Saturated fatty acids-induced neutrophil extracellular traps contribute to exacerbation and biologic therapy resistance in obesity-related psoriasis

Saturated fatty acids-induced neutrophil extracellular traps contribute to exacerbation and biologic therapy resistance in obesity-related 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 Article Saturated fatty acids-induced neutrophil extracellular traps contribute to exacerbation and biologic therapy resistance in obesity-related psoriasis Juan Tao, Yuting Xia, Jiajia Lan, Jing Yang, Shijie Yuan, Xiaorong Xie, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4940299/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Cellular & Molecular Immunology → Version 1 posted 9 You are reading this latest preprint version Abstract Psoriasis patients with obesity tend to show a serious clinical manifestation and have poor responses to various biological agents in most cases. However, the mechanisms in obesity-exacerbated psoriasis remain enigmatic. In this study, we found that the abundance of systemic and localized cutaneous neutrophil extracellular traps (NETs) in obesity-induced aggravation of psoriasis was positively correlated with disease severity, and inhibition of NETs alleviated psoriatic dermatitis in obese mice. Mechanistically, we found that changes of fatty acid composition in obese subjects resulted in the deposit of saturated fatty acids (SFAs), which promoted the release of NETs via the TLR4-MD2/ROS signaling pathway. We further found that NETs potentiated IL-17 inflammation in obesity-exacerbated psoriasis, especially γδT17-mediated immune responses. Moreover, SFAs induced decreased response to anti-IL17A treatment in psoriasis-like mice, whereas inhibition of NETs improved the beneficial effects of anti-IL17A in psoriasis-like mice with lipid metabolism disorders. Our findings collectively suggest that SFAs-induced NETs play a critical role in the exacerbation of obesity-related psoriasis, and provide potential new strategies for the treatment of refractory psoriasis patients with lipid metabolism disorders clinically. Biological sciences/Immunology/Innate immune cells/Granulocytes/Neutrophils Biological sciences/Immunology/Cell death and immune response Biological sciences/Immunology/Inflammation/Chronic inflammation Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Immunology/Translational immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Psoriasis is a common chronic inflammatory skin disease that is induced by hereditary and environmental factors, affecting approximately 125 million people worldwide ( 1 , 2 ). Patients with psoriasis often have systemic metabolic disorders, including obesity, cardiometabolic diseases, metabolic syndrome, type 2 diabetes, and dyslipidemia, which can lead to aggravation of patients' condition and deterioration of their life quality ( 3 – 6 ). It has been reported that obesity doubles the risk of psoriasis and higher body mass index (BMI) is independently associated with the development of psoriasis ( 7 , 8 ). Besides, several clinical studies have shown that psoriasis patients with obesity have poor responses to various biological agents ( 9 , 10 ). Secukinumab has been reported to exhibit better psoriasis area and severity index (PASI) 75 responses in psoriasis patients weighing < 90 kg than heavier patients after 12 weeks of treatment and obesity-related factors could adversely affect its efficacy ( 11 , 12 ). And ustekinumab has been found to show worse PASI 75 and PASI 90 responses in psoriasis patients weighing > 100 kg ( 13 ). In addition, studies have indicated the exacerbation of psoriatic dermatitis in obese mice ( 14 , 15 ). As the product of neutral fat metabolism, saturated fatty acids (SFAs) have been found to be major risk factors for the amplification of psoriasis inflammation ( 14 ). However, the particular mechanisms of lipid metabolic disorder exacerbating psoriasis is not well understood. Neutrophil extracellular traps (NETs) are fibrous weblike structures which protrude from the membranes of activated neutrophils and are coated with histones, proteases, and granular cytosolic proteins ( 16 , 17 ). Accumulative