SYK gain-of-function mutation regulates neutrophil functions to drive spontaneous psoriasis

SYK gain-of-function mutation regulates neutrophil functions to drive spontaneous psoriasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SYK gain-of-function mutation regulates neutrophil functions to drive spontaneous psoriasis Zhiyang Zeng, Yuxuan Lai, Haozhe Guo, Weihua Yang, Xiya Cao, Wanqing Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8234680/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Psoriasis is a chronic, complex autoimmune disease characterized by massive immune cells infiltration including neutrophils. Spleen tyrosine kinase (SYK), a cytoplasmic non-receptor tyrosine kinase, has been reported to be essential to maintain the pro- inflammatory phenotype of neutrophils. Nevertheless, the influence of SYK on neutrophil function under psoriasis remains largely unknown. Here, we employed a SYK gain-of-function (GOF) mutant (SYK S544Y ) mouse model to investigate the mechanistic function of SYK activation in the progression of psoriasis. Our findings demonstrate that SYK S544Y mice exhibit increased susceptibility to Imiquimod (IMQ) -induced psoriasis and spontaneously develop spontaneous psoriasis-like skin inflammation. SYK activation promotes myeloid cells, particularly neutrophils, infiltration in lesional skin and augments their recruitment activity. Furthermore, inhibition of SYK by R406 has demonstrated therapeutic efficacy on psoriasis. Consequently, these results suggest that SYK activation may contribute to the pathogenesis of psoriasis by augmenting neutrophil recruitment, and targeting SYK may be an attractive strategy to treat psoriasis. Psoriasis syk neutrophils immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Psoriasis is a chronic inflammatory skin disease mediated by an innate and adaptive immune system disorder and affects approximately 2–3% of the global population [ 1 , 2 ] . The histological features of psoriasis are mainly characterized by hyperproliferative epidermal keratinocytes, excessive infiltration of immune cells and exaggerated angiogenesis in the dermis [ 3 ] . Despite the development of multiple treatment options including topical treatments, phototherapy, systemic agents and biologic agents, psoriasis remains a formidable challenge [ 4 ] . Drug resistance, high costs, clinical relapse and adverse effects limit their application and efficacy [ 5 ] . In conclusion, it is imperative to explore novel treatment strategies for psoriasis. Neutrophils, the most abundant cells in the innate immune system, are critical for the body's defense mechanisms. The primary defensive mechanisms of neutrophils involve respiratory burst accompanied by reactive oxygen species (ROS) generation, degranulation (release of granules), and the formation of neutrophil extracellular traps (NETs), modulating inflammatory responses in various immune-mediated diseases. In recent studies, the role of neutrophils in autoimmune conditions, such as psoriasis, has garnered increasing attention. Beyond classical antigen-presenting cells (APCs) [ 6 ] , the continuous neutrophil infiltration within lesional skin is considered as one of the major hallmarks of psoriasis. The sustained infiltration of neutrophils at the lesion interacts with endothelial cells, thereby facilitating the recruitment and extravasation of inflammatory cells. Additionally, the release of NETs activates key immune cells, including keratinocytes, dendritic cells, and T helper 17 (Th17) cells, which further initiate and perpetuate the adaptive immune response, exacerbating the inflammatory cascade characteristic of psoriasis [ 7 , 8 ] . However, the precise mechanisms by which activated neutrophils modulate T-cell responses and contribute to immune dysregulation remain incompletely understood. Spleen tyrosine kinase (SYK) is a 72 kDa non-receptor tyrosine kinase, which is expressed on hematopoietic and nonhematopoietic cells, such as T lymphocytes, B lymphocytes, macrophages, DCs, mast cells, and intestinal epithelial cells [ 9 , 10 ] . Immunoreceptor tyrosine-based activation motifs (ITAMs) are rapidly phosphorylated following receptor engagement, mediating SYK recruitment and activation. Indeed, SYK has emerged as an excellent therapeutic target for various diseases. This is because signaling functions as a link to various downstream signaling molecules including NF-κB, p38MAPK, and Jak-Stat, which subsequently enhance the inflammatory signals initiated at the cell surface. Previous studies have reported that macrophage-specific knockout of SYK can influence the initiation and progression of psoriasis, underscoring its potential role in the disease. However, it is unclear which cell types, such as neutrophils, dendritic cells, or keratinocytes, are primarily responsible for SYK activation in the context of psoriasis, and the precise mechanisms through which SYK drives disease progression are still under investigation. In our previous study, we reported that gain-of-function variants in SYK lead to immune deficiency and systemic inflammation, including colitis, arthritis, and skin inflammatory [ 11 , 12 ] . A GOF knock-in SYK (p.Ser544Tyr) mouse model (hereafter denoted SYK S544Y ) was generated, which provided a very potent and unique animal model to study SYK function in psoriasis-like skin inflammation. In this study, we found that SYK S544Y mice exhibit increased susceptibility to Imiquimod (IMQ)-induced psoriasis and develop spontaneous psoriasis at about 8 months. Additionally, neutrophils emerge as critical mediators of spontaneous psoriasis in SYK S544Y mice, potentially through recruiting T cells. Blocking SYK activity by R406 demonstrated therapeutic efficacy on psoriasis. These results collectively contribute to comprehending the role of SYK in neutrophils and the underlying mechanism involved in the progression of psoriasis and suggest that SYK may be an attractive target to treat psoriasis. Materials and Methods Animal Animal experiments were conducted in accordance with the regulations of the Association of Assessment and Accreditation of Laboratory Animal Care in Shanghai and were approved by the East China Normal University Center for Animal Research (Notification m20241002). All mice were housed at the Animal Centre of East China Normal University. Each group in the experiments included at least three six- to eight-month-old male mice. Human skin sample To detect pSYK expression by immunofluorescence and total SYK expression by immunohistochemistry, skin samples from psoriasis patients and healthy volunteers were obtained from Shanghai Jiaotong University Affiliated Sixth People’s Hospital South Campus (No. 6600, Nanfeng Highway, Fengxian District, Shanghai) with informed consent of anonymous healthy donors. All procedures involving human tissues were approved by the Ethics Committee of Shanghai Jiaotong University Affiliated Sixth People’s Hospital South Campus (Notification 2025-KY-01-03) Bioinformatics Analysis Human psoriasis datasets for RNA-seq (GSE13355, GSE14905, and GSE54456) were downloaded from GEO (Gene Expression Omnibus databases). Transcriptomic datasets GSE11903 and GSE53552 were interrogated to assess SYK expression level following therapeutic blockade of TNF-α and IL-17 receptor inhibition. All statistical analysis were performed by using R software. Imiquimod-Induced Mouse Psoriasis Model One-month-old C57BL/6 wild-type or SYK S544Y mice were used to establish the IMQ-induced psoriasis-like model [13] . Each mouse had a 2cm×3cm area of skin shaved or ear hair cut short before IMQ treatment under avertin (1.25%, MeilunBio). Backs were treated with 62.5mg of imiquimod cream (5%, Sichuan MED-SHINE Pharmaceutical) daily for 6d, while ears were treated with 25mg daily for 6d. Mice were were sacrificed 24 hours post-induction. The severity of disease was measured using the clinical Psoriasis Area and Severity Index (PASI), included checking for erythema, scaling, and skin thickening (0, none; 1, mild; 2, moderate; 3, more severe; 4, severe) [14] . Ear thickness was measured by vernier caliper daily. Flow cytometry analysis Ears were collected minced and digested in ear digestion buffer consisted of 1640 (Gibco), 1.25% FBS, collagenase type Ⅳ (2 mg/mL, Gibco), dispase Ⅱ (100 μg/mL, Beyotime) and DNase (1 mg/mL, Beyotime). Tissues were placed on a shaker and incubated for 60 minutes at 37 °C. Following 1h digestion, the ear tissues was terminated by quenching buffer (PBS supplemented with 10% FBS) and through 40 um cell strainer. Then the digested cells were collected by centrifugation (600 g, 5 min, 4 ℃) and resuspended in washing buffer. Spleens were aseptically harvested, mechanically ground and through 40 μm cell strainers. Subsequently , cell suspensions treated with erythrocyte lysis buffer (Gibco) for 3 minute and centrifuged at 600g for 5 minutes. Cells were resuspended in cold washing buffer. Single-cell suspensions were stained with Zombie Aqua™ Fixable Viability Dye (BioLegend, 423101) for 15 minutes at room temperature with gentle agitation (100 rpm), maintaining light-protected conditions. Surface marker staining was performed by incubating cells with fluorochrome-conjugated antibodies for 20 minutes at 4°C under light-protected conditions. The following antibodies were employed for flow cytometric analysis. BioLegend: PerCP/Cyanine5.5 anti-mouse CD45, APC/Cyanine7 anti-mouse CD3, Brilliant Violet 605™ anti-mouse CD8a, PE anti-mouse CD4, Brilliant Violet 650™ anti-mouse CD11b, PE anti-mouse Ly-6G, FITC anti-mouse Ly-6C. After completing the staining, cell suspensions were pelleted by centrifugation (600g, 5 min, 4°C) and resuspended in PBS, then subjected to flow cytometry (BD LSR Fortessa). Bone Marrow-Derived Myeloid (BMDM) and