Abstract

   Psoriasis is a chronic inflammatory dermatosis driven by excessive
   activation of the immune system. Recent studies have demonstrated the
   therapeutic potential of mesenchymal stem cell‐derived extracellular
   vesicles (MSC‐EVs) to psoriasis because of their immunomodulation
   functions. Yet, the outcome of MSC‐EVs alone is still unsatisfactory
   and the underlying mechanisms are also unclear. Here, it is uncovered
   that arginase1 (Arg1)/polyamine is overexpressed in psoriasis patients
   and murine, inducing the in‐situ accumulation of self‐antigens.
   Engineered nor@MSC‐EVs are fabricated by loading Arg1 inhibitor
   nor‐NOHA into MSC‐EVs for studying the therapeutic effect and mechanism
   of psoriasis. The nor@MSC‐EVs exhibited profound suppression of the
   NF‐κB signaling pathway by targeting Arg1/polyamine‐mediated DCs/Th17
   axis through scavenging self‐antigens, resulting in superior mitigation
   of skin lesions and modulation of local and systemic metabolic and
   immunological disorders compared to the MSC‐EVs and clinically used
   anti‐IL17A both in vitro and in vivo. Together, the results highlight a
   novel perspective for psoriasis therapy by nor@MSC‐EVs with broad
   clinical translational potential.

   Keywords: arginase1, mesenchymal stem cell‐derived extracellular
   vesicles, metabolic and immunological modulation, psoriasis therapy,
   self‐antigens
     __________________________________________________________________

   An engineered nor@MSC‐EVs are fabricated by encapsulating arginase1
   inhibitor nor‐NOHA into MSC‐EVs. The nor@MSC‐EVs exhibited profound
   suppression of the NF‐κB signaling pathway by targeting
   Arg1/polyamine‐mediated DCs/Th17 axis through scavenging self‐antigens,
   resulting in superior mitigation of skin lesions and modulation of
   local and systemic metabolic and immunological disorders.

   graphic file with name ADVS-12-2410067-g005.jpg

1. Introduction

   Psoriasis is a chronic inflammatory dermatosis characterized by
   erythematous plaques and silvery scales.^[ [52]^1 ^] It is primarily
   driven by excessive activation of both the innate and adaptive immune
   systems. Particularly, psoriasis‐specific self‐antigens presented by
   dendritic cells (DCs) initiate the activation and clonal expansion of
   antigen‐specific CD4+ T cells, which subsequently secrete excessive
   cytokines. This dysregulation of the IL23/Th17 axis ultimately leads to
   hyperproliferation and inflammation of the epidermis.^[ [53]^2 ^]

   Traditional therapeutic approaches, such as methotrexate and
   cyclosporine, have been utilized to manage psoriasis by inhibiting
   systemic immunity since the 1980s.^[ [54]^3 ^] However, their
   applications are hindered due to the associated risks, including
   infections and malignancies.^[ [55]^1 , [56]^4 ^] Recently developed
   bioagents targeting key cytokines involved in psoriasis pathogenesis,
   such as IL‐17A, and IL‐23, have shown efficacy in alleviating psoriatic
   symptoms.^[ [57]^5 ^] Despite their benefits, completely curing the
   disease by merely neutralizing cytokines from the overactive immune
   system remains challenging.^[ [58]^6 ^]

   Mesenchymal stem cells (MSCs) and their derived extracellular vesicles
   (EVs) have emerged as novel strategies for psoriasis treatment due to
   their remarkable immunomodulatory and anti‐inflammatory properties.^[
   [59]^7 ^] A phase 1/2a clinical trial reported that MSC treatment
   resulted in a 40% improvement in 47% of the patients.^[ [60]^7 , [61]^8
   ^] Similarly, recent studies have demonstrated that MSC‐EVs injections
   reduce skin inflammation in psoriasis mice model by regulating
   cytokines expression.^[ [62]^9 ^] Indeed, MSC‐EVs have shown
   significant promise in pre‐clinical studies of other autoimmune
   diseases,^[ [63]^10 ^] highlighting their potential in psoriasis
   treatment. Nevertheless, recent studies have also identified metabolic
   pathways as crucial contributors to the epithelial and immune
   mechanisms underlying psoriasis.^[ [64]^11 ^] Lou et al. revealed that
   the RNA+polyamine+Hnrnpa[1226‐237] complex^[ [65]^12 ^] is considered
   as a self‐antigen, with aberrant polyamine metabolism driven by the
   overexpression of arginase‐1 (Arg1) in psoriatic keratinocytes
   enhancing the sensing of this complex by DCs.^[ [66]^13 ^] Therefore,
   targeting metabolic and immunological disorders via eliminating
   psoriasis‐related self‐antigens could offer a synergistic strategy for
   psoriasis therapy.^[ [67]^14 ^]

   Here, we reported a novel engineered MSC‐EVs (nor@MSC‐EVs), which are
   loaded with Arg1 inhibitor (nor‐NOHA) through electroporation for the
   synergistic therapy of psoriasis via modulating both local and systemic
   metabolic and immunological disorders (Scheme [68]1 ). We initially
   investigated the specific overexpression of Arg1 in psoriasis by
   examining patient samples with varied scaly erythematous skin diseases.
   Upon intravenous administration in psoriasis mice, nor@MSC‐EVs
   outperformed MSC‐EVs, nor‐NOHA, and the clinical biological agent
   anti‐IL17A in decreasing polyamines, inhibiting DC maturation via
   reducing self‐antigen generation, Th1/17 cells activation, and
   keratinocyte inflammation, thereby modulating both local and systemic
   metabolic and immunological disorders. We believe this study provided a
   prospective translational practice for psoriasis treatment with
   nor@MSC‐EVs.

Scheme 1.

   Scheme 1
   [69]Open in a new tab

   Schematic illustration of nor@MSC‐EVs for synergistic therapy against
   psoriasis by modulating both local and systemic metabolic and
   immunological disorders.

2. Results

2.1. Arg1/Polyamine Mediated the Development of Psoriasis

   The key enzymes of amino acid metabolism pathways are pivotal
   checkpoints involved in regulating autoimmune disease. To investigate
   the association between Arg1 and psoriasis, Arg1 expression in skin
   sections from psoriasis patients with different severities was
   determined via immunohistochemical staining (IHC). Consistent with
   previous reports,^[ [70]^15 ^] we identified that Arg1 was
   overexpressed in the epidermis of psoriasis patients than healthy
   controls (Figure [71] 1A). Linear regression analysis demonstrated a
   positive correlation between Arg1 expression (H score) and with
   psoriasis area and severity index (PASI score) (Figure [72]1B),
   implying that Arg1 was involved in the psoriasis pathogenesis.
   Additionally, an immunohistochemical analysis of Arg1 on skin sections
   from patients with scaly erythematous skin diseases similar to
   psoriasis was conducted. Unlike psoriasis, conditions such as
   parapsoriasis, atopic dermatitis, lichen planus, pityriasis rosea, and
   neurodermatitis have shown negligible Arg1 expression despite epidermis
   hyperplasia (Figure [73]1C,D). In contrast, Arg1 overexpression was
   observed in lupus erythematosus, seborrheic dermatitis, and psoriasis
   due to the varying degrees of involvement in dysregulation of
   Th17/IL‐17A^[ [74]^13 , [75]^16 ^] (Figure [76]1C,D). For further
   validation, an in vitro psoriatic keratinocytes model was established
   by stimulating HaCaT cells with different cytokines and subsequently
   the expression of Arg1 was analyzed. It was found that only IL‐17A was
   able to drive Arg1 up‐regulation (Figure [77]1E) and increasing Arg1
   expression exhibited a concentration‐dependent manner of IL‐17A (Figure
   [78]S1, Supporting Information). It was known that Arg1 catalyzes
   arginine hydrolysis to direct products (urea and ornithine) and end
   products (polyamine). Thus, the level of plasma polyamine (putrescine,
   PUT and spermidine, SPD) in different groups was examined by
   High‐Performance Liquid Chromatography (HPLC). It revealed that the PUT
   and SPD were higher in plasma from psoriasis patients compared to
   atopic dermatitis patients, while no robust difference was observed
   between atopic dermatitis and healthy control (Figure [79]1F),
   demonstrating the overexpression of Arg1 was psoriasis‐specific and
   could lead to the perturbations of polyamine metabolism.