evidence has revealed the important roles of NETs in inflammatory diseases and metabolic disorders. The capabilities of neutrophils to migrate and produce NETs are elevated in obese subjects ( 18 , 19 ). In non-alcoholic steatohepatitis, free fatty acids (FFAs) stimulate NETs formation, which facilitate the crosstalk between innate and adaptive immunity by promoting Treg activity through metabolic reprogramming and promote development of hepatocellular carcinoma ( 20 , 21 ). Cholesterol crystals trigger neutrophils to release NETs in cardiometabolic diseases, which prime macrophages and Th17 cells to produce cytokines that amplify immune cell recruitment in atherosclerotic plaques ( 22 ). As a hallmark of psoriatic skin lesions, neutrophils infiltration appears early in developing lesions ( 23 , 24 ). Studies show that NETs are prominent within psoriasis lesions and in peripheral blood, and the positive correlation between the amount of NETotic cells in the peripheral blood and psoriasis disease severity has also been reported ( 25 – 27 ). Thus, it would be interesting to clarify the functional link between NETs and the aggravation of psoriasis induced by metabolic disorders. In this study, we identified the increase of systemic NETs and localized cutaneous NETs in both obese psoriasis patients and imiquimod (IMQ)-induced psoriasis-like mice, which was positively correlated with disease severity. And NETs inhibitor alleviated psoriatic dermatitis in obese mice. We also present experimental evidence that the increased SFAs promoted the release of NETs through TLR4-MD2/ROS pathway. Moreover, NETs amplified IL-17 inflammation in obesity-exacerbated psoriasis, especially γδT17-mediated immune responses. Interestingly, we demonstrated that SFAs induced decreased response to anti-IL17A treatment in psoriasis-like mice, whereas inhibition of NETs improved the beneficial effects of anti-IL17A in psoriasis-like mice with lipid metabolism disorder. Thus, our research indicates that NETs play a critical role in obesity-exacerbated psoriasis and brings new insights for the treatment of psoriasis patients with lipid metabolism disorders clinically. Results NETs elevated systemically and skin locally in obesity-exacerbated psoriasis We compared disease severity in lean psoriasis patients (BMI < 24) (n = 47) and obese psoriasis patients (BMI ≥ 28) (n = 31). A significantly increased severity of psoriasis was observed in obese patients (Supplemental Fig. 1A) and there was a positive association between PASI and BMI (Supplemental Fig. 1B). In addition, we observed more patients showing poor responses to biological agents in BMI ≥ 28 group than that in BMI < 24 group. Histologic analysis showed increased epidermal hyperplasia and neutrophils infiltration in the skin lesions of obese psoriasis patients (Supplemental Fig. 1C). Mice were fed with either a control diet (CD) or high fat diet (HFD) for 16 weeks and HFD consumption led to significant weight gain in mice (Supplemental Fig. 2A). Then daily application of IMQ for 6 days was conducted to develop psoriasis-like dermatitis, with Vaseline treatment in sham group (Supplemental Fig. 2B). As expected, HFD/IMQ-treated mice exhibited significantly more severe cutaneous manifestations and local pathological changes than CD/IMQ-treated mice (Supplemental Fig. 2, C-E). And HFD/IMQ-treatment led to significantly increased proportion of IL-17A + , IL-17F + , IL-22 + , and RORγt + CD3 + T cells and decreased proportion of Foxp3 + CD3 + T cells in skin-draining lymph node (dLN) (Supplemental Fig. 2F). Psoriatic lesions are characterized by neutrophil infiltration, the activation of which leads to release of web-like structures named NETs ( 23 , 28 , 29 ). Previous reports have shown that NETs exist in skin lesions of both psoriasis patients and IMQ-induced psoriasis-like mice ( 29 , 30 ). To identify NETs within psoriasis lesions, immunofluorescent co-staining for MPO and NETs marker, citrullinated histone H3 (citH3), was performed. We found that NETs were present in abundance within obesity-exacerbated inflamed skin of psoriasis patients (Fig. 1 A). Sera from psoriasis patients with BMI < 24 and BMI ≥ 28 were analyzed