Neutrophil (BMDN) Isolation Bone marrow cells were isolated from murine femurs and tibias by flushing with 10 mL of ice-cold PBS. The suspension was centrifuged at 600 g for 5 min at 4 °C and erythrocytes were removed using erythrocytes lysis buffer (BD Biosciences, 555899). The reaction was quenched with buffer (PBS and 10%FBS), and the cell suspension was filtered through a 40μm cell strainer. CD11b⁺ cells were isolated using a specialized magnetic bead-based cell sorting kit (Biolegend 480110). Sorted cells were centrifuged and resuspended in culture medium (1640 and 10%FBS). After acquiring a single-cell suspension from bone marrow, cells were stained with fluorescently labeled anti-CD45 and anti-Ly6G antibodies for 20 minutes at 4°C, followed through Flow cytometer (BD) to obtain the neutrophil. T Cell Migration Assay Spleen tissues were ground and through a 40 μm cell strainer. After erythrocyte lysis, single-cell suspensions were subjected to negative selection using a CD4 + T Cell Isolation Kit (Biolegend). Purified CD4 + T cells were cultured in T cell medium (RPMI-1640 and 10% FBS) containing 1% T cell activator (Stemcell, 100-1572). Half-medium changes were performed at 48h, with cells harvested at 72h for counting. Cell migration was assessed using a 3 μm transwell chamber (Absin, abs7281). Freshly isolated 100,000 CD4 + T cells were plated to the upper chamber with T complete medium. WT or SYK S544Y BMDMs and BMDNs were added in the lower chamber at varying concentrations in RPMI-1640 medium. After 24-hour chemotaxis assay, migrated cells in the lower chamber were collected and quantified. Cells were then stained with anti-CD45 and anti-CD4 fluorescent antibodies for 20 minutes at 4°C and analyzed by flow cytometry. Histological analysis Tissue specimens preserved in paraformaldehyde and embedded in paraffin were sectioned at 5 μm thickness and collected onto adhesion microscopy slides (Citotest). For histopathological analysis, tissue slides were stained with hematoxylin and eosin (H&E) and visualized using an Olympus BX51 microscope. For immunohistochemistry staining, the human and mouse skin paraffin sections were deparaffinized and rehydrated. Next, endogenous peroxidases of the tissue slides were quenched by 3% H 2 O 2 treatment for 10 minutes. Antigen retrieval was performed by heat-mediated epitope unmasking in citrate buffer (20 min, 95 °C). Subsequent specimens were blocked with 3% goat serum and incubated with primary antibody overnight at 4°C. Appropriate secondary antibodies were incubated according to experimental objectives and then were visualized with DAB reagent according to the manufacturer's protocol. Tissue sections were subsequently counterstained with hematoxylin and then dehydrated and mounted in neutral resins. Final results were visualized utilizing an Olympus BX51 microscope. Primary antibodies and their dilutions were as follows: Ki67 (1:200, Abcam, ab16667), Vegf (1:200, Abcam, ab317030). For immunofluorescence, paraffin-embedded human and mouse skin sections were deparaffinized and rehydrated. Subsequently, antigen retrieval was performed with 1 mM EDTA (pH 8.0) for 20 min in boiling water and cooled to room temperature. The slides were then incubated with 2.5% normal goat serum(VECTOR) diluted in PBS (pH 7.4) at room temperature for 30 min to block non-specific binding sites. Samples were incubated with fluorescent antibody overnight at 4℃. After counterstained with DAPI (1:1000; Sigma) for 10 min, samples were oriented and mounted in Fluorescent Mounting Medium and visualized using an Olympus BX51 microscope. qPCR Total RNA was extracted from mouse skin and ear samples using 1ml Trizol reagent in RNA-free EP tubes. Then specimens RNA reverse transcribed into cDNA using Hifair ® Ⅲ 1st Strand cDNA Synthesis SuperMix (Yeasen). Quantitative real-time PCR was conducted using Hieff qPCR SYBR Green Master Mix (Yeasen Biotechnology) following manufacturer's protocols. Resluts were read with 7500 Real-Time PCR Instrument (Thermo Fisher Scientific). The primers used in this study were listed in Supplementary Table 1. Bone marrow transplantation Femurs and tibias were isolated from WT and SYK S544Y mice, and bone marrow cells were flushed with PBS followed washed twice with DPBS. Recipient mice were subjected to lethal-dose irradiation using irradiator prior to transplantation. Isolated bone marrow cells (5×10 6 ) from donor mice were intravenously injected into irradiated recipients. Seven months after bone marrow transplantation skin phenotypes indicative of psoriasis were assessed through histological analysis. Pharmacologic inhibition of SYK in vivo Eight-month-old SYK mice and age-matched WT mice were treated by intragastric administration with 100ul R406 (dissolved in 5% DMSO and 95% corn oil) daily at 2 mg/mL for consecutive 14 days, while mice in the control group were treated with 100 μL vehicle (5% DMSO and 95% corn oil) daily. After 15 consecutive days of treatment, ear thickness in mice was measured using vernier calipers. Statistical Analysis All results are presented as the mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism 7 (GraphPad Software, Inc.), employing either unpaired two-tailed t-tests or one-way ANOVA as appropriate. All experiments were independently biological repeated at least three times. Throughout all figures: * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001. Significance was concluded at p < 0.05. Results SYK expression is elevated in psoriatic patients. Our previous investigations revealed that SYK gain-of-function (GOF) mutations in patients induce immune dysregulation accompanied by multi-organ inflammatory manifestations, variably affecting cutaneous, gastrointestinal, articular, hepatic, and neurological tissues [11] . Interestingly, five of six reported SYK-GOF cases demonstrated consistent skin inflammation [11] , prompting our investigation into the exploration of the role of SYK in skin inflammation, such as psoriasis. Analysis of three independent GEO datasets (GSE13355, GSE14905, GSE54456) showed that SYK mRNA expression was significantly higher in the skin lesions of psoriatic patients (n=58) compared to normal controls (n=63) and non-lesional skin (n=58) (GSE13355) (Fig 1A, B, C). More importantly, the expression of SYK progressively reduced upon TNF-α inhibitor and anti-IL-17R antibody treatment (Fig 1D). Furthermore, Immunohistochemical (IHC) analysis of skins also revealed significantly increased SYK protein abundance in the skin lesions of psoriasis patients compared to that of healthy controls (Fig 1E). Accordingly, immunofluorescence staining of patient biopsies showed phos-SYK (p-SYK) levels were enriched 6-fold in psoriatic skin compared to healthy donors, consistent with hyperactivated SYK signaling in disease progression (Fig 1F). These findings suggest that SYK activation may contribute to the progression of psoriasis. Next, we employed an imiquimod (IMQ)-induced psoriasis mouse model and found that p-SYK was greatly elevated following IMQ induction, while in the control group, p-SYK was almost not expressed (Fig 1G). These data collectedly demonstrated that SYK may be an important regulator in the initiation and development of psoriasis. SYK S544Y mice exhibit enhanced sensitivity to IMQ-induced psoriasis To study the role of SYK in psoriasis, the SYK S544Y GOF moues model [11] built by our team was used. The SYK S544Y mice did not display obvious swelling and redness of ears as well as erythemato-squamous plaques of skin at the age of two month. (Fig 2A). However, H&E staining revealed a thicker epidermis and increased dermal immune cell infiltration in SYK S544Y mice compared to WT controls (Fig 2B). To further evaluate whether the SYK S544Y alters responsiveness to stimulation compared to WT controls, we employed an IMQ-induced Ps mouse model (Fig 2C). Of note, SYK S544Y mice exhibited heightened vulnerability to IMQ-induced inflammation. Ear thickness measurements demonstrated a 1.19-fold increase compared to the control group (Fig 2D). Additionally, SYK S544Y mice exhibited significantly elevated Psoriasis Area and Severity Index (PASI) scores (Fig 2E). In alignment with observed phenomena, H&E assessment of SYK S544Y mice revealed exacerbated swelling, keratinocyte proliferation, dilated capillaries, and dermal inflammatory cell infiltration in the ears (Fig 2F). Furthermore, immunofluorescence analysis also demonstrated a 1.55-fold elevation of p-SYK levels in IMQ-induced SYK mice compared to that of IMQ-induced WT mice (Figure 2G). Splenomegaly, an indicator of systemic inflammation, was increased by 2.36-fold in SYK S544Y mice compared to WT controls (Fig 2H). These results demonstrate that activation of SYK in mice may accelerates the progression of IMQ induced psoriasis. SYK S544Y Mice Spontaneously exhibit Psoriasis-like skin inflammation Although we did not observe a psoriasis phenotype in 2-month-old mice, there is epidemiological evidence delineates an age-dependent escalation in psoriasis prevalence, peaking during midlife [16] , so we speculate that SYK S544Y mice may develop spontaneous psoriasis as they age. As expected, eight-month-old SYK S544Y mice developed prominent psoriasis-like skin inflammation affecting multiple regions, including the dorsal skin, ears, and tail (Fig 3A). Moreover, SYK S544Y mice exhibited significantly increased auricular thickness, with 1.3-fold augmentation compared to WT controls (Fig 3B). According to the standardized clinical scoring system for assessing skin lesion severity, the PASI score revealed a near 3.8-fold amplification in SYK mice compared to age-matched WT counterparts (Fig 3B). H&E staining also revealed that erythema, abnormal proliferation and differentiation of keratinocytes, and excessive