Figure 1.

   Figure 1
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   Arg1/polyamine mediated the development of psoriasis. A) IHC staining
   depicting Arg1 expression in skin sections from healthy controls and
   psoriasis patients. Scale bar = 200 µm. B) Linear regression analysis
   demonstrating the correlation between Arg1 expression levels (H score)
   and the severity of psoriasis (PASI score), n = 16. C) IHC staining
   depicting Arg1 expression in skin sections from patients with a range
   of erythematous scaly skin diseases. Scale bar = 200 µm. D)
   Quantification of Arg1 expression (H score) of different erythematous
   scaly skin diseases (n = 3 for the neurodermatitis group, n = 28 for
   psoriasis group, and n = 5 for other groups). E) Flow cytometry
   analysis of Arg1 expression in HaCaT cells treated with TNF‐α or IL‐17A
   in vitro (n = 3). F) Analysis of polyamine (putrescine and spermidine)
   levels in plasma from healthy controls (HC), atopic dermatitis (AD),
   and psoriasis (Pso) patients (n = 3 for HC, n = 6 for AD and Pso). G)
   and H) Linear regression analysis of the H score of Arg1 of mouse skin
   (IHC staining) with immunological markers of mouse skin (flow
   cytometry): MFI of CD80+ in DCs (G) and Th17% cells (H) (n = 9). I)
   Statistical analysis of CD80+CD86+ cells and MHC II+ cells in BMDCs (n
   = 3). J) Representative flow cytometry of CD80+CD86+ cells in BMDCs
   treated with indicated reagents. K) Statistical analysis and L)
   representative flow cytometry of the percentage of CD4+IL‐17A+ cells in
   co‐cultured naïve CD4+ T cells and immature/mature BMDCs (n = 3). M)
   Schematic diagram depicting the mechanism of Arg1/polyamine mediated
   development of psoriasis. All data are expressed as mean ± S.D. (n≥3).
   Linear regression analysis was performed (B, G, and H). Statistical
   significance was calculated via one‐way ANOVA with a Tukey's test (D‐F,
   and I) or unpaired two‐tailed t test (K). ns, not significant; *p <
   0.05, **p < 0.01, ***p < 0.001.

   We further verified the above findings in an imiquimod (IMQ)‐induced
   psoriasis mouse model. A linear regression analysis (Figure [81]1G,H)
   was performed for the skin samples between the expression of Arg1 by
   IHC (H score) and immunological markers (CD80 in DCs, and Th17 in CD4+
   T cells) via flow cytometry. Positive correlations were identified
   between CD80 (Figure [82]1G), Th17 (Figure [83]1H) and Arg1 H score.
   Together, these data demonstrated an inner link between Arg1 and innate
   immune cells (DCs) and adaptive immune cells (Th17), which might be
   IL‐17A secreted by DC/Th17A axis driving the Arg1 upregulation in
   psoriasis.^[ [84]^3 , [85]^13 , [86]^16 ^]

   Nevertheless, the effect of Arg1 and its metabolites (such as urea and
   polyamines) on immune cells of psoriasis was unclear. Previous studies
   have shown that the RNA from psoriatic keratinocytes complexed with
   peptide could activate monocyte‐derived DCs.^[ [87]^17 ^] The
   maturation of DCs subsequent activation of DCs/Th17 axis indicated
   there was a loop of metabolic and immunological disorders mediated by
   Arg1/polyamine. To this, we investigated the impact of Arg1/polyamine
   on the DCs/Th17 axis by assessing the maturation markers of bone marrow
   derived DCs (BMDCs). Stimulation with RNA+polyamine+peptide complex
   significantly increased the maturation markers of BMDCs compared to
   other single or combination stimulators (Figure [88]1I,J), emphasizing
   the role of this complex in immunological disorder modulation.
   Co‐culturing naïve CD4+ T cells with BMDCs pre‐treated with or without
   RNA+polyamine+peptide complex revealed that mature DCs (mDCs) promoted
   the differentiation of CD4 + IL17A + cells (Th17) (Figure [89]1K,L). In
   summary, the above results demonstrated the psoriasis‐specific
   overexpression of Arg1 and its contribution to the cyclic relationship
   between metabolic and immunological disorders mediated by
   Arg1/polyamine, as depicted in Figure [90]1M.

2.2. Reducing Psoriasis‐Specific Self‐Antigens Decreased the Maturation of
DCs and Activation of Th17 Cells

   Self‐antigens can bind to Toll‐like receptors on the surface of DCs in
   the dermis of patients with psoriasis, triggering DCs activation and
   subsequent clonal expansion of antigen‐specific CD4+ T cells.^[ [91]^3
   ^] To investigate the impact of decreasing self‐antigens on BMDCs
   maturation, we incubated BMDCs with RNA+low/mid/high polyamine+peptide
   before the assessment of the maturation markers (Figure [92] 2A).
   Stimulated by RNA+polyamine+peptide complex, robust elevations of
   maturation markers expression were observed with the increasing of
   polyamine concentration. Notably, the RNA+high polyamine+peptide
   complex exhibited the highest increase in MHC II expression
   (Figure [93]2B), and elevation of CD80 and CD86 (Figure [94]2B),
   compared to other lower polyamine concentration groups, demonstrating
   the reduced polyamine concentration correlated with diminished
   maturation state of BMDCs.

Figure 2.

   Figure 2
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   Reducing psoriasis‐specific self‐antigens decreased the maturation of
   DCs and activation of Th17 cells. A) Schematic diagram of co‐culture of
   naïve CD4+ T cells and BMDCs pre‐treated with psoriasis‐specific
   antigens (RNA+low/mid/high+polyamine+peptide complex). B) Flow
   cytometry analysis of MHC II and CD80+CD86+ expression in BMDCs treated
   with PBS, LPS, RNA+low/mid/high polyamine+peptide complex. C,D) Bright
   field microscope images (C) and schematic diagram (D) of co‐culture of
   naïve CD4+ T cells and BMDCs with different degrees of maturation. E)
   Flow cytometry analysis of IL17A+CD4+ T cells activated by BMDCs
   pre‐treated with RNA+low/mid/high polyamine+peptide complex. All data
   are expressed as mean ± S.D. (n = 3). Statistical significance was
   calculated via one‐way ANOVA with a Tukey's test. ns, not significant;
   *p < 0.05, **p < 0.01, ***p < 0.001.