for myeloperoxidase (MPO) -DNA complexes and double-stranded DNA (dsDNA). Concentrations of serum NETs in obese psoriasis patients were significantly higher than that in lean psoriasis patients (Fig. 1 B) and the abundance of serum NETs was positively correlated with disease severity (Fig. 1 C). Consistently, NETs markers were significantly increased within psoriasis-like lesions and in serum of HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Fig. 1 , D and E). In addition, flow cytometry analysis of dLN single-cell suspensions revealed the significant increase of neutrophil recruitment and NETs formation in HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Supplemental Fig. 2F and Fig. 1 F). Notably, concentrations of serum NETs in HFD/Vas-treated mice were significantly increased compared with those in CD/Vas-treated mice (Fig. 1 E). And HFD alone promoted neutrophil infiltration and NETs generation in mouse skin and dLN (Fig. 1 , D and F). To further investigate the effect of obesity on NETs production, neutrophils from CD-fed and HFD-fed mouse bone marrow were isolated. NETs extension was evaluated by confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). Neutrophils from HFD-fed mouse exhibited an increased capacity of releasing more web-like structure to extracellular space compared with those from CD-fed mouse (Fig. 1 G). The enhanced spontaneous NETs of HFD-fed mouse neutrophils in the absence of specific stimulation ex vivo suggested that NETs have already proceeded in obesity subjects. The result was further validated with flow cytometry analysis. Neutrophils from HFD-fed mouse bone marrow displayed significantly increased mean fluorescence intensity (MFI) of SYTOX Red and citH3 and increased proportion of citH3 + SYTOX Red + populations with or without LPS stimulation (Fig. 1 H). Taken together, these data implicate that obesity leads to exacerbated psoriasis and increased NETs may contribute to obesity-exacerbated psoriasis. Targeting NETs alleviated obesity-exacerbated psoriasis in mice To further verify our hypothesis that enhanced NETs generation may contribute to aggravated inflammation caused by obesity, CD-fed or HFD-fed mice were daily treated with DNase Ⅰ, a nuclease that dismantles NETs in vivo , or equivalent vehicle starting 3 d prior to the imiquimod treatment (Fig. 2 A). DNase Ⅰ treatment resulted in the expected reduction of NETs markers in skin, serum, and dLN, demonstrating the efficacy of DNase Ⅰ at eliminating NETs (Fig. 2 , B-D). DNase Ⅰ treatment had no significant impact on the pathogenesis of IMQ alone according to clinical severity and histologic analysis (Fig. 2 , E and F). However, NETs inhibition significantly reduced inflammation induced by HFD prior to IMQ based on the ameliorated symptoms, reduced epidermal thickening and downregulated infiltration of neutrophils (Fig. 2 , E and F, Supplemental Fig. 3A). Flow cytometry analysis further revealed that DNase Ⅰ treatment significantly reduced leukocytes infiltration in skin and neutrophil accumulation in dLN of HFD/IMQ-treated mice compared with CD/IMQ-treated mice ((Supplemental Fig. 3, B and C). Collectively, our data suggest that NETs contribute to obesity-exacerbated psoriasis and inhibition of NETs remarkably relieve the amplified inflammation. Deposit of SFAs in obesity-exacerbated psoriasis promotes the release of NETs SFAs are considered as the link between lipid metabolism and the activation of various inflammatory pathways in obese individuals ( 31 , 32 ). We next assessed whether the disturbance of fatty acid metabolism was related to obesity-exacerbated psoriasis. The total amount of FFAs in the serum of obese psoriasis patients was significantly increased compared with lean psoriasis patients (Fig. 3 A). And the blood FFAs concentration was positively correlated with disease severity (Fig. 3 B). Moreover, serum FFAs level of psoriasis patients showed positive correlation with serum NETs concentrations (Fig. 3 C). We also investigated the changes in the amount of FFAs in mouse serum after intake of HFD. After 16 weeks of HFD feeding, the total amount of FFAs in mouse serum was significantly elevated (Fig. 3 D). Targeted lipidomic