angiogenesis were histologically evident in SYK S544Y mice tissues (Fig 3C). Nevertheless, psoriasis in human patients is characterized not only by epidermal thickening but also by prominent inflammatory histopathological features. To determine whether SYK S544Y mice exhibit pathological features resembling human psoriasis, we stained the dorsal skin of mice. Immunohistochemical analysis demonstrated a pronounced elevation of Ki67 and Vegf protein in the dorsal skin of SYK S544Y mice (Fig 3D, E). Numerous previous research highlighted the critical role of diverse pro-inflammatory cytokines and chemokines in modulating the cutaneous microenvironment. Consistent with previous reports [17,18] , we found that the mRNA expression levels of S100a9, S100a8, Il1β, Cxcl1, Tnf, Il6, Il23, and Il17a were significantly elevated in the skin lesions of SYK S544Y mice (Fig 4A). In addition to confirming the involvement of lesions in the ears, we systematically quantified mRNA expression of key inflammatory mediators in the skin, revealing a consistent upregulation pattern that paralleled the disease progression (Fig 4B). Together, these data suggest that SYK activation alone is sufficient to lead to the occurrence of spontaneous psoriasis. Gain-of-Function variant in SYK promotes neutrophil recruitment capacity The current academic consensus holds that the pathogenesis of psoriasis primarily arises from dysregulated interactions between epidermal keratinocytes and immune cells [19] . Among these, specific immune subsets play crucial roles in both disease initiation and amplification of the inflammatory cascade [20,21] . Thus, to verify whether the spontaneous psoriasis-like skin inflammation in SYK S544Y mice is mediated by dysfunctional immune cell populations, bone marrow transplantation (BMT) experiments were performed. (Fig 5A). As expected, recipients of SYK S544Y bone marrow developed visible swelling and scaling on the ears, tail, and skin of the back at 8 months (Fig 5B). In contrast, recipients received WT bone marrow remained phenotypically normal. Histological analyses revealed marked epidermal hyperplasia and pronounced immune cell infiltration in the skin, ear, and tail of WT mice receiving SYK S544Y bone marrow (Fig 5C). To delineate the immune landscape shaped by SYK activation, flow cytometric analysis of ear tissue was conducted, the frequency of CD45⁺ cells in SYK S544Y mice was elevated by 57.72% compared with WT controls (Fig 5D). Specifically, the frequencies of myeloid cells and neutrophils in SYK S544Y mice were significantly elevated, with increases 2.35-fold and 48.47-fold, respectively (Fig 5E). In addition, CD4 + T cell frequency markedly increased by 2.21-fold, while no differences were observed in CD8 + T cells (Fig 5F). Given that psoriasis is a CD4 + T cell-driven inflammatory disorder [22,23] and that neutrophils can release various chemokines (such as CCL20, CCL2, CXCL10) and cytokines to recruit T cells [24] , we aim to investigate whether SYK activation augments neutrophil-mediated T cell recruitment. Bone marrow-derived neutrophils (BMDN) were isolated for in vitro chemotaxis assays (Fig 5G). Notably BMDNs from SYK S544Y mice displayed enhanced recruitment capacity, showing a 1.54-fold and a 1.25-fold increase at different neutrophil densities compared with WT BMDNs (Fig 5H). In addition, we found that the capacity of myeloid cells to recruit T cells was also enhanced (Fig 5I). Collectively, these results demonstrate that SYK hyperactivation may promotes sustained neutrophil infiltration and enhances their recruitment capacity, contributing to psoriasis-like inflammation. BMT and SYK inhibitor ameliorated psoriasis-like skin inflammation of mice Considering that WT mice receiving SYK S544Y bone marrow spontaneously developed psoriasis, we wondered whether SYK S544Y mice that received WT bone marrow transplantation could alleviate the psoriasis phenotype (Fig 6A). Eight months post-transplantation, psoriatic SYK S544Y mice reconstituted with WT bone marrow displayed complete resolution of skin inflammation. (Fig 6B). H&E assessment revealed marked improvement, including reduced epidermal hyperplasia and partial restoration of dermal architecture with diminished immune cell infiltration (Fig 6C). These findings indicate that SYK activation within hematopoietic cells is a key driver of psoriatic inflammation. SYK inhibitors have been extensively investigated in various autoimmune and inflammatory diseases. For instance, Fostamatinib (R788), an oral SYK inhibitor, has been approved for the treatment of chronic immune thrombocytopenia and has shown clinical efficacy in rheumatoid arthritis (RA) and other inflammatory disorders [25,26] . Additionally, R406, is widely used in preclinical studies to suppress SYK-mediated signaling [27] . Previous studies have reported that SYK inhibitors attenuate psoriasis-like inflammation by blocking the dendritic cell-Th17 inflammatory axis [28,29] . We next evaluated the effect of pharmacological SYK inhibition to further validate the therapeutic potential of targeting SYK signaling. Administration R406 (10mg/kg) for two weeks significantly mitigated psoriasis-like pathology, with reduced erythema and scaling in treated mice (Fig 6D). Histopathological results further confirmed attenuation of epidermal thickening and immune infiltration. Following intervention, the hyperkeratotic and parakeratotic plaques covering the epidermis in SYK S544Y mice were substantially reduced. (Fig 6E). Flow cytometric analysis was then performed to examine the impact of the SYK inhibitor on different immune cell populations. A 25.2% decrease in the frequency of CD45⁺ cells was observed in R406-treated mice compared to placebo-treated mice (Figure 6F). Following R406 treatment, SYK S544Y mice exhibited a reduced proportion of myeloid cells. Notably, the proportion of neutrophils decreased by 93.03%. (Figure 6G). Taken together, these findings collectively demonstrate that pharmacological inhibition of SYK effectively ameliorates psoriasis-like skin inflammation, curtails immune infiltration, and improves the cutaneous immune microenvironment. DISCUSSION A growing body of evidence confirms that psoriasis is a multifactorial immune-mediated disorder driven by the dysregulation of both innate and adaptive immune responses, in which autoimmune and inflammatory mechanisms synergistically promoting disease progression [30,31] . Animal models have been indispensable in elucidating pathogenic pathways and evaluating novel therapeutic strategies [32] . However, the widely used IMQ-induced model primarily reflects acute inflammation rather than the chronic, relapsing course characteristic of clinical psoriasis [33] . Thus, there is a pressing need to develop more clinically relevant mouse models. In the present study, CRISPR-Cas9 technology was employed to generate a gain-of-function SYK mutant mouse model. Intriguingly, these mice spontaneously developed psoriasis-like skin inflammation at 8 months, closely mimicking key clinical features and histopathological hallmarks of human psoriasis. Mechanistically, SYK hypoactivation trigger robust immune cell infiltration, particularly sustained neutrophil accumulation in lesional skin. Furthermore, SYK S544Y bone marrow-derived neutrophils and myeloid cells exhibited enhanced chemotactic and recruitment capacities. Collectively, these findings highlight a critical role for SYK hyperactivation in driving psoriatic pathology. SYK, a widely expressed non-receptor tyrosine kinase, orchestrates multiple signaling pathways across diverse immune cell lineages. Prior studies have established its contribution to psoriasis pathogenesis, particularly in macrophages and neutrophils [34,35] . Consistent with these findings, we observed markedly elevated SYK and p-SYK expression in both human lesions and mouse model skin. Moreover, SYK S544Y mice displayed heightened susceptibility to IMQ-induced psoriasis and developed more severe systemic chronic inflammation, underscoring the critical role of SYK hyperactivation in disease progression. SYK regulates signal transduction and cytokine production in innate immune cells, including neutrophils, monocytes, DCs and B cells. Functional interrogation through bone marrow transplantation further demonstrated that SYK activation within hematopoietic cells was sufficient to drive psoriatic skin inflammation. Notably, reciprocal transplantation with WT bone marrow completely reversed disease manifestations, highlighting the immune cell-intrinsic contribution of SYK signaling. Consistent with previous reports showing that SYK inhibition suppresses the IL-23/IL-17A axis in DCs to alleviate psoriasis, we found that the SYK inhibitor R406 significantly ameliorated skin inflammation and reduced immune cell infiltration, particularly neutrophils, in our model. These findings hint SYK in neutrophils as a promising therapeutic target for psoriasis. While our findings established a critical role for SYK activation in neutrophils in psoriasis progression, several limitations should be acknowledged. Future investigations employing neutrophil-specific SYK mutant models will be essential to delineate the precise contribution of SYK GOF variants to psoriasis development. Additionally, neutrophils contribute to psoriatic pathology not only through recruitment but also via ROS production and IL-17 dependent mechanisms [36,37] . Whether SYK directly modulates these effector functions remains to be elucidated. These aspects have been described more deeper in our subsequent studies. Declarations Acknowledgment The authors express their gratitude to Shanghai Jiaotong University Affiliated Sixth People’s Hospital South Campus and East China Normal University. We are grateful for the support