   We then co‐cultured the above BMDCs and naïve CD4+ T cells for 72 h
   (Figure [96]2A). Microscope images revealed decreased interactions
   between CD4+ T cells and BMDCs due to the reduced MHC II expression on
   DCs, leading to diminished cell‐cell interaction (Figure [97]2C,D).^[
   [98]^18 ^] Flow cytometry analysis also demonstrated a decrease in
   IL17A+CD4+ T cells percentage when co‐cultured with BMDCs pre‐treated
   with RNA+low/mid polyamine+peptide complex (1.77% and 2.67%
   respectively) compared to the high polyamine group (3.36%)
   (Figure [99]2E), indicating that reducing polyamine concentration can
   attenuate DC‐T cell interactions and lead to lower Th17
   differentiation. Together, targeting Arg1‐mediated polyamine production
   provides a novel strategy to interfere the DCs/Th17 axis and may offer
   therapeutic benefits for psoriasis therapy.

2.3. Engineering and Characterizations of nor@MSC‐EVs

   To address the metabolic and immunological disorder of psoriasis, we
   developed MSC‐EVs loaded with nor‐NOHA, an Arg1 inhibitor. First, human
   umbilical cords MSCs were isolated and cultured, demonstrating
   fibroblast‐like morphology (Figure [100]S2A, Supporting Information).
   Their stemness was further verified by Oil Red O and Alizarin Red S
   staining after osteogenesis and adipogenesis differentiation (Figure
   [101]S2B, Supporting Information). Flow cytometry analysis validated
   the MSC identity by both the positive (CD73 and CD105) and negative
   markers (CD34 and CD45) (Figure [102]S2C, Supporting Information).

   Subsequently, MSC‐EVs were isolated and loaded with nor‐NOHA via
   electroporation to obtain the engineered MSC‐EVs (nor@MSC‐EVs) (Figure
   [103]S3, Supporting Information). Both the MSC‐EVs and nor@MSC‐EVs
   showed the double concave discs as typical shapes of EVs under
   transmission electron microscope (TEM) (Figure [104] 3A). Nanoflow
   analysis revealed a peak diameter of 60.2 nm for MSC‐EVs, and 67.7 nm
   for nor@MSC‐EVs (Figure [105]3B). Notably, the incorporation of
   nor‐NOHA, a positively charged small molecule, resulted in a
   significant zeta potential change, shifting from ‐27.8 mV of MSC‐EVs to
   ‐15.2 mV of nor@MSC‐EVs (Figure [106]3C), confirming the successful
   loading of nor‐NOHA into MSC‐EVs. We then quantified the unloaded
   nor‐NOHA via a ninhydrin reaction and calculated the encapsulation
   efficiency and drug loading efficiency, which were 52% and 34%,
   respectively (Table [107]S1, Supporting Information). Moreover, western
   blotting analysis revealed that both MSC‐EVs and nor@MSC‐EVs expressed
   the common EV biomarkers, such as CD9 and TSG101, while showing
   negative expression of calnexin (Figure [108]3D), further validating
   the successful purification and modification of MSC‐EVs. Stability
   assessments of MSC‐EVs and nor@MSC‐EVs in PBS at 4 °C in a 5‐day period
   showed no significant alteration in physicochemical properties
   (Figure [109]3E). Briefly, our results convincingly demonstrated the
   successful isolation of MSC‐EVs and the subsequent engineering of
   nor@MSC‐EVs.

Figure 3.

   Figure 3
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   Engineering and characterizations of nor@MSC‐EVs and its modulation
   effect of metabolic disorder and psoriatic phenotype in keratinocytes
   in vitro. A) TEM images of MSC‐EVs and nor@MSC‐EVs (scale bar = 200
   nm). B) Size of MSC‐EVs and nor@MSC‐EVs measured by nanoflow. C) Zeta
   potentials of MSC‐EVs and nor@MSC‐EVs measured by DLS. D) Expression of
   the marker proteins in the MSC, MSC‐EVs, and nor@MSC‐EVs analyzed by
   western blotting. E) size and zeta potential changes of MSC‐EVs and
   nor@MSC‐EVs in PBS, 4 °C analyzed via DLS. F) Schematic diagram of the
   NF‐κB signaling pathway activated by TNF‐α and IL‐17A in HaCaT and the
   inhibitory effect of nor@MSC‐EVs. G) Western blotting analysis of p‐p65
   and p65 (the ratio of p‐p65/p65 indicated the activation degree of the
   NF‐κB signaling pathway) in HaCaT treated with different groups for 12
   h, following TNF‐α and IL‐17A stimulation for another 2 h. H) Schematic
   diagram illustrating the induction of the HaCaT psoriatic phenotype. I)
   and J) Flow cytometry analysis depicting Arg1 expression in psoriatic
   HaCaT following treatment with nor@MSC‐EVs (n = 3). K) and L) HPLC
   analysis of the concentration of intracellular PUT (K) and SPD (L) in
   psoriatic HaCaT treated with nor@MSC‐EVs for 12 h (n = 3). M) and N)
   Flow cytometry analysis illustrating the cell cycle of psoriatic HaCaT
   treated with nor@MSC‐EVs (n = 3). O‐R) qPCR evaluated the effect of
   nor@MSC‐EVs on the psoriatic HaCaT, including proliferation (O),
   antimicrobial peptides (P and Q), and chemokines (R). All data are
   expressed as mean ± S.D. (n = 3). Statistical significance was
   calculated via one‐way ANOVA with a Tukey's test. ns, not significant;
   *p < 0.05, **p < 0.01, ***p < 0.001.

2.4. Nor@MSC‐EVs Alleviated NF‐κB Signaling Pathway‐Mediated Arg1/Polyamine
Metabolic Disorder and Psoriatic Phenotype of Keratinocytes In Vitro

   The combination of TNF‐α and IL‐17A has a pivotal role in the
   inflammatory cascade of psoriatic keratinocytes,^[ [111]^19 ^] where
   they activate the NF‐κB signaling pathway, leading to the
   overexpression of downstream genes, including Arg1 and
   psoriasis‐related genes. It results in dysregulation of polyamine
   metabolism and manifests the psoriatic phenotype of keratinocytes^[
   [112]^13 , [113]^20 ^] (Figure [114]3F). Therefore, this cytokine
   combination has often been employed to establish an in vitro model of
   psoriatic keratinocytes.^[ [115]^21 ^] HaCaTs were first incubated with
   TNF‐α and IL‐17A to trigger NF‐κB activation, then they were treated
   with PBS, nor‐NOHA, MSC‐EVs, and nor@MSC‐EVs. Western blotting analysis
   demonstrated a noteworthy increase in the p‐p65/p65 of untreated group
   compared to PBS group. Also, significant reduction in the p‐p65/p65 in
   both the MSC‐EVs group and the nor@MSC‐EVs group was observed compared
   to the PBS group and nor‐NOHA group, underlying the inhibitory effect
   of both MSC‐EVs and nor@MSC‐EVs on NF‐κB activation (Figure [116]3G).
   Further, this attenuation of NF‐κB might contribute to the decrease in
   Arg1‐mediated polyamine production and expression of genes associated
   with psoriatic phenotype (Figure [117]3F).