analysis was further performed and we found HFD consumption changed the composition of FFAs in mouse serum (Fig. 3 E; Supplemental Fig. 4, A and B). SFAs such as palmitic acid (PA) and stearic acid (SA) were among the most enriched FFAs in the serum of HFD-fed mice (Fig. 3 F). Independent of obese phenotype, SFAs act as major risk factors for the amplification of skin inflammation ( 14 , 15 ). To investigate whether SFAs functioned as a stimulus for NETs formation, neutrophils from bone marrow of CD-fed mice at 6–8 weeks of age were treated with different concentrations of PA and SA for 3 h. We found that PA induced NETs ex vivo in a dose-dependent manner and PA-treated neutrophils exhibited the greatest capacity to generate NETs when the concentration of PA was at 200 µM (Fig. 3 G). Consistently, SA also induced NETs production ex vivo in a dose-dependent manner, which exhibited the greatest ability for NETs generation at 400 µM (Fig. 3 H). The results of CLSM and SEM further proved that PA promoted the release of web-like structure from neutrophils with PMA and DNase Ⅰ as positive and negative control respectively (Fig. 3 I). In summary, HFD feeding leads to an increase of long-chain SFAs, which promote NETs generation during obesity. SFA promotes NETs generation through TLR4–MD2/ROS signaling pathway in obesity-exacerbated psoriasis To further investigate the effects of abnormal lipid metabolism on neutrophils, RNA sequencing (RNA-seq) was performed on neutrophils isolated from CD-fed and HFD-fed mouse bone marrow. The results revealed differential gene expression, with 2394 genes upregulated and 1918 genes downregulated (FC > 1.5, P value < 0.05) in neutrophils from HFD-fed mice compared to CD-fed mice (Fig. 4 A). And neutrophils from HFD-fed mice had increased expression of genes involved in ROS generation (i.e., Cgas, Nox1, and Junb) and NETs production (i.e., Selp, Tlr2 and Nlrp3) (Fig. 4 B). Myeloid cell–mediated inflammation has been linked to TLR4 in obesity and insulin resistance, which can be activated by long-chain SFAs ( 33 – 35 ). Endocytosis of palmitate/TLR4–MD2 complex generates NADPH oxidase (NOX) -mediated superoxide generation in endothelial cells, cardiomyocytes and hepatic macrophages ( 36 – 38 ). Flow cytometry analysis of skin single-cell suspensions from mice showed significantly increased TLR4–MD2 complex formation in neutrophils from HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Fig. 4 C). To investigate the role of TLR4–MD2/ROS signaling in SFA-induced NETs, neutrophils from CD-fed and HFD-fed mouse bone marrow were isolated respectively. Flow cytometry analysis and CLSM revealed that neutrophils from HFD-fed mouse bone marrow displayed significantly increased TLR4–MD2 complex formation and ROS generation with or without LPS stimulation (Fig. 4 , D and E; Supplemental Fig. 5, A and B). We then treated neutrophils isolated from bone marrow of CD-fed mice at 6–8 weeks of age with 200 µM PA or 400 µM SA. Both of PA and SA were able to promote TLR4–MD2 complex formation and ROS generation as measured by flow cytometry analysis and CLSM (Fig. 4 , F and G; Supplemental Fig. 5, C, D and E). TAK-242, a TLR4 specific inhibitor, and DPI, a NOX inhibitor, were used to further identify the involvement of TLR4–MD2/ROS signaling in SFA-induced NETs formation. We found that TAK-242 significantly reduced NOX-derived ROS (Fig. 4 H; Supplemental Fig. 5F). Flow cytometry analysis revealed that treatment with TAK-242 and DPI significantly abrogated the increase of NETs formation induced by PA and SA while treatment with TAK-242 combined with H 2 O 2 reversed this effect (Fig. 4 H; Supplemental Fig. 5F). Moreover, immunofluorescent co-staining for NETs, TLR4, and ROS was performed. The results showed that not only citH3 + neutrophils infiltration was increased in skin lesions of obese psoriasis patients and mice but also that these cells showed higher TLR4 expression and more ROS generation (Fig. 4 , I and J). In total, our results implicate that TLR4–MD2/ROS signaling pathway is crucial for SFAs-induced NETs generation. Obesity-induced generation of NETs promotes γδT17-mediated inflammation in psoriasis Immunological