from East China Normal University Public Platform for innovation (011). We thank Y. Zhang from the Flow Cytometry Core Facility of School of Life Sciences in ECNU. Author contributions Z.Z., X.Z., D.L. and Z.S. conceived and designed the project. Y.L conduct the research under the guidance of D.L. and Z.Z., Y.L. and H.G collected and analyzed the data. W.Y. was involved in conducting the histochemical and qPCR experiments. Z.Z and Y.L. wrote the manuscript with contributions from all authors. Funding This work was partially supported by grants from the National Key R&D Program of China (2023YFC3403400), the National Natural Science Foundation of China (32200732), grants from the Shanghai Municipal Commission for Science and Technology (22YF1437700), the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054), the Key Medical Disciplines of Shanghai Municipal Health Commission (2024ZDXK0045)，the Shanghai Fengxian District Central Hospital Research Project (YJKT-25-01-001). Conflicts of interest The authors report no conflict of interest in this work. Data availability All data are available from the corresponding author upon request. Clinical Trial This project does not involve clinical trials. Clinical trial number: not applicable. References Griffiths C E M, Armstrong A W, Gudjonsson J E, et al. Psoriasis[J]. The Lancet, 2021, 397(10281): 1301-1315. Xue M, Yang R, Li G, et al. LGR4 Deficiency Aggravates Skin Inflammation and Epidermal Hyperplasia in Imiquimod-Induced Psoriasis[J]. Immunology, 2025, 174(2): 213-225. Stern R S. Psoriasis[J]. The Lancet, 1997, 350(9074): 349-353. Ghoreschi K, Balato A, Enerbäck C, et al. Therapeutics targeting the IL-23 and IL-17 pathway in psoriasis[J]. Lancet (London, England), 2021, 397(10275): 754-766. Lee H J, Kim M. Challenges and Future Trends in the Treatment of Psoriasis[J]. International Journal of Molecular Sciences, 2023, 24(17): 13313. Whitley S K, Li M, Kashem S W, et al. 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1","display":"","copyAsset":false,"role":"figure","size":707713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSYK expression is upregulated in psoriasis skin lesions.\u003c/strong\u003e (A, B, C) Analysis of SYK mRNA expression in normal human skin tissues versus non-lesion and lesion tissues of patients with clinical psoriasis; datasets from the Gene Expression Omnibus (GEO). (D) Analysis of SYK mRNA expression in patients with psoriasis after retreatment with IL-17R antibody (left) (GSE53552) or TNF-α inhibitor (right) (GSE11903); data from GEO. (E) Immunohistochemical staining and quantitative analysis of SYK in patients and donors in each group. Scale bars, 50μm. (n=3). (F)Immunofluorescence images and quantitative analysis of psoriasis patients and healthy donors skin stained with pSYK (red) and DAPI (blue). Scale bars, 100μm. (n=3). (G)Immunofluorescence staining of pSYK (red) and DAPI (blue) in skin tissue from PBS-induced WT and IMQ-induced WT groups. Error bars show the mean±SEM. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001; determined using two-tailed unpaired Student's t-tests (E, F, and G) or one-way ANOVA (A, B, and C).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/543ddbe3f54a019afb42ae9b.png"},{"id":97894162,"identity":"42d04371-2e24-43fe-82f7-46f2169d7824","added_by":"auto","created_at":"2025-12-10 15:31:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":937971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSYK activation enhanced sensitivity to IMQ-induced psoriasis.\u003c/strong\u003e (A) Representative images of ear from two months wild-type and SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;mice. (B) Representative images of back skin samples, ear tissue and tail tissue stained with haematoxylin and eosin (H\u0026amp;E) and epidermal thickness quantitated via epidermal thickness measurements. Scale bar 100μm. (n=6). (C) Diagram showing the construction of IMQ-induced psoriasis mouse model. (D) Daily thickness changes of the ear from IMQ induced WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;mice (n=4 per group).\u0026nbsp;(E) Daily psoriasis scores of ear tissue from IMQ-induced WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e mice. (F) H\u0026amp;E staining of the ear samples from IMQ-induced WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;mice. Scale bar: 100μm. (G) Immunofluorescence staining and quantitative analysis of pSYK in each group. Scale bars, 50 μm (H) Representative images of IMQ-treated mouse spleens and the spleen weight of mice in each group was quantified. (n=4). Error bars show the mean±SEM. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001; determined using two-tailed unpaired Student's t-tests (B, F, G, and H).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/9357d0bb5c27bf19b65bc10b.png"},{"id":97709630,"identity":"82fb65e8-d6fa-4a22-af5b-ff6649679808","added_by":"auto","created_at":"2025-12-08 13:31:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1008693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSYK\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eS544Y\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;mice developed spontaneous psoriasis-like skin inflammation. \u003c/strong\u003e(A) Macroscopic views of the ear, back skin, and tail from an 8-month-old SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;and WT mice. (B) Ear thickness of diseased SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;mice compared with WT mice. (n = 6). (C) H\u0026amp;E staining of the skin lesions, ear sample, and tail tissue from the SYK\u003csup\u003eS544Y\u003c/sup\u003e and WT mice. Ear sample image scale bar, 200μm; Skin and tail sample image 100μm. (D, E) Representative immunohistochemical staining and quantitation of Ki67 (upper) and Vegf (lower) in ears (n = 3). Scale bar, 100μm. Data are representative of two (B-E) independent experiments. Data are mean ± SEM. P values are determined by two-tailed Student’s t test (B-E). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/386c7d03d7903e2f849b3d95.png"},{"id":97709631,"identity":"f5c9aa37-8c2f-47eb-9f8f-f0c9b9fd07c5","added_by":"auto","created_at":"2025-12-08 13:31:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated expression of inflammatory cytokines in the lesion area of SYK\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eS544Y\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003e(A, B) Quantitative PCR analysis of mRNA from ear (upper) and skin (lower) encoding chemokines, cytokines, and antimicrobial peptides. Results (calculated by the change-in-cycling-threshold (2\u003csup\u003e－△△Ct\u003c/sup\u003e) method) are normalized to internal control gene Gapdh. Data are representative of two (A-B) independent experiments. n=3. Data are mean ± SEM. P values are determined by two-tailed Student’s t test (B-G). *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001;\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/d74ce5bfa2365d2ffc6e12eb.png"},{"id":97709632,"identity":"d99b8e47-b66f-49f4-bcae-3d984d62c5f2","added_by":"auto","created_at":"2025-12-08 13:31:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":977949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSYK promotes neutrophil recruitment capacity.(A) \u003c/strong\u003eSchematic of mouse bone-marrow transplantation model. (B) Representative photographs of bone marrow transplantation in WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;mice 8 months after transplantation. (C) Representative H\u0026amp;E staining of the ear, skin and tail. Scale bar, 100μm. (n=6). (D) Flow cytometry of CD45\u003csup\u003e+\u003c/sup\u003e cells in ear. Numbers adjacent to outlined areas indicate the percentage of CD45\u003csup\u003e+\u003c/sup\u003e cells among live cells. (n=4). (E,F) Flow cytometry of ear myeloid cells (CD45\u003csup\u003e+\u003c/sup\u003eCD11B\u003csup\u003e+\u003c/sup\u003e), CD3 (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e), CD4 (CD4\u003csup\u003e+\u003c/sup\u003e), CD8(CD8\u003csup\u003e+\u003c/sup\u003e), and neutrophils (Ly6G\u003csup\u003e+\u003c/sup\u003e) from the SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;and WT mice. (n=4). (G) Schematic of in vitro Chemotaxis Assay. (H)After 24 hours of co-culture, the number of CD4\u003csup\u003e+\u003c/sup\u003e T cells attracted by 2×10\u003csup\u003e5\u003c/sup\u003e, 4×10\u003csup\u003e5\u003c/sup\u003e, and 8×10\u003csup\u003e5\u003c/sup\u003e BM-derived neutrophils from WT or SYK\u003csup\u003eS544Y\u003c/sup\u003e mice (n=3). (I)After 24 hours of co-culture, the number of CD4\u003csup\u003e+\u003c/sup\u003e T cells attracted by 2×10\u003csup\u003e5\u003c/sup\u003e, 4×10\u003csup\u003e5\u003c/sup\u003e, and 8×10\u003csup\u003e5\u003c/sup\u003e BM-derived myeloid cells from WT or SYK\u003csup\u003eS544Y\u003c/sup\u003e mice (n=3). Data are mean ± SEM. P values are determined by two-tailed Student’s t test (C-G). *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001;\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/d5fda7d955bda21033e94ce7.png"},{"id":97709637,"identity":"e1606f2a-9ff9-41ab-9281-9649735b84f7","added_by":"auto","created_at":"2025-12-08 13:31:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1006089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSYK inhibitor R406 and BMT ameliorated psoriasis-like skin inflammation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of mouse bone-marrow transplantation model. (B) Representative images of ear and tail from SYK\u003csup\u003eS544Y\u003c/sup\u003e mice receiving WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e bone marrow after 8 months. (C) Representative histological sections (left) of dorsal back, ear and tail from transplanted mice stained by hematoxylin