   To validate the influence of nor@MSC‐EVs on Arg1‐mediated polyamine
   synthesis and psoriasis‐related gene expression, psoriatic HaCaT cells
   were incubated with different treatments before comprehensive analysis
   (Figure [118]3H). Flow cytometry results demonstrated both MSC‐EVs and
   nor@MSC‐EVs effectively suppressed the expression of Arg1 compared to
   other groups (Figure [119]3I,J). No evident difference was observed
   between the PBS group and the nor‐NOHA group, suggesting that nor‐NOHA
   alone cannot inhibit Arg1 overexpression. HPLC analysis further
   validated the decrease of intracellular polyamine levels in the
   nor‐NOHA group and MSC‐EVs group (Figure [120]3K,L). Notably, both PUT
   and SPD were clearly reduced in the nor@MSC‐EVs group, while it was not
   the same case for nor‐NOHA and MSC‐EVs groups, suggesting that
   nor@MSC‐EVs exhibited the most potent inhibition of both the function
   and expression of Arg1 as illustrated in Figure [121]3F.

   Keratinocytes, as the targets of inflammatory cytokine cascade
   triggered by the over‐activated immune system, were marked by aberrant
   proliferation and significantly elevated levels of antimicrobial
   peptides, chemokines, cytokines, and inflammatory proteins^[ [122]^22
   ^] (Figure [123]3H). To test the impact of nor@MSC‐EVs on the
   hyperproliferation of keratinocytes, a cell cycle analysis of the S
   phase was conducted.^[ [124]^20 ^] Our results showed an evident
   reduction in the percentage of cells in the S phase, from 35.8% in the
   PBS group to 30.7% in the nor@MSC‐EVs group, indicating that
   nor@MSC‐EVs effectively restricted the progression from G1 to S phase
   (Figure [125]3M,N). Additionally, qPCR results demonstrated an
   upregulation of the PPP6C gene, known to negatively regulate the G1‐S
   phase transition, increasing from 0.6‐fold in the PBS group to 1.0‐fold
   in the nor@MSC‐EVs group (Figure [126]3O), validating the observed
   decrease in S phase percentage. Together, these findings indicated that
   nor@MSC‐EVs inhibited keratinocyte hyperproliferation, offering
   potential therapeutic implications for psoriasis. Antimicrobial
   peptides produced by keratinocytes, such as S100A8 and S100A9,
   modulated psoriatic skin inflammation by regulating the expression of
   complement factor C3.^[ [127]^23 ^] A detailed analysis using qPCR
   revealed a robust decrease in the expression of both S100A8 and S100A9
   in the nor@MSC‐EVs group (Figure [128]3P,Q). Psoriatic keratinocytes
   can secrete chemokines like CCL2 to recruit innate immune cells,
   including CCR2+ DCs and macrophages to the dermis.^[ [129]^24 ^] In
   this study, CCL2 expression decreased to almost 50% in the nor@MSC‐EVs
   group than the PBS group (Figure [130]3R). Additionally, inflammatory
   cytokines, such as TNF‐α and IL‐1β, secreted by psoriatic keratinocytes
   were known to exacerbate skin inflammation.^[ [131]^22 ^] Our results
   demonstrated a significant downregulation of TNF and IL1B genes in the
   nor@MSC‐EVs group than the PBS group (Figure [132]S4A,B, Supporting
   Information). We also assessed the expression of inflammatory proteins
   CAMP and CEBP/β, which contributed to the pathogenesis of psoriasis by
   amplifying inflammatory signaling pathways in keratinocytes.^[ [133]^13
   , [134]^21 ^] The gene expression of both CAMP and CEBPB markedly
   decreased to 0.7‐fold of CAMP (Figure [135]S4C, Supporting Information)
   and 1.2‐fold of CEBPB (Figure [136]S4D, Supporting Information) in the
   nor@MSC‐EVs group compared to 1.6‐fold and 1.4‐fold in the PBS group.
   Collectively, these findings provided compelling evidence that
   nor@MSC‐EVs mitigated the psoriatic phenotype by inhibiting the
   expression of associated antimicrobial peptides, chemokines, cytokines,
   and inflammatory proteins, as well as those related to polyamine
   production and cell proliferation.

2.5. Nor@MSC‐EVs Directly Modulated Immunological Disorder In Vitro

   In addition to modulating polyamine metabolic disorder by scavenging
   self‐antigen to reduce BMDCs maturation, previous studies have also
   reported that MSC‐EVs can directly suppress BMDCs maturation.^[
   [137]^25 ^] To this, BMDCs were stimulated with RNA+polyamine+peptide
   complex, then incubated with PBS, nor‐NOHA, MSC‐EVs, or nor@MSC‐EVs
   (Figure [138] 4A). Maturation markers of BMDCs were assessed via flow
   cytometry (gating strategy was shown in Figure [139]S5, Supporting
   Information). Upon stimulation, a robust increase of MHC II was found
   in PBS group compared to the blank control, suggesting the successful
   induction of BMDCs maturation. The expression of MHC II decreased from
   55.2% in PBS group to 44.2% in MSC‐EVs and 37.2% in nor@MSC‐EVs
   (Figure [140]4B). Additionally, co‐stimulatory molecules CD80 and CD86
   were also significantly reduced after the treatment of MSC‐EVs and
   nor@MSC‐EVs compared to the PBS group (Figure [141]4C; Figure [142]S6,
   Supporting Information). Nor‐NOHA alone had negligible impact on BMDCs
   maturation compared to the PBS group.

Figure 4.

   Figure 4
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   Nor@MSC‐EVs directly modulated immunological disorder in vitro. A)
   Schematic diagram of isolation, culture, stimulation, and treatment of
   BMDCs. B) Flow cytometry analysis of the expression MHC II+ in BMDCs
   treated with different groups. C) Flow cytometry analysis of the
   expression of CD80+CD86+ in BMDCs treated by different groups. D)
   Schematic diagram of the isolation, culture, treatment of splenic cells
   from psoriasis mice. E) and F) Flow cytometry analysis of the
   percentage of IL‐17A+CD4+ cells (E) and IFN‐γ+ cells (F) treated with
   different groups. All data are expressed as mean ± S.D. (n = 3).
   Statistical significance was calculated via one‐way ANOVA with a
   Tukey's test. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

   The pathogenesis of psoriasis is distinctively characterized by the
   aberrant activation of the adaptive immune system, including Th1 and
   Th17 cells, culminating in an inflammatory cascade that leads to
   hyperproliferation and erythema of the epidermis.^[ [144]^3 ^] To this,
   splenic cells isolated from IMQ‐induced psoriasis mice were incubated
   with nor‐NOHA, MSC‐EVs, or nor@MSC‐EVs in the presence of anti‐CD3/28
   antibody (Figure [145]4D). It revealed that nor@MSC‐EVs significantly
   decreased the percentage of IL‐17A+CD4+ T cells (Th17) from 7.1% to
   2.8% compared to the untreated control (Figure [146]4E, gating strategy
   was shown in Figure [147]S7, Supporting Information). Besides, the
   percentage of IFNγ+CD4+ T cells (Th1 cells) notably reduced in both
   MSC‐EVs and nor@MSC‐EVs groups, from 8.5% to 4.0% and 3.9% respectively
   (Figure [148]4F). Conversely, nor‐NOHA alone had negligible effects on
   the differentiation of these T help cells. Overall, these findings
   supported that MSC‐EVs and nor@MSC‐EVs can directly inhibit the
   maturation of BMDCs and the differentiation of Th1 and Th17 in vitro,
   exerting direct immunomodulatory effects. The engineering process of
   MSC‐EVs didn't alter their immunomodulatory capability.