studies have identified IL-17 as one of the key drivers of psoriasis pathogenesis ( 1 ). As our results showed that IL-17A + cells infiltration was increased in psoriatic lesions of obese patients and mice, while NETs inhibition remarkably relieved this phenomenon (Supplemental Fig. 6, A-C), we hypothesized that NETs promoted IL-17-mediated inflammation in obesity-exacerbated psoriasis. Immunofluorescent co-staining for MPO, citH3, and IL-17A revealed that the abundance of NETs within obesity-exacerbated inflamed skin of psoriasis patients and psoriasis-like mice was accompanied by increased IL-17A + cell infiltration (Supplemental Fig. 6, D and E). In addition, NETs inhibition in mouse skin with DNase Ⅰ was paralleled by a reduction of IL-17A + cells (Supplemental Fig. 6F). We next sought to further dissect the main source of NETs-promoted IL-17 in obesity-exacerbated psoriasis. Immunofluorescent co-staining for citH3, IL-17A, TCRγδ, and CD4 confirmed that NETs and dermal γδT17 were often found in close proximity while there was a considerable distance between NETs and CD4 + T cells, implying potential interactions between NETs and γδT17 in obesity-exacerbated psoriasis (Fig. 5 A). In addition, HFD/IMQ-treatment brought about significantly increased proportion of γδT cells in CD3 + T cells from dLN (Fig. 5 B) and NETs inhibition reversed this effect (Fig. 5 C). Flow cytometry analysis also revealed significant increased proportion of IL-17A + , IL-17F + , and RORγt + γδT, IL-17A + , IL-17F + , and RORγt + CD4 + T cells in CD3 + T cells from dLN of HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Fig. 5 , E and F). Additionally, treatment with DNase I led to a significant decrease proportion of IL-17A + , IL-17F + , and RORγt + γδT, IL-17A + , IL-17F + , and RORγt + CD4 + T cells in CD3 + T cells from dLN of HFD/IMQ-treated mice, while that of CD/IMQ-treated mice remained comparable (Fig. 5 , F and G). Thus, NETs promote the induction of IL-17 inflammation in obesity-exacerbated psoriasis, especially γδT17-mediated immune responses. DNase Ⅰ potentiates therapeutic effect of anti-IL17A in PA-treated psoriasis-like mice As SFA-induced NETs heightened IL-17 inflammation in obesity-exacerbated psoriasis, we supposed that SFAs might induce insensitivity to anti-IL17A therapy for psoriasis and NETs inhibition could reverse its effect. Thus, mice were daily treated with vehicle, anti-IL17A, PA and anti-IL17A, or PA and anti-IL17A combined with DNase Ⅰ 3 d prior to the imiquimod treatment (Fig. 6 A). The curative effect of anti-IL17A was apparent with significantly relieved cutaneous manifestations in anti-IL17A-treated psoriatic mice compared with psoriatic mice without treatment (Fig. 6 , B and C). When interfered with PA at the same time, anti-IL17A alone failed to alleviate skin symptoms, including erythema, thickness, and scaling, in psoriasis-like mice, while DNase Ⅰ combined with anti-IL17A treatment significantly remedied these conditions (Fig. 6 , B and C). Consistently, histologic analysis of mouse dorsal skin showed that anti-IL17A failed to alleviate local pathological changes when psoriasis-like mice were interfered with PA at the same time, while DNase Ⅰ combined with anti-IL17A treatment significantly reduced epidermal thickness, keratinocyte proliferation, and neutrophil infiltration (Fig. 6 C, Supplemental Fig. 7A). In agreement with our hypothesis, we found that PA intervention promoted IL-17A + cell and citH3 + cell infiltration in mouse psoriatic lesions (Supplemental Fig. 7A). Moreover, anti-IL17A therapy alone failed to inhibit the production and transcription of IL-17A and IL-17F in mouse dLN when interfered with PA at the same time, while DNase Ⅰ combined with anti-IL17A treatment showed reversal effects with decreased proportion of IL-17A + , IL-17F + , and RORγt + γδT, IL-17A + and IL-17F + CD4 + T cells in CD3 + T cells from mouse dLN (Fig. 6 , D and E). We also found that PA intervention resulted in increased proportion of γδT cells in CD3 + T cells from dLN and NETs inhibition reversed this effect (Supplemental Fig. 7B). Overall, SFAs lead to decreased response to anti-IL17A treatment in psoriasis and inhibition of NETs