and eosin, and epidermal thickness (right) quantitated via epidermal thickness measurements. Scale bar: 100μm. (n=6). (D, E) Psoriatic phenotype and hematoxylin and eosin (H\u0026amp;E) staining from WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e\u0026nbsp;mice treated with R406 or PBS. Scale bar: 100μm. (n=3). (F, G) Flow cytometry (top) of CD45\u003csup\u003e+\u003c/sup\u003e cell, myeloid cells, and neutrophils (lower) suspensions from ear. Numbers adjacent to outlined areas indicate the percentage of CD45\u003csup\u003e+\u003c/sup\u003e cells, myeloid cells, and neutrophils among live cells. Data are mean ± SEM. P values are determined by two-tailed Student’s t test (D-F). *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/67212b7dcb3ec0efda7ca4fd.png"},{"id":105928079,"identity":"1b5e88ec-83b8-4008-98b8-c5c037c63467","added_by":"auto","created_at":"2026-04-01 13:44:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6034375,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/f6cb062e-b1b9-4157-99ef-7836ba93c16d.pdf"},{"id":97895538,"identity":"91929294-a2d9-4859-b324-8aa237a65e64","added_by":"auto","created_at":"2025-12-10 15:34:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13403,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8234680/v1/9b207d63b497f40386fd1dc2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SYK gain-of-function mutation regulates neutrophil functions to drive spontaneous psoriasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePsoriasis is a chronic inflammatory skin disease mediated by an innate and adaptive immune system disorder and affects approximately 2\u0026ndash;3% of the global population\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The histological features of psoriasis are mainly characterized by hyperproliferative epidermal keratinocytes, excessive infiltration of immune cells and exaggerated angiogenesis in the dermis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Despite the development of multiple treatment options including topical treatments, phototherapy, systemic agents and biologic agents, psoriasis remains a formidable challenge\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Drug resistance, high costs, clinical relapse and adverse effects limit their application and efficacy\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In conclusion, it is imperative to explore novel treatment strategies for psoriasis.\u003c/p\u003e\u003cp\u003eNeutrophils, the most abundant cells in the innate immune system, are critical for the body's defense mechanisms. The primary defensive mechanisms of neutrophils involve respiratory burst accompanied by reactive oxygen species (ROS) generation, degranulation (release of granules), and the formation of neutrophil extracellular traps (NETs), modulating inflammatory responses in various immune-mediated diseases. In recent studies, the role of neutrophils in autoimmune conditions, such as psoriasis, has garnered increasing attention. Beyond classical antigen-presenting cells (APCs) \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, the continuous neutrophil infiltration within lesional skin is considered as one of the major hallmarks of psoriasis. The sustained infiltration of neutrophils at the lesion interacts with endothelial cells, thereby facilitating the recruitment and extravasation of inflammatory cells. Additionally, the release of NETs activates key immune cells, including keratinocytes, dendritic cells, and T helper 17 (Th17) cells, which further initiate and perpetuate the adaptive immune response, exacerbating the inflammatory cascade characteristic of psoriasis\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. However, the precise mechanisms by which activated neutrophils modulate T-cell responses and contribute to immune dysregulation remain incompletely understood.\u003c/p\u003e\u003cp\u003eSpleen tyrosine kinase (SYK) is a 72 kDa non-receptor tyrosine kinase, which is expressed on hematopoietic and nonhematopoietic cells, such as T lymphocytes, B lymphocytes, macrophages, DCs, mast cells, and intestinal epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Immunoreceptor tyrosine-based activation motifs (ITAMs) are rapidly phosphorylated following receptor engagement, mediating SYK recruitment and activation. Indeed, SYK has emerged as an excellent therapeutic target for various diseases. This is because signaling functions as a link to various downstream signaling molecules including NF-κB, p38MAPK, and Jak-Stat, which subsequently enhance the inflammatory signals initiated at the cell surface. Previous studies have reported that macrophage-specific knockout of SYK can influence the initiation and progression of psoriasis, underscoring its potential role in the disease. However, it is unclear which cell types, such as neutrophils, dendritic cells, or keratinocytes, are primarily responsible for SYK activation in the context of psoriasis, and the precise mechanisms through which SYK drives disease progression are still under investigation. In our previous study, we reported that gain-of-function variants in SYK lead to immune deficiency and systemic inflammation, including colitis, arthritis, and skin inflammatory\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. A GOF knock-in SYK (p.Ser544Tyr) mouse model (hereafter denoted SYK\u003csup\u003eS544Y\u003c/sup\u003e) was generated, which provided a very potent and unique animal model to study SYK function in psoriasis-like skin inflammation.\u003c/p\u003e\u003cp\u003eIn this study, we found that SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibit increased susceptibility to Imiquimod (IMQ)-induced psoriasis and develop spontaneous psoriasis at about 8 months. Additionally, neutrophils emerge as critical mediators of spontaneous psoriasis in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice, potentially through recruiting T cells. Blocking SYK activity by R406 demonstrated therapeutic efficacy on psoriasis. These results collectively contribute to comprehending the role of SYK in neutrophils and the underlying mechanism involved in the progression of psoriasis and suggest that SYK may be an attractive target to treat psoriasis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were conducted in accordance with the regulations of the Association of Assessment and Accreditation of Laboratory Animal Care in Shanghai and were approved by the East China Normal University Center for Animal Research (Notification m20241002). All mice were housed at the Animal Centre of East China Normal University. Each group in the experiments included at least three six- to eight-month-old male mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman skin sample\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo detect pSYK expression by immunofluorescence and total SYK expression by immunohistochemistry, skin samples from psoriasis patients and healthy volunteers were obtained from Shanghai Jiaotong University Affiliated Sixth People\u0026rsquo;s Hospital South Campus (No. 6600, Nanfeng Highway, Fengxian District, Shanghai) with informed consent of anonymous healthy donors. All procedures involving human tissues were approved by the Ethics Committee of Shanghai Jiaotong University Affiliated Sixth People\u0026rsquo;s Hospital South Campus (Notification 2025-KY-01-03)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman psoriasis datasets for RNA-seq (GSE13355, GSE14905, and GSE54456) were downloaded from GEO (Gene Expression Omnibus databases).\u0026nbsp;Transcriptomic datasets GSE11903 and GSE53552 were interrogated to assess SYK expression level\u0026nbsp;following therapeutic blockade of TNF-\u0026alpha; and IL-17 receptor inhibition. All statistical analysis were performed by using R software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImiquimod-Induced Mouse Psoriasis Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-month-old C57BL/6 wild-type or SYK\u003csup\u003eS544Y\u0026nbsp;\u003c/sup\u003emice were used to establish the IMQ-induced psoriasis-like model\u003csup\u003e[13]\u003c/sup\u003e.\u0026nbsp;Each mouse had a 2cm\u0026times;3cm area of skin shaved or ear hair cut short before IMQ treatment under avertin (1.25%, MeilunBio). Backs were treated with 62.5mg of imiquimod cream (5%, Sichuan MED-SHINE Pharmaceutical) daily for 6d, while ears were treated with 25mg daily for 6d. Mice were were sacrificed 24 hours post-induction.\u0026nbsp;The severity of disease was measured using the clinical Psoriasis Area and Severity Index (PASI), included checking for erythema, scaling, and skin thickening (0, none; 1, mild; 2, moderate; 3, more severe; 4, severe)\u003csup\u003e[14]\u003c/sup\u003e. Ear thickness was measured by vernier caliper daily.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEars were collected minced and digested in ear digestion buffer consisted of 1640 (Gibco), 1.25% FBS, collagenase type Ⅳ (2 mg/mL, Gibco), dispase Ⅱ (100 \u0026mu;g/mL, Beyotime) and DNase (1 mg/mL, Beyotime).\u0026nbsp;Tissues were placed on a shaker and incubated for 60\u0026thinsp;minutes at 37 \u0026deg;C.\u0026nbsp;Following 1h digestion, the ear tissues was terminated by quenching buffer (PBS supplemented with 10% FBS) and through 40 um cell strainer. Then the digested cells were collected by centrifugation (600 g, 5 min, 4 ℃) and resuspended in washing buffer.\u003c/p\u003e\n\u003cp\u003eSpleens were aseptically harvested, mechanically ground and through 40 \u0026mu;m cell strainers. Subsequently\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003ecell suspensions treated with erythrocyte lysis buffer (Gibco) for 3 minute and centrifuged at 600g for 5 minutes. Cells were resuspended in cold washing buffer.