2.6. In Vivo Bioidistribution and Therapy Study of nor@MSC‐EVs in IMQ‐Induced
Psoriasis Mice

   To evaluate the distribution of nor@MSC‐EVs, we first use DiR dye to
   label nor@MSC‐EVs. In vivo imaging system (IVIS) was then applied to
   visualize the distribution of DiR‐nor@MSC‐EVs in both normal and
   IMQ‐induced psoriasis mice. Consistent with previous reports,^[ [149]^9
   ^] we observed that DiR‐nor@MSC‐EVs accumulated in the lesional skins
   of psoriasis mice and rarely appeared in healthy skins in normal mice
   (Figure [150]S8, Supporting Information). Compared with free DiR, the
   DiR‐nor@MSC‐EVs showed 2.3‐fold accumulation in the lesional skin of
   psoriasis mice (Figure [151]S9B,C, Supporting Information), indicating
   the targeted accumulation of nor@MSC‐EVs at the inflammation site. To
   further investigate whether nor@MSC‐EVs could be taken up by
   keratinocytes in psoriasis mice, we collected the lesional skin and
   performed immunofluorescence analysis 48 h after tail vein injection of
   free Dil and Dil‐nor@MSC‐EVs (Figure [152]S9A, Supporting Information).
   The Dil‐nor@MSC‐EVs (pink) were found in the epidermis, especially in
   pan‐keratin‐positive keratinocytes (green) (Figure [153]S9D,E,
   Supporting Information).

   To assess the in vivo therapeutic efficacy of nor@MSC‐EVs, IMQ‐induced
   mice were administered with PBS, nor‐NOHA, MSC‐EVs, nor@MSC‐EVs, or
   anti‐IL17A (a clinically used bioagent for psoriasis therapy)
   intravenously on day 1, 3, 5^[ [154]^26 ^] (Figure [155] 5A). The
   biosafety was first assessed. Histological analysis of heart, liver,
   spleen, lung, and kidney tissues stained with H&E showed no signs of
   tissue or organ damage in all groups (Figure [156]S10A, Supporting
   Information). To further evaluate hepatotoxicity and nephrotoxicity, we
   assessed serum levels of alanine transaminase (ALT), aspartate
   transaminase (AST), urea, and creatinine (CRE) (Figure [157]S10B–E,
   Supporting Information). These markers in the nor@MSC‐EVs groups were
   close to those in the untreated group, indicating excellent biosafety
   and biocompatibility of nor@MSC‐EVs in psoriasis mice. The mouse body
   weight change, clinical score, spleen index, H&E‐staining, and
   Ki67‐staining were evaluated pre‐and post‐treatment (Figure [158]5A).

Figure 5.

   Figure 5
   [159]Open in a new tab

   In vivo therapy study of nor@MSC‐EVs in IMQ‐induced psoriasis mice. A)
   Experimental design for therapy study in IMQ‐induced psoriasis mouse
   model. B) Body weight change (%) during the treatment (n = 4). C)
   Representative images of mice dorsal skins from day 1 to 6. D‐F)
   Corresponding clinical scores including erythema (D), scaling (E), and
   total (PASI) (F) measured from days 1 to 6 (n = 4). G) Spleen index of
   different groups after treatment (n = 4). H) Analysis of epidermis
   thickness in H&E‐stained sections. Scale bar = 200 µm (n = 4). I)
   Immunostaining of Ki67 in skin lesions and quantification of Ki67+
   cells in epidermis. Scale bar = 200 µm (n = 3). All data are expressed
   as mean ± S.D. (n≥3). Statistical significance was calculated via
   one‐way ANOVA with a Tukey's test. ns, not significant; *p < 0.05, **p
   < 0.01, ***p < 0.001.

   No evident body weight changes were observed among the PBS, nor‐NOHA,
   MSC‐EVs, nor@MSC‐EVs, and anti‐IL17A groups after topical application
   of IMQ cream for 6 days (Figure [160]5B). The MSC‐EVs, nor@MSC‐EVs, and
   anti‐IL17A groups displayed notable improvements in skin lesions
   compared to the PBS and nor‐NOHA groups (Figure [161]5C), characterized
   by reduced redness (Figure [162]5D) and scaling (Figure [163]5E) as
   well as lower PASI scores (Figure [164]5F),^[ [165]^27 ^] suggesting a
   potential alleviation in the hyperproliferation and inflammation of
   keratinocytes. Spleen enlargement, indicating immune cell clonal
   expansion, served as a sign of systemic immune response.^[ [166]^26 ^]
   Although the spleen index (spleen weight/body weight) trended downward
   in the nor‐NOHA, MSC‐EVs, nor@MSC‐EVs, and anti‐IL17A groups, only the
   nor@MSC‐EVs group exhibited a statistically significant reduction (P <
   0.05) compared to the PBS group (Figure [167]5G), highlighting the
   effective inhibition of systemic inflammation by nor@MSC‐EVs.

   Histological analysis revealed a marked increase in average epidermis
   thickness of 147.2 µm in the PBS group than 12.9 µm in the control
   group (Figure [168]5H). Post‐treatment, all groups showed significant
   reduction in epidermis thickness, with nor@MSC‐EVs group (81.0 µm) and
   anti‐IL17A group (79.6 µm) displaying the most potent suppression of
   epidermis hyperplasia. Furthermore, quantification of Ki67+ cells in
   the epidermis, a marker of cell proliferation, revealed a substantial
   decrease in the MSC‐EVs and nor@MSC‐EVs groups compared to the PBS
   group, while no statistical difference was found among other groups
   (Figure [169]5I), suggesting that the hyperproliferation of basal
   keratinocytes was significantly attenuated in the presence of MSC‐EVs
   or nor@MSC‐EVs. Collectively, a comprehensive evaluation including
   clinical scores, spleen index, and histological analysis indicated that
   nor@MSC‐EVs were the most efficacious therapeutic option for mitigating
   psoriasis symptoms in the IMQ‐induced mouse model.