potentiates therapeutic effect of anti-IL17A in psoriasis-like mice with lipid metabolism disorder. Discussion Obesity is a major risk factor for psoriasis ( 6 ). Previous work has indicated that disease severity of psoriasis relates to heightened NETs formation, but the regulatory roles and internal mechanism of NETs in psoriasis still remain elusive ( 25 , 29 ). Herein, we delineated that increased NETs induced by abnormal fatty acid metabolism remodulated skin immune microenvironment and it was essential for obesity-exacerbated psoriasis. Elevated SFAs in obese individuals promoted release of NETs through TLR4–MD2/ROS signaling pathway, which further amplified type 17 immune responses. Moreover, our results provided evidence that SFAs drove the abrogation of anti-IL17A therapeutic effect in psoriasis with lipid metabolism disorder and that NETs inhibition combined with anti-IL17A treatment could reverse this phenomenon. Therefore, we document that NETs inhibition may serve as a potential therapeutic avenue in defined psoriatic settings with lipid metabolism disorder clinically (Fig. 7 ). The existence of NETs in skin lesions and blood of both psoriasis patients and imiquimod-induced psoriasis-like mice has been addressed ( 25 , 29 , 30 ). Here, we found that systemic NETs and localized cutaneous NETs in both obese psoriasis patients and IMQ-induced psoriasis-like mice were significantly higher than lean subjects. Inhibition of NETs significantly alleviated the inflammation in obesity-aggravated psoriasis but failed to influence the pathogenesis of imiquimod alone. In line with our study, NETs inhibition ameliorates skin inflammation in psoriasis exacerbated by IL-36 receptor antagonist deficiency and fungi-aggravated psoriasis but exhibits no significant impact on mice treated by imiquimod alone ( 39 , 40 ). Moreover, studies have reported that NETs directly promote psoriasis exacerbation by mediating keratinocytes activation, plasmacytoid dendritic cells stimulation, and release of IL-17A ( 25 , 29 , 30 , 41 , 42 ). However, little is known about the precise mechanism regulating the production of NETs in psoriasis. According to our results, changes in fatty acid metabolism led to the increase of NETs generation. Others have reported that the release of keratinocyte exosomes activates neutrophils and induces NETs formation ( 43 ). Moreover, the self-propagating vicious cycle mediated by RNA and LL37 via TLRs recognition and NETs formation contributes to chronic inflammation in psoriasis ( 44 ). Thus, it’s plausible that NETs formation is vital for the exacerbation of psoriatic inflammation induced by obesity. According to our results, neutrophils from obese subjects are more prone to generate NETs. Other investigators have shown similar results that obesity/lipid metabolism disorder leads to greater NETs formation in influenza pneumonia mouse model, vascular dysfunction mouse model, the STAM mouse model, and ApoE-deficient mice ( 20 , 22 , 45 – 47 ). However, the exact mechanism of obesity/lipid metabolism disorder promoting NETs generation awaits further study. Metabolic pathways that regulate glycolysis and energy supply have been proved to be tightly linked to the capacity of neutrophils to produce NETs ( 48 – 50 ). Moreover, SFAs such as PA and SA are among the most enriched FFAs in both serum and skin from HFD-fed mice and SFAs have been regarded as major risk factors for the amplification of skin inflammation ( 14 , 15 ). Thus, we focused on SFAs as a contributor for augmented NETs formation in obesity-exacerbated psoriasis. Our study indicates that NETs have already proceeded in obesity subjects and SFAs promote NETs production in a dose-dependent manner. Mechanistically, our data reveal that SFA-induced TLR4-MD2 complex formation further promote NOX-derived ROS, which leads to NETs generation in obesity-exacerbated psoriasis. SFAs directly stimulate inflammatory gene expression by way of TLR4 signaling, while MUFAs and PUFAs fails to activate TLR4 signaling ( 51 , 52 ). In hepatic macrophages of an HFD-induced hepatic steatosis mouse model, endocytosis of palmitate/TLR4–MD2 complex generates NOX2-mediated ROS ( 37 ). In obesity-