\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions were stained with Zombie Aqua\u0026trade; Fixable Viability Dye (BioLegend, 423101) for 15 minutes at room temperature with gentle agitation (100 rpm), maintaining light-protected conditions. Surface marker staining was performed by incubating cells with fluorochrome-conjugated antibodies for 20 minutes at 4\u0026deg;C under light-protected conditions. The following antibodies were employed for flow cytometric analysis. BioLegend: PerCP/Cyanine5.5 anti-mouse CD45, APC/Cyanine7 anti-mouse CD3, Brilliant Violet 605\u0026trade; anti-mouse CD8a, PE anti-mouse CD4, Brilliant Violet 650\u0026trade; anti-mouse CD11b, PE anti-mouse Ly-6G, FITC anti-mouse Ly-6C. After completing the staining, cell suspensions were pelleted by centrifugation (600g, 5 min, 4\u0026deg;C) and resuspended in PBS, then subjected to flow cytometry (BD LSR Fortessa).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone Marrow-Derived Myeloid (BMDM) and Neutrophil (BMDN) Isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow cells were isolated from murine femurs and tibias by flushing with 10 mL of ice-cold PBS. The suspension was centrifuged at 600\u003cem\u003eg\u003c/em\u003e for 5 min at 4 \u0026deg;C and erythrocytes were removed using erythrocytes lysis buffer (BD Biosciences, 555899). The reaction was quenched with buffer (PBS and 10%FBS), and the cell suspension was filtered through a 40\u0026mu;m cell strainer. CD11b⁺ cells were isolated using a specialized magnetic bead-based cell sorting kit (Biolegend 480110). Sorted cells were centrifuged and resuspended in culture medium (1640 and 10%FBS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter acquiring a single-cell suspension from bone marrow, cells were stained with fluorescently labeled anti-CD45 and anti-Ly6G antibodies for 20 minutes at 4\u0026deg;C, followed through Flow cytometer (BD) to obtain the neutrophil.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT Cell Migration Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpleen tissues were ground and through a 40\u0026nbsp;\u0026mu;m cell strainer. After erythrocyte lysis, single-cell suspensions were subjected to negative selection using a CD4\u003csup\u003e+\u003c/sup\u003e T Cell Isolation Kit (Biolegend). Purified CD4\u003csup\u003e+\u003c/sup\u003e T cells were cultured in T cell medium (RPMI-1640 and 10% FBS) containing 1% T cell activator (Stemcell, 100-1572). Half-medium changes were performed at 48h, with cells harvested at 72h for counting.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell migration was assessed using a 3 \u0026mu;m transwell chamber (Absin, abs7281).\u0026nbsp;Freshly isolated 100,000 CD4\u003csup\u003e+\u003c/sup\u003e T cells were plated to the upper chamber with T complete medium. WT or SYK\u003csup\u003eS544Y\u003c/sup\u003e BMDMs and BMDNs were added in the lower chamber at varying concentrations in RPMI-1640 medium. After 24-hour chemotaxis assay, migrated cells in the lower chamber were collected and quantified. Cells were then stained with anti-CD45 and anti-CD4 fluorescent antibodies for 20 minutes at 4\u0026deg;C and analyzed by flow cytometry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue specimens preserved in paraformaldehyde and embedded in paraffin were sectioned at 5 \u0026mu;m thickness and collected onto adhesion microscopy slides (Citotest). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor histopathological analysis,\u0026nbsp;tissue slides were stained with hematoxylin and eosin (H\u0026amp;E) and visualized using an Olympus BX51 microscope. For immunohistochemistry staining, the human and mouse skin paraffin sections were deparaffinized and rehydrated. Next, endogenous peroxidases of the tissue slides were quenched by 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 10 minutes. Antigen retrieval was performed by heat-mediated epitope unmasking in citrate buffer (20 min, 95 \u0026deg;C). Subsequent specimens were blocked with 3% goat serum and incubated with primary antibody overnight at 4\u0026deg;C. Appropriate secondary antibodies were incubated according to experimental objectives and then were visualized with DAB reagent according to the manufacturer\u0026apos;s protocol. Tissue sections were subsequently counterstained with hematoxylin and then dehydrated and mounted in neutral resins. Final results were visualized utilizing an Olympus BX51 microscope. Primary antibodies and their dilutions were as follows: Ki67 (1:200, Abcam, ab16667), Vegf (1:200, Abcam, ab317030).\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence, paraffin-embedded human and mouse skin sections were deparaffinized and rehydrated.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSubsequently, antigen retrieval was performed with 1 mM EDTA (pH 8.0) for 20\u0026thinsp;min in boiling water and cooled to room temperature. The slides were then incubated with 2.5% normal goat serum(VECTOR) diluted in PBS (pH 7.4) at room temperature for 30 min to block non-specific binding sites. Samples were incubated with fluorescent antibody overnight at 4℃. After counterstained with DAPI (1:1000; Sigma) for 10 min, samples were oriented and mounted in Fluorescent Mounting Medium and visualized using an Olympus BX51 microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from mouse skin and ear samples using 1ml Trizol reagent in RNA-free EP tubes. Then specimens RNA reverse transcribed into cDNA using Hifair\u003csup\u003e\u0026reg;\u003c/sup\u003e Ⅲ 1st Strand cDNA Synthesis SuperMix (Yeasen). Quantitative real-time PCR was conducted using Hieff qPCR SYBR Green Master Mix (Yeasen Biotechnology) following manufacturer\u0026apos;s protocols. Resluts were read with 7500 Real-Time PCR Instrument (Thermo Fisher Scientific). The primers used in this study were listed in Supplementary Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone marrow transplantation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemurs and tibias were isolated from WT and SYK\u003csup\u003eS544Y\u003c/sup\u003e mice, and bone marrow cells were flushed with PBS followed washed twice with DPBS. Recipient mice were subjected to lethal-dose irradiation using irradiator prior to transplantation. Isolated bone marrow cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e) from donor mice were intravenously injected into irradiated recipients. Seven months after bone marrow transplantation skin phenotypes indicative of psoriasis were assessed through histological analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacologic inhibition of SYK in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-month-old SYK mice and age-matched WT mice were treated by intragastric administration with 100ul R406 (dissolved in 5% DMSO and 95% corn oil) daily at 2 mg/mL for consecutive 14 days, while mice in the control group were treated with 100 \u0026mu;L vehicle (5% DMSO and 95% corn oil) daily. After 15 consecutive days of treatment, ear thickness in mice was measured using vernier calipers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll results are presented as the mean \u0026plusmn; standard deviation. Statistical analyses were conducted using GraphPad Prism 7 (GraphPad Software, Inc.), employing either unpaired two-tailed t-tests or one-way ANOVA as appropriate. All experiments were independently biological repeated at least three times. Throughout all figures: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Significance was concluded at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSYK expression is elevated in psoriatic patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous investigations revealed that SYK gain-of-function (GOF) mutations in patients induce immune dysregulation accompanied by multi-organ inflammatory manifestations, variably affecting cutaneous, gastrointestinal, articular, hepatic, and neurological tissues\u003csup\u003e[11]\u003c/sup\u003e. Interestingly, five of six reported SYK-GOF cases demonstrated consistent skin inflammation\u003csup\u003e[11]\u003c/sup\u003e, prompting our investigation into the exploration of the role of SYK in skin inflammation, such as psoriasis. Analysis of three independent GEO datasets (GSE13355, GSE14905, GSE54456) showed that SYK mRNA expression was significantly higher in the skin lesions of psoriatic patients (n=58) compared to normal controls (n=63) and non-lesional skin (n=58) (GSE13355) (Fig 1A, B, C). More importantly, the expression of SYK progressively reduced upon TNF-\u0026alpha; inhibitor and anti-IL-17R antibody treatment (Fig 1D). Furthermore, Immunohistochemical (IHC) analysis of skins also revealed significantly increased SYK protein abundance in the skin lesions of psoriasis patients compared to that of healthy controls (Fig 1E). Accordingly, immunofluorescence staining of patient biopsies showed phos-SYK (p-SYK) levels were enriched 6-fold in psoriatic skin compared to healthy donors, consistent with hyperactivated SYK signaling in disease progression (Fig 1F). These findings suggest that SYK activation may contribute to the progression of psoriasis. Next, we employed an imiquimod (IMQ)-induced psoriasis mouse model and found that p-SYK was greatly elevated following IMQ induction, while in the control group, p-SYK was almost not expressed (Fig 1G). These data collectedly demonstrated that SYK may be an important regulator in the initiation and development of psoriasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibit enhanced sensitivity to IMQ-induced psoriasis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the role of SYK in psoriasis, the SYK\u003csup\u003eS544Y\u003c/sup\u003e GOF moues model\u003csup\u003e[11]\u003c/sup\u003e built by our team was used. The SYK\u003csup\u003eS544Y\u003c/sup\u003e mice did not display obvious swelling and redness of ears as well as erythemato-squamous plaques of skin at the age of two month. (Fig 2A). However, H\u0026amp;E staining revealed a thicker epidermis and increased dermal immune cell infiltration in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice compared to WT controls (Fig 2B). To further evaluate whether the SYK\u003csup\u003eS544Y\u003c/sup\u003e alters responsiveness to stimulation compared to WT controls, we employed an IMQ-induced Ps mouse model (Fig 2C). Of note, SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibited heightened vulnerability to IMQ-induced inflammation. Ear thickness measurements demonstrated a 1.19-fold increase compared to the control group (Fig 2D). Additionally, SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibited significantly elevated Psoriasis Area and Severity Index (PASI) scores (Fig 2E). In alignment with observed phenomena, H\u0026amp;E assessment of SYK\u003csup\u003eS544Y\u003c/sup\u003e mice revealed exacerbated swelling, keratinocyte proliferation, dilated capillaries, and dermal inflammatory cell infiltration in the ears (Fig 2F).