2.7. Nor@MSC‐EVs Suppressed NF‐κB and Cytokines Signaling Pathway by Bulk
RNA‐Sequencing Analysis

   To elucidate the mechanisms underlying the therapeutic effects of
   nor@MSC‐EVs in a psoriasis mouse model, bulk RNA sequencing was
   conducted on skin lesions from psoriasis mice treated with PBS,
   nor@MSC‐EVs, and anti‐IL17A (Figure [170] 6A). Principal components
   analysis (PCA) revealed distinct gene expression patterns among the
   groups (Figure [171]6B). In total, we identified 5378 significantly
   differentially expressed genes (DEGs), including 2193 upregulated and
   3185 downregulated genes in the nor@MSC‐EVs group compared to the PBS
   group (Figure [172]6C). Among the top 10 downregulated and top 10
   upregulated DEG (Figure [173]S11, Supporting Information), we
   identified several key genes related to psoriasis, including Krt25,
   Krt8, Muc16, Slc5a7, Reg3b. Krt25 and Krt8, which are critical
   structural proteins in the epidermis, essential for the proliferation
   and keratosis of keratinocytes.^[ [174]^28 ^] Their downregulation
   indicated that nor@MSC‐EVs treatment normalized epidermal thickening
   and keratosis, aligning with histological observations
   (Figure [175]5H,I). Epidermis barrier depends on the proper progression
   of the keratosis of keratinocytes. Further analysis of epidermis
   barrier function revealed that Muc16,^[ [176]^29 ^] which encodes an
   epithelial protein marked as compromised barrier function in psoriasis,
   was significantly downregulated following nor@MSC‐EVs treatment
   compared to PBS. Patients suffering from guttate psoriasis have shown
   positive responses to anti‐cholinergic treatment, which inhibits mast
   cell degranulation triggered by acetylcholine (ACh). Slc5a7, involved
   in the synthesis and transportation of ACh,^[ [177]^30 ^] was
   downregulated after nor@MSC‐EVs treatment, suggesting nor@MSC‐EVs might
   inhibit mast cell degranulation by reducing Slc5a7‐mediated ACh
   synthesis. Moreover, Reg3b, which promotes and sustains STAT3
   activation in a self‐amplifying feed‐forward loop,^[ [178]^31 ^] was
   downregulated following nor@MSC‐EVs treatment compared to the control.
   It suggested that nor@MSC‐EVs may inhibit STAT3‐mediated Th17
   activation,^[ [179]^32 ^] offering a reasonable explanation for the
   observed reduction in splenomegaly of psoriasis mice (Figure [180]5G)
   and further hint at investigating the immunological modulation
   mechanism of nor@MSC‐EVs treatment for psoriasis.

Figure 6.

   Figure 6
   [181]Open in a new tab

   Treatment with nor@MSC‐EVs resulted in transcriptomic alterations and
   suppression of NF‐κB and cytokines signaling pathway in psoriasis mice
   skin. A) Experimental schedule for analyzing the transcriptome of
   psoriasis mouse skin. B) PCA of genes in PBS, nor@MSC‐EVs, and
   anti‐IL17A groups, n = 3. C) Volcano plot of DEGs in nor@MSC‐EVs and
   PBS groups (P<0.05 and |log2(fold change) |>1). D) and E) KEGG pathway
   enrichment analysis by DEGs in environmental information processing (D)
   and organismal systems (E), orb size indicates the number of genes
   enriched in the pathway. F) Heatmap of DEGs involved in NF‐κB signaling
   pathway, TNF‐α signaling pathway, IL‐17 signaling pathway, and
   cytokine‐cytokine receptor interactions.

   To gain deeper insights, we subjected the DEGs between the nor@MSC‐EVs
   and PBS groups to Kyoto Encyclopedia of Genes and Genomes (KEGG)
   annotation analysis. This revealed an enrichment of the top 10
   signaling pathways in environmental information processing
   (Figure [182]6D) and organismal systems (Figure [183]6E). It showed
   that the significant enrichment of DEGs in pathways was associated with
   the NF‐κB, TNF‐α, IL‐17 signaling pathways and cytokine‐cytokine
   receptor interactions (Figure [184]6D,E). Upstream genes of the NF‐κB
   signaling pathway, including Cxcl12, Lbp, Atm, Tlr4, Syk, Llr1, Tlrap,
   and Ticam2,^[ [185]^33 ^] as illustrated in Figure [186]6F, were
   markedly downregulated following treatment with nor@MSC‐EVs and
   anti‐IL‐17A. This indicated a reduced activation of the NF‐κB signaling
   pathway which further inhibited the synthesis of Arg1 and the psoriatic
   phenotype of keratinocytes, as shown in Figure [187]3F. Additionally,
   both TNF‐α and IL‐17 signaling pathways are crucial in the inflammatory
   processes of psoriasis, leading to hyperinflammation in keratinocytes
   and attracting additional immune cells to the skin. The heatmap
   demonstrated a predominant downregulation of genes associated with
   these pathways in response to nor@MSC‐EVs and anti‐IL‐17A treatment
   (Figure [188]6F), suggesting a suppressive effect on TNF‐α and IL‐17A
   signaling and cytokine processes. These findings offer insights into
   the therapeutic mechanisms of nor@MSC‐EVs, partially explaining their
   efficacy in reducing erythema and scaling of psoriatic skin
   (Figure [189]5C–F) and the infiltration of immune cells observed in H&E
   staining from psoriasis mice (Figure [190]5H). Subsequently, we
   verified the expression of DEGs in NF‐κB (Figure [191]S12, Supporting
   Information), IL‐17 (Figure [192]S13, Supporting Information), and TNF
   (Figure [193]S14, Supporting Information) signaling pathways, as well
   as cytokine‐cytokine receptor interaction (Figure [194]S15, Supporting
   Information) by RT‐QPCR. The expression trends of most differentially
   expressed genes were consistent with the bulk RNA‐seq, although some
   genes didn't show statistic difference.

2.8. Decreased Polyamine Levels in Epidermis and Plasma Following nor@MSC‐EVs
Treatment

   To investigate the local and systemic metabolic alterations of
   psoriasis mice, we conducted a comprehensive analysis, including
   non‐targeted metabolomics of the epidermis, IHC of the whole skin, and
   HPLC analysis of the plasma samples (Figure [195] 7A).

Figure 7.

   Figure 7
   [196]Open in a new tab

   Decreased polyamine levels in epidermis and plasma following
   nor@MSC‐EVs treatment. A) Experimental schedule for analyzing local and
   systemic metabolites in the epidermis and plasma of IMQ‐induced
   psoriasis mice. B) PCA illustrating global sample distribution profiles
   and relationships among the PBS, nor@MSC‐EVs, and anti‐IL17A groups. n
   = 3. C) Volcano plot representing differentially regulated metabolites
   (DRMs) for the nor@MSC‐EVs group compared to the PBS group, with
   statistical significance defined as P < 0.05, |Log2(fold change)| > 2,
   and VIP > 1. D) Top 20 enriched metabolic pathways identified through
   KEGG annotation of DRMs between the nor@MSC‐EVs and PBS groups, the
   number of differential metabolites contained in each pathway was
   indicated by the size of the circle, and significance was indicated by
   the different color. E) Diagram illustrating arginine biosynthesis,
   downstream polyamine metabolism, and the influence on immune
   homeostasis. F) Volcano plot of DRMs of amino acids and their
   metabolites for nor@MSC‐EVs compared to PBS, with statistical
   significance defined as P < 0.05, |Log2(fold change)| > 2, and VIP > 1.
   G) IHC of Arg1 in the epidermis from IMQ‐induced psoriasis mice. Scale
   bar, 200 µm. n = 3. H‐K) Results from LC‐MS/MS and HPLC showing the
   fold change in local (epidermis, n = 3) and systemic (plasma, n = 4)
   polyamines (PUT and SPD). L) and M) Linear regression analysis
   demonstrating the correlation between Arg1 expression levels (Arg1 mRNA
   relative expression and Arg1 H score) and the PUT level. N) and O)
   Linear regression analysis demonstrating the correlation between Arg1
   expression levels (Arg1 mRNA relative expression and Arg1 H score) and
   the SPD level. All data are expressed as mean ± S.D. (n ≥ 3). Linear
   regression analysis was performed (L‐O). Statistical significance was
   calculated via one‐way ANOVA with a Tukey's test (H‐K). ns, not
   significant; *p < 0.05, **p < 0.01, ***p < 0.001.

