficacy of JAK inhibitors (tofacitinib, ruxolitinib) [58, 59], which have emerged as promising therapeutic options in psoriasis trials. Our antibody array analysis of hUSC-CM revealed predominant cytokine enrichment in this signaling pathway. Functional studies confirmed hUSCs markedly inhibit JAK2/STAT3 activation in both skin tissues and macrophages, indicating their macrophage polarization-modulating capacity through this pathway, which positioned hUSCs as a promising biologic intervention.

TGF-β is a critical immunomodulatory factor. Some studies indicated that overexpression of TGF-β1 led to a severe psoriasis-like skin disease [60, 61]. However, it has been reported that adipose-derived mesenchymal stromal cells infusion attenuates psoriatic inflammation through elevating the secretion of TGF-β that directly curbs keratinocyte hyperproliferation [62, 63]. Of note, MSCs can secrete TGF-β, which drives macrophage polarization toward an M2-like phenotype, thereby alleviating hyperinflammation [32, 64]. In the detection of hUSC-CM’s cytokine profiling, we also found a high expression of TGF-β1. Consistent with the above, experimental validation confirms that recombinant TGF-β1 recapitulated hUSC-CM’s dual effects on macrophage polarization-potently inhibiting M1 while enhancing M2 phenotypes. This activity was specifically abrogated by LY2109761, providing direct evidence to confirm that TGF-β1 played a central role in the treatment of psoriasis, which was consistent with the observation that TGF-β significantly attenuated IL-6-induced JAK phosphorylation degradation, consequently reducing STAT3 tyrosine phosphorylation in epithelial cells [65, 66]. The therapeutic effects we observed are not attributable to systemic TGF-β1 overexpression. Rather, the paracrine delivery of TGF-β1 by hUSCs represents a more physiological, localized, and tightly controlled regulatory mechanism. Furthermore, TGF-β suppressed IL-6 signaling in articular chondrocytes through downregulating IL-6 receptor expression, ultimately inhibiting both STAT3 phosphorylation and nuclear translocation to maintain cartilage homeostasis [67]. To deeply investigate the role and the underlying mechanism of TGF-β1 in modulating macrophage polarization, we further demonstrated that TGF-β1 or hUSC-CM protected skin from psoriasis by selectively inhibiting JAK2/STAT3 signaling pathway, and in turn, inhibiting M1 and promoting M2 macrophage polarization in BMDM and RAW264.7. Obviously, our study further validated that hUSCs were an alternative approach for the treatment of psoriasis, and furthermore, TGF-β1 might be potential therapeutic target for psoriasis.

There are still several limitations in this study. First, given highly heterogeneous and plastic of macrophages, distinct subsets may differentially contribute to psoriasis pathogenesis. Beyond the classical M1/M2 types, intermediate or non-canonical macrophage subpopulations exist whose roles in psoriasis remain to be defined. Second, the IMQ-induced psoriasis model used here has relatively low specificity, and future studies should employ K5-STAT3C or JunB/c-Jun transgenic mouse models to precisely identify target genes through which hUSCs and their secreted TGF-β1 ameliorate psoriatic symptoms via macrophage polarization. Besides, the potential synergistic role between TGF-β1 and other cytokines in hUSC-CM requires further elucidation.

Conclusions

In summary, our data demonstrated that hUSCs ameliorated psoriatic lesions via paracrine effects, in which hUSCs-derived TGF-β1 suppressed JAK2/STAT3-mediated M1 macrophage polarization and enhanced M2 polarization (Fig. 8), suggesting that hUSCs may hold great promise for clinical adoption due to their easy preparation, low cost, feasibility, and excellent biosafety.

Fig. 8.

Mechanism of hUSCs alleviating IMQ-induced psoriasis in mice. hUSCs alleviated IMQ-induced psoriasis by suppressing the expression of skin inflammatory factors, promoted M2 and suppressed M1 polarization of macrophages, and in tern, inhibiting NETs production in the skin lesion sites. Mechanistically, hUSCs-derived TGF-β1 inhibitd the JAK2/STAT3 pathway to shift M1 to M2 macrophage polarization, mitigating IMQ-induced psoriasis

Abbreviations

hUSCs

Human urine-derived stem cells

IMQ

Imiquimod

NETs

Neutrophil extracellular traps

MSCs

Mesenchymal stem cells

PBS

Phosphate buffered saline

ITS-X

Insulin-transferrin-selenium ethanolamine

NEAA

Non-essential amino acids

EGF

Epidermal growth factor

SFM

Serum-free medium

SM

Serum medium

KSFM

Keratinocyte serum-free medium

H-DMEM

High Glucose-Dulbecco’s Modified Eagle Medium

DMEM/F12

Dulbecco’s Modified Eagle Medium/Nutrient Mixture

FBS

Fetal bovine serum

HL-60

Human acute promyelocytic leukemia-60

hUSC-CM

hUSCs-derived conditioned medium

BMDM

Bone marrow-derived macrophages

BMDC

Bone marrow-derived dendritic cells

BSA

Bovine serum albumin

i.v.

Intravenously

s.c.

Subcutaneously

PASI

Psoriasis Area and Severity Index

H&E

Hematoxylin and eosin

WBC

White blood cells

Lymph

Lymphocytes

Mid

Mid cells

Gran

Granulocytes

RBC

Red blood cell

HGB

Hemoglobin

MCH

Mean corpuscular hemoglobin

RDW-CV

Red blood cell distribution width-coefficient of variation

RDW-SD

Red blood cell distribution width-standard deviation

ELISA

Enzyme-linked immunosorbent assay

qRT‑PCR

Quantitative reverse transcription‑polymerase chain reaction

BCA

Bicinchoninic acid

PVDF

Polyvinylidene fluoride

ROS

Reactive oxygen species

Author contributions

H.B.X. and K.Y.D. supervised this study, with contributions to conceptualization, experimental design, data analysis/interpretation, and manuscript revision. Y.Q.Z. performed most of experimental work and data analysis. Q.M.H, H.C.G, and L.F.W performed animal experiments. D. T., X.Y.W and Z.H.Z. conducted cell experiments. Y.Q.Z. wrote the manuscript draft. All authors contributed to manuscript review and approved the final version.

Funding

Funding for this project was made possible through the National Key Research and Development Program of China (2022YFA1104300 to Hong-Bo Xin and Ke-Yu Deng), the National Natural Science Foundation of China (82470454, 82270302 to Hong-Bo Xin, 81970256 to Ke-Yu Deng) and the Jiangxi Provincial Department of Science and Technology, China (20252BAC240701 to Ling-Fang Wang).

Data availability

Relevant data described in this study can be obtained from the corresponding author following a reasonable request.

Declarations

Ethics approval and consent to participate

This research was performed in accordance with the ethical principles set forth in the Declaration of Helsinki (2013 revision), with written informed consent obtained from all participating patients for tissue donation. The animal experiments were performed in accordance with the principles outlined in the Basel Declaration. This study received dual institutional ethical approvals: human subject protocols were authorized by the Institutional Review Board of the First Affiliated Hospital of Nanchang University (Approval no. 2023CDYFYYLK08-015, “Aging and diabetic nephropathy’s influence on the biological characteristics and functions of human urine-derived stem cells”; August 11, 2023), while animal experimental procedures were ratified by the Animal Research Ethics Board of Nanchang University (Approval no. 20250508001, “The Potential and Molecular Mechanism of Human Urine-Derived Stem Cells in the Treatment of Psoriasis”; May 8, 2025) in strict compliance with the Guidelines for the Care and Use of Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.