\u003ca id=\"_anchor_2\" href=\"#_msocom_2\" language=\"JavaScript\" name=\"_msoanchor_2\"\u003e\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, immunofluorescence analysis also demonstrated a 1.55-fold elevation of p-SYK levels in IMQ-induced SYK mice compared to that of IMQ-induced WT mice (Figure 2G). Splenomegaly, an indicator of systemic inflammation, was increased by 2.36-fold in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice compared to WT controls (Fig 2H). These results demonstrate that activation of SYK in mice may accelerates the progression of IMQ induced psoriasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSYK\u003csup\u003eS544Y\u003c/sup\u003e Mice Spontaneously exhibit Psoriasis-like skin inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough we did not observe a psoriasis phenotype in 2-month-old mice, there is epidemiological evidence delineates an age-dependent escalation in psoriasis prevalence, peaking during midlife\u003csup\u003e[16]\u003c/sup\u003e,\u0026nbsp;so we speculate that SYK\u003csup\u003eS544Y\u003c/sup\u003e mice may develop spontaneous psoriasis as they age. As expected, eight-month-old SYK\u003csup\u003eS544Y\u003c/sup\u003e mice developed prominent psoriasis-like skin inflammation affecting multiple regions, including the dorsal skin, ears, and tail (Fig 3A). Moreover, SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibited significantly increased auricular thickness, with 1.3-fold augmentation compared to WT controls (Fig 3B). According to the standardized clinical scoring system for assessing skin lesion severity,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe PASI score revealed a near 3.8-fold amplification in SYK mice compared to age-matched WT counterparts (Fig 3B).\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E staining also revealed that erythema, abnormal proliferation and differentiation of keratinocytes, and excessive angiogenesis were histologically evident in SYK\u003csup\u003eS544Y\u0026nbsp;\u003c/sup\u003emice tissues (Fig 3C). Nevertheless, psoriasis in human patients is characterized not only by epidermal thickening but also by prominent inflammatory histopathological features. To determine whether SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibit pathological features resembling human psoriasis, we stained the dorsal skin of mice. Immunohistochemical analysis demonstrated a pronounced elevation of Ki67 and Vegf protein in the dorsal skin of SYK\u003csup\u003eS544Y\u003c/sup\u003e mice (Fig 3D, E). Numerous previous research highlighted the critical role of diverse pro-inflammatory cytokines and chemokines in modulating the cutaneous microenvironment. Consistent with previous reports\u003csup\u003e[17,18]\u003c/sup\u003e,\u0026nbsp;we found that the mRNA expression levels of S100a9, S100a8, Il1\u0026beta;, Cxcl1, Tnf, Il6, Il23, and Il17a were significantly elevated in the skin lesions of SYK\u003csup\u003eS544Y\u003c/sup\u003e mice (Fig 4A). In addition to confirming the involvement of lesions in the ears, we systematically quantified mRNA expression of key inflammatory mediators in the skin, revealing a consistent upregulation pattern that paralleled the disease progression (Fig 4B). Together, these data suggest that SYK activation alone is sufficient to lead to the occurrence of spontaneous psoriasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGain-of-Function variant in SYK promotes neutrophil\u003cem\u003e\u0026nbsp;\u003c/em\u003erecruitment capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current academic consensus holds that the pathogenesis of psoriasis primarily arises from dysregulated interactions between epidermal keratinocytes and immune cells\u003csup\u003e[19]\u003c/sup\u003e. Among these, specific immune subsets play crucial roles in both disease initiation and amplification of the inflammatory cascade\u003csup\u003e[20,21]\u003c/sup\u003e. Thus, to verify whether the spontaneous psoriasis-like skin inflammation in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice is mediated by dysfunctional immune cell populations, bone marrow transplantation (BMT) experiments were performed. (Fig 5A). As expected, recipients of SYK\u003csup\u003eS544Y\u003c/sup\u003e bone marrow developed visible swelling and scaling on the ears, tail, and skin of the back at 8 months (Fig 5B). In contrast, recipients received WT bone marrow remained phenotypically normal. Histological analyses revealed marked epidermal hyperplasia and pronounced immune cell infiltration in the skin, ear, and tail of WT mice receiving SYK\u003csup\u003eS544Y\u003c/sup\u003e bone marrow (Fig 5C). To delineate the immune landscape shaped by SYK activation, flow cytometric analysis of ear tissue was conducted, the frequency of CD45⁺ cells in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice was elevated by 57.72% compared with WT controls (Fig 5D). Specifically, the frequencies of myeloid cells and neutrophils in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice were significantly elevated, with increases 2.35-fold and 48.47-fold, respectively (Fig 5E). In addition, CD4\u003csup\u003e+\u003c/sup\u003e T cell frequency markedly increased by 2.21-fold, while no differences were observed in CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig 5F).\u003c/p\u003e\n\u003cp\u003eGiven that psoriasis is a CD4\u003csup\u003e+\u003c/sup\u003e T cell-driven inflammatory disorder\u003csup\u003e[22,23]\u003c/sup\u003e and that neutrophils can release various chemokines (such as CCL20, CCL2, CXCL10) and cytokines to recruit T cells\u003csup\u003e[24]\u003c/sup\u003e, we aim to investigate whether SYK activation augments neutrophil-mediated T cell recruitment. Bone marrow-derived neutrophils (BMDN) were isolated for in vitro chemotaxis assays (Fig 5G). Notably\u003cu\u003e\u0026nbsp;\u003c/u\u003eBMDNs from SYK\u003csup\u003eS544Y\u0026nbsp;\u003c/sup\u003emice displayed enhanced recruitment capacity, showing a 1.54-fold and a 1.25-fold increase at different neutrophil densities compared with WT BMDNs (Fig 5H). In addition, we found that the capacity of myeloid cells to recruit T cells was also enhanced (Fig 5I). Collectively, these results demonstrate that SYK hyperactivation may promotes sustained neutrophil infiltration and enhances their recruitment capacity, contributing to psoriasis-like inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBMT and SYK inhibitor ameliorated psoriasis-like skin inflammation of mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering that WT mice receiving SYK\u003csup\u003eS544Y\u0026nbsp;\u003c/sup\u003ebone marrow spontaneously developed psoriasis, we wondered whether SYK\u003csup\u003eS544Y\u003c/sup\u003e mice that received WT bone marrow transplantation could alleviate the psoriasis phenotype (Fig 6A). Eight months post-transplantation, psoriatic SYK\u003csup\u003eS544Y\u003c/sup\u003e mice reconstituted with WT bone marrow displayed complete resolution of skin inflammation. (Fig 6B). H\u0026amp;E assessment revealed marked improvement, including reduced epidermal hyperplasia and partial restoration of dermal architecture with diminished immune cell infiltration (Fig 6C). These findings indicate that SYK activation within hematopoietic cells is a key driver of psoriatic inflammation. SYK inhibitors have been extensively investigated in various autoimmune and inflammatory diseases. For instance, Fostamatinib (R788), an oral SYK inhibitor, has been approved for the treatment of chronic immune thrombocytopenia and has shown clinical efficacy in rheumatoid arthritis (RA) and other inflammatory disorders\u003csup\u003e\u0026nbsp;[25,26]\u003c/sup\u003e. Additionally, R406, is widely used in preclinical studies to suppress SYK-mediated signaling\u003csup\u003e\u0026nbsp;[27]\u003c/sup\u003e. Previous studies have reported that SYK inhibitors attenuate psoriasis-like inflammation by blocking the dendritic cell-Th17 inflammatory axis\u003csup\u003e[28,29]\u003c/sup\u003e. We next evaluated the effect of pharmacological SYK inhibition to further validate the therapeutic potential of targeting SYK signaling. Administration R406 (10mg/kg) for two weeks significantly mitigated psoriasis-like pathology, with reduced erythema and scaling in treated mice (Fig 6D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistopathological results further confirmed attenuation of epidermal thickening and immune infiltration. Following intervention, the hyperkeratotic and parakeratotic plaques covering the epidermis in SYK\u003csup\u003eS544Y\u003c/sup\u003e mice were substantially reduced. (Fig 6E). Flow cytometric analysis was then performed to examine the impact of the SYK inhibitor on different immune cell populations. A 25.2% decrease in the frequency of CD45⁺ cells was observed in R406-treated mice compared to placebo-treated mice (Figure 6F). Following R406 treatment, SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibited a reduced proportion of myeloid cells. Notably, the proportion of neutrophils decreased by 93.03%. (Figure 6G). Taken together, these findings collectively demonstrate that pharmacological inhibition of SYK effectively ameliorates psoriasis-like skin inflammation, curtails immune infiltration, and improves the cutaneous immune microenvironment.