   Metabolite profiling is an effective approach to uncover
   genotype–phenotype interactions and their underlying mechanisms.^[
   [197]^34 ^] PCA clearly distinguished the classifications between the
   PBS, nor@MSC‐EVs, and anti‐IL17A groups (Figure [198]7B). A subsequent
   volcano plot analysis revealed significantly differential regulated
   metabolites (DRMs) in the nor@MSC‐EVs group, with 37 metabolites
   upregulated and 1456 downregulated compared to the PBS group
   (Figure [199]7C), demonstrating the profound influence of nor@MSC‐EVs
   on the epidermis metabolites in psoriasis mice. Subsequently, KEGG
   enrichment analysis was employed to display the enrichment of
   differential metabolites in metabolic pathways (Figure [200]7D).
   Notably, DRMs were significantly enriched in the D‐amino acid
   metabolism, central carbon metabolism, arginine biosynthesis pathway,
   etc. A differential abundance score (DA score) analysis showed low DA
   scores (less than 0) among the top 20 enriched metabolic pathways,
   indicating downregulation of these pathways, particularly the arginine
   biosynthesis pathway, in the nor@MSC‐EVs group (Figure [201]S16,
   Supporting Information). Integrated transcriptome and metabolome
   analysis revealed significant alterations in multiple biochemical
   pathways, particularly in arginine, proline metabolism, and arginine
   biosynthesis (Figure [202]S17, Supporting Information), highlighting
   consistent changes in arginine metabolism, encompassing both the
   transcription of key metabolic pathways and their associated
   metabolites. To gain a deeper understanding, we mapped the arginine
   metabolism pathways, highlighting the key metabolites involved,
   particularly the substrates (argininosuccinate and arginine) and
   products (ornithine and citrulline) of Arg1 (Figure [203]7E). Volcano
   plot analysis confirmed the downregulation of these substrates and
   products in the nor@MSC‐EVs group compared to the PBS group
   (Figure [204]7F), with statistical significance defined as P<0.05,
   |Log2(fold change)|>2, and VIP (Variable Importance in Projection) >1.
   We further investigated the expression of Arg1 in the epidermis of
   psoriasis mice by IHC. The results showed a significant decrease in the
   H score of Arg1 in both the MSC‐EVs and nor@MSC‐EVs groups compared to
   the PBS group. Additionally, a decreasing trend was also observed in
   the nor‐NOHA and anti‐IL17A groups (Figure [205]7G; Figure [206]S18,
   Supporting Information). These findings suggested that nor@MSC‐EVs
   effectively inhibited Arg1 expression in the epidermis of psoriasis
   mice, further validating their inhibitory effects observed in vitro
   (Figure [207]3I,J).

   Arg1‐mediated polyamine production in keratinocytes contributed to the
   formation of self‐antigens, which disrupted immune homeostasis by
   activating the DCs (Figure [208]7E). To evaluate the impact of
   nor@MSC‐EVs on both local and systemic polyamine metabolism, we
   quantified PUT and SPD levels in the epidermis and the plasma. Notably,
   following the treatment with nor@MSC‐EVs and anti‐IL17A, a significant
   reduction in PUT and SPD levels was observed in the epidermis
   (Figure [209]7H,I). The application of nor@MSC‐EVs further resulted in
   a substantial decline in plasma PUT and SPD levels, decreasing to
   1.8‐fold and 0.9‐fold, respectively, compared to 3.9‐fold and 2.1‐fold
   observed in the PBS group (Figure [210]7J,K). To confirm whether the
   decrease in polyamines was associated with the reduced expression of
   Arg1 in the epidermis under the treatment with nor@MSC‐EVs, we
   performed a correlation analysis integrating metabolome data (PUT and
   SPD levels), transcriptome data (Arg1 mRNA expression), and IHC results
   (H score of Arg1). The transcriptome analysis revealed a greater
   reduction in Arg1 mRNA expression following nor@MSC‐EVs treatment
   compared to the anti‐IL17A group and PBS group (Figure [211]S19,
   Supporting Information), consistent with the H score of Arg1 in Figure
   [212]S18, Supporting Information. Positive correlations were observed
   between polyamine levels and Arg1 expression (Figure [213]7L–O). It
   suggested concordant alterations in the transcription and translation
   of the key metabolic enzyme Arg1 and its associated metabolites
   polyamines.^[ [214]^35 ^] Therefore, nor@MSC‐EVs significantly
   decreased local and systemic polyamine levels due to their synergistic
   effects for Arg1, as indicated in Figure [215]3F.

2.9. Therapeutic Effect of nor@MSC‐EVs on Modulating Local and Systemic
Immunological Disorder of IMQ‐Induced Psoriasis Mice

   To further explore the impact of nor@MSC‐EVs on immunological
   modulation in vivo, skin lesions, spleens, and draining lymph nodes
   (dLNs, inguinal lymph nodes) were harvested post‐treatment on day 7 to
   analyze the infiltrated immune cells (Figure [216] 8A).

Figure 8.

   Figure 8
   [217]Open in a new tab

   Therapeutic effect of nor@MSC‐EVs on modulating local and systemic
   immunological disorder of IMQ‐induced psoriasis mice. A) Experimental
   design for assessing the local (skin) and systemic (spleen and dLN)
   immunological modulation effect of PBS, nor‐NOHA, MSC‐EVs, nor@MSC‐EVs,
   and anti‐IL17A in IMQ‐induced psoriasis mice. Flow cytometry analysis
   of the percentage of CD80+CD86+ in CD45+CD11c+ cells (mDCs) in skin
   lesion B), spleen C), and dLN D), respectively. n = 4. Flow cytometry
   analysis of the percentage of IFNγ+ cells in CD45+CD3+CD4+ cells (Th1
   cells) in skin lesion E), spleen F), and dLN G), respectively. n = 3.
   Flow cytometry analysis of the percentage of IL‐17A+ cells in
   CD45+CD3+CD4+ cells (Th17 cells) in skin lesion H), spleen I), and dLN
   J), respectively. n = 3. All data are expressed as mean ± S.D. (n≥3).
   Statistical significance was calculated via one‐way ANOVA with a
   Tukey's test. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

   To evaluate the local innate immune response, skin lesions were
   collected and processed through enzymatic digestion for flow cytometry
   analysis (gating strategies were shown in Figure [218]S20, Supporting
   Information). A notable reduction in the proportion of CD80+CD86+ of
   CD11c+ cells in skin was presented, from 59.5% in the PBS group to
   49.1% and 44.2% in the MSC‐EVs and nor@MSC‐EVs groups, respectively (P
   < 0.05) (Figure [219]8B). Further analysis revealed a pronounced
   decrease of the CD80 and CD86 expression in the nor@MSC‐EVs group,
   whereas the reductions observed in other groups were not statistically
   significant (Figure [220]S21, Supporting Information). To assess
   systemic innate immune response, spleen, and dLN were collected for
   flow cytometry analysis (gating strategies were shown in Figure
   [221]S22, Supporting Information). Compared to the PBS group, both
   MSC‐EVs and nor@MSC‐EVs groups displayed a reduction in the percentage
   of CD80+CD86+ cells of CD11c+ cells in the spleen, decreasing from
   56.6% to 37.0% and 29.1%, respectively (Figure [222]8C). Similarly, in
   the dLN, this percentage declined from 16.0% to 7.9% and 6.1%,
   respectively (Figure [223]8D), indicating a significant inhibitory
   effect of MSC‐EVs and nor@MSC‐EVs on the maturation of DCs. Although
   treatment with anti‐IL17A displayed a decreasing trend in DC
   maturation, there was no statistical significance (Figure [224]8B–D).

   Further investigation on the local and systemic adaptive immune
   response revealed a reduction of the percentage of IFNγ+CD4+ cells in
   skin lesion from 30.0% in the PBS group to 4.0% in the nor@MSC‐EVs
   group (Figure [225]8E). Similar phenomenon was also observed in the
   spleen, where IFNγ +CD4+ cells decreased from 5.8% to 3.3%
   (Figure [226]8F), and in the dLN, where they fell from 2.2% to 1.1%
   (Figure [227]8G), compared to the PBS group. Moreover, a declining
   trend in the percentage of IL‐17A+CD4+ cells in skin lesions was
   observed across the treatment groups. While the nor‐NOHA, MSC‐EVs, and
   anti‐IL17A groups all showed a reduction compared to the PBS group,
   only nor@MSC‐EVs achieved a significant decrease to 1.8%
   (Figure [228]8H). Similarly, nor@MSC‐EVs also inhibited the
   differentiation of IL‐17A+CD4+ cells both in spleen, from 0.9% to 0.8%
   (Figure [229]8I) and dLN, from 1.6% to 0.4% (Figure [230]8J) compared
   to the PBS group. These findings demonstrated the potent efficacy of
   nor@MSC‐EVs in suppressing Th1/17 cell differentiation in the skin
   lesion, spleen, and dLN. In summary, nor@MSC‐EVs effectively modulated
   both local and systemic immunological disorders in psoriasis mouse
   model.

3. Discussion

   In our study, we show that Arg1 is specifically overexpressed in
   psoriasis by analyzing skin and plasma samples from patients with scaly
   erythematous dermatosis. We also demonstrate the elevation of
   Arg1/polyamines could lead to the overactivation of the DCs/Th17 axis
   triggered by excess psoriasis‐specific self‐antigens. Meanwhile, the
   activated Th17 cells secrete more IL‐17A and drive the overexpression
   of Arg1, further exacerbating the loop of Arg1/polyamine and DCs/Th17
   axis (Figure [231]1M). To this, we introduce the engineered
   nor@MSC‐EVs, fabricated by incorporating Arg1 inhibitor nor‐NOHA into
   MSC‐EVs. We confirm first in vitro that nor@MSC‐EVs can reduce the
   generation of Arg1‐mediated polyamines and suppress the overactivated
   innate and adaptive immune responses via inhibiting DC maturation and
   the following Th1/17 cell differentiation. Further in vivo study with
   IMQ‐induced psoriasis mice indicates that nor@MSC‐EVs alleviate
   erythema, scaling, and thickness of skin lesions, showing a more potent
   therapeutic effect than the currently used clinical bioreagent
   anti‐IL17A. Integrating data of multi‐omics and flow cytometry show
   that nor@MSC‐EVs inhibit the NF‐κB signaling pathway and attenuate
   local and systemic metabolic and immunological disorders. Our results
   highlight the potential of targeting the Arg1/polyamine‐mediated
   DCs/Th17 axis by scavenging self‐antigens with nor@MSC‐EVs in
   clinically ameliorating psoriasis symptoms and providing an alternative
   for psoriasis therapy.

   Previously, it has implied the overexpression of Arg1‐mediated the
   limiting of the activity of inducible nitric oxide synthase may be a
   molecular mechanism underlying the hyperproliferation of psoriasis.^[
   [232]^15 ^] Lou et al. also demonstrates a rewiring of the
   urea‐metabolic pathway that results in increased Arg1, augmenting the
   release of polyamines that stabilize self‐antigen complexes, which
   further activate DCs.^[ [233]^13 ^] However, some questions remain
   unsolved. For instance, it is vital to prove that targeting
   Arg1‐mediated metabolic pathways to treat psoriasis is safe and
   superior to currently used clinical treatments that provide solutions
   not only to ameliorate but also to cure psoriasis. In consideration of
   the above issues to achieve synergistic therapeutic effects, we
   incorporate MSC‐EVs in the study design as MSC‐EVs have illustrated
   their great immunomodulation efficacies in treating autoimmune
   diseases,^[ [234]^10 ^] including psoriasis.^[ [235]^9 ^] The newest
   phase I/II clinical trial (ID: IRCT20080901001159N2) reveals that a
   dosage of 200 µg MSC‐EVs significantly improves psoriatic symptoms.^[
   [236]^36 ^] The biological therapies targeting the IL‐23/IL‐17 axis
   have shown partially effective for ameliorating psoriasis symptoms,
   however, patients can stop responding to prolonged treatment in some
   cases and often find difficulties in getting fully cured, suggesting
   there is a need for developing alternative therapy strategies. In
   addition, IL‐23/IL‐17 blockade is associated with some side effects,
   e.g. increasing susceptibility to infections and unpredictable immune
   shift caused by the disrupted cytokine networks.^[ [237]^37 ^] Compared
   to the above clinically used biologic therapies, nor@MSC‐EVs could
   potentially avoid the overactive immune system and achieve complete
   healing of psoriasis by combined modulation of both metabolic and
   immunological disorders in psoriasis.

   Careful consideration of the biosafety of nor@MSC‐EVs via intravenous
   administration is essential for their further translational
   applications. As a fact, numbers of clinical trials in humans have
   already evaluated the safety of intravenous MSC‐EVs administration in
   respiratory distress syndrome, complex regional pain syndrome, etc.^[
   [238]^41 , [239]^42 ^] No adverse events or safety issues were
   reported, indicating the safety profile of MSC‐EVs. Together with our
   data, which assess the biosafety of intravenous administration of
   nor@MSC‐EVs, it provides a basis of using MSC‐EVs for psoriasis as a
   safe therapeutic approach.

   Nevertheless, we also acknowledge that our findings are limited due to
   the following concerns. First, we have used only one animal model,
   which is not replicable to all human psoriasis features. Moreover, it
   is critical to thoroughly evaluate the biosafety of nor@MSC‐EVs on
   human. Another knotty issue is the stability of nor@MSC‐EVs for
   long‐term storage. Our data suggested that the physicochemical
   properties of nor@MSC‐EVs are not significantly altered in PBS at 4 °C
   for 5 days. However, the long‐term stability including the biological
   functions of nor@MSC‐EVs still needs to be evaluated. Collectively,
   future studies and clinical trials using nor@MSC‐EVs for targeting Arg1
   for evaluation of both efficacies and translational possibilities for
   the treatment of psoriasis may be warranted.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   X.Z. and B.T. contributed equally to this work. L.X., R.O., Y.X., and
   D.L. supervised and administrated the project. X.Z., L.X., R.O., Y.X.,
   and D.L. conceived and designed the experiments. X.Z., B.T., Q.H.,
   S.Y., Y.J., W.C., X.L., G.S., and Z.Y. performed the experiments. X.Z.,
   B.T., Q.H., S.Y., Y.J., W.C., and C.Z. analyzed the data. X.Z., B.T.,
   L.X., R.O., Y.X., and D.L. wrote the original manuscript and revised
   the final manuscript. All authors reviewed and given approval to the
   final version of the manuscript.

Supporting information

   Supporting Information
   [240]ADVS-12-2410067-s001.docx^ (28.1MB, docx)

Acknowledgements