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eA growing body of evidence confirms that psoriasis is a multifactorial immune-mediated disorder driven by the dysregulation of both innate and adaptive immune responses, in which autoimmune and inflammatory mechanisms synergistically promoting disease progression\u003csup\u003e[30,31]\u003c/sup\u003e. Animal models have been indispensable in elucidating pathogenic pathways and evaluating novel therapeutic strategies\u003csup\u003e[32]\u003c/sup\u003e. However, the widely used IMQ-induced model primarily reflects acute inflammation rather than the chronic, relapsing course characteristic of clinical psoriasis\u003csup\u003e[33]\u003c/sup\u003e. Thus, there is a pressing need to develop more clinically relevant mouse models. In the present study, CRISPR-Cas9 technology was employed to generate a gain-of-function SYK mutant mouse model. Intriguingly, these mice spontaneously developed psoriasis-like skin inflammation at 8 months, closely mimicking key clinical features and histopathological hallmarks of human psoriasis. Mechanistically, SYK hypoactivation trigger robust immune cell infiltration, particularly sustained neutrophil accumulation in lesional skin. Furthermore, SYK\u003csup\u003eS544Y\u003c/sup\u003e bone marrow-derived neutrophils and myeloid cells exhibited enhanced chemotactic and recruitment capacities. Collectively, these findings highlight a critical role for SYK hyperactivation in driving psoriatic pathology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSYK, a widely expressed non-receptor tyrosine kinase, orchestrates multiple signaling pathways across diverse immune cell lineages. Prior studies have established its contribution to psoriasis pathogenesis, particularly in macrophages and neutrophils\u003csup\u003e[34,35]\u003c/sup\u003e. Consistent with these findings, we observed markedly elevated SYK and p-SYK expression in both human lesions and mouse model skin. Moreover, SYK\u003csup\u003eS544Y\u003c/sup\u003e mice displayed heightened susceptibility to IMQ-induced psoriasis and developed more severe systemic chronic inflammation, underscoring the critical role of SYK hyperactivation in disease progression.\u003c/p\u003e\n\u003cp\u003eSYK regulates signal transduction and cytokine production in innate immune cells, including neutrophils, monocytes, DCs and B cells. Functional interrogation through bone marrow transplantation further demonstrated that SYK activation within hematopoietic cells was sufficient to drive psoriatic skin inflammation.\u0026nbsp;Notably, reciprocal transplantation with WT bone marrow completely reversed disease manifestations, highlighting the immune cell-intrinsic contribution of SYK signaling. Consistent with previous reports showing that SYK inhibition suppresses the IL-23/IL-17A axis in DCs to alleviate psoriasis, we found that the SYK inhibitor R406 significantly ameliorated skin inflammation and reduced immune cell infiltration, particularly neutrophils, in our model. These findings hint SYK in neutrophils as a promising therapeutic target for psoriasis.\u003c/p\u003e\n\u003cp\u003eWhile our findings established a critical role for SYK activation in neutrophils in psoriasis progression, several limitations should be acknowledged. Future investigations employing neutrophil-specific SYK mutant models will be essential to delineate the precise contribution of SYK GOF variants to psoriasis development. Additionally, neutrophils contribute to psoriatic pathology not only through recruitment but also via ROS production and IL-17 dependent mechanisms\u003csup\u003e[36,37]\u003c/sup\u003e. Whether SYK directly modulates these effector functions remains to be elucidated. These aspects have been described more deeper in our subsequent studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to Shanghai Jiaotong University Affiliated Sixth People\u0026rsquo;s Hospital South Campus and East China Normal University. We are grateful for the support from East China Normal University Public Platform for innovation (011). We thank Y. Zhang from the Flow Cytometry Core Facility of School of Life Sciences in ECNU.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.Z., X.Z., D.L. and Z.S. conceived and designed the project. Y.L conduct the research under the guidance of D.L. and Z.Z., Y.L. and H.G collected and analyzed the data. W.Y. was involved in conducting the histochemical and qPCR experiments. Z.Z and Y.L. wrote the manuscript with contributions from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by grants from the National Key R\u0026amp;D Program of China (2023YFC3403400), the National Natural Science Foundation of China (32200732), grants from the Shanghai Municipal Commission for Science and Technology (22YF1437700), the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054), the Key Medical Disciplines of Shanghai Municipal Health Commission (2024ZDXK0045)，the Shanghai Fengxian District Central Hospital Research Project (YJKT-25-01-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project does not involve clinical trials. Clinical trial number: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGriffiths C E M, Armstrong A W, Gudjonsson J E, et al. Psoriasis[J]. The Lancet, 2021, 397(10281): 1301-1315.\u003c/li\u003e\n\u003cli\u003eXue M, Yang R, Li G, et al. LGR4 Deficiency Aggravates Skin Inflammation and Epidermal Hyperplasia in Imiquimod-Induced Psoriasis[J]. Immunology, 2025, 174(2): 213-225.\u003c/li\u003e\n\u003cli\u003eStern R S. Psoriasis[J]. The Lancet, 1997, 350(9074): 349-353.\u003c/li\u003e\n\u003cli\u003eGhoreschi K, Balato A, Enerb\u0026auml;ck C, et al. Therapeutics targeting the IL-23 and IL-17 pathway in psoriasis[J]. Lancet (London, England), 2021, 397(10275): 754-766.\u003c/li\u003e\n\u003cli\u003eLee H J, Kim M. Challenges and Future Trends in the Treatment of Psoriasis[J]. International Journal of Molecular Sciences, 2023, 24(17): 13313.\u003c/li\u003e\n\u003cli\u003eWhitley S K, Li M, Kashem S W, et al. 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The Journal of Allergy and Clinical Immunology, 2021, 147(2): 439-455.\u003c/li\u003e\n\u003cli\u003eKamata M, Tada Y. Dendritic Cells and Macrophages in the Pathogenesis of Psoriasis[J]. Frontiers in Immunology, 2022, 13: 941071.\u003c/li\u003e\n\u003cli\u003eShao S, Fang H, Dang E, et al. Neutrophil Extracellular Traps Promote Inflammatory Responses in Psoriasis via Activating Epidermal TLR4/IL-36R Crosstalk[J]. Frontiers in Immunology, 2019, 10: 746.\u003c/li\u003e\n\u003cli\u003eGlennon-Alty L, Hackett A P, Chapman E A, et al. Neutrophils and redox stress in the pathogenesis of autoimmune disease[J]. Free Radical Biology \u0026amp; Medicine, 2018, 125: 25-35.\u003c/li\u003e\n\u003cli\u003eLiu X ting, Shi Z rui, Lu S yao, et al. Enhanced Migratory Ability of Neutrophils Toward Epidermis Contributes to the Development of Psoriasis via Crosstalk With Keratinocytes by Releasing IL-17A[J]. Frontiers in Immunology, 2022, 13: 817040. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Psoriasis, syk, neutrophils, immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-8234680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8234680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePsoriasis is a chronic, complex autoimmune disease characterized by massive immune cells infiltration including neutrophils. Spleen tyrosine kinase (SYK), a cytoplasmic non-receptor tyrosine kinase, has been reported to be essential to maintain the pro- inflammatory phenotype of neutrophils. Nevertheless, the influence of SYK on neutrophil function under psoriasis remains largely unknown. Here, we employed a SYK gain-of-function (GOF) mutant (SYK\u003csup\u003eS544Y\u003c/sup\u003e) mouse model to investigate the mechanistic function of SYK activation in the progression of psoriasis. Our findings demonstrate that SYK\u003csup\u003eS544Y\u003c/sup\u003e mice exhibit increased susceptibility to Imiquimod (IMQ) -induced psoriasis and spontaneously develop spontaneous psoriasis-like skin inflammation. SYK activation promotes myeloid cells, particularly neutrophils, infiltration in lesional skin and augments their recruitment activity. Furthermore, inhibition of SYK by R406 has demonstrated therapeutic efficacy on psoriasis. Consequently, these results suggest that SYK activation may contribute to the pathogenesis of psoriasis by augmenting neutrophil recruitment, and targeting SYK may be an attractive strategy to treat psoriasis.\u003c/p\u003e","manuscriptTitle":"SYK gain-of-function mutation regulates neutrophil functions to drive spontaneous psoriasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 13:31:28","doi":"10.21203/rs.3.rs-8234680/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b2c322f-5e13-4db4-9f7b-c54f8e7bb79e","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T05:55:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 13:31:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8234680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8234680","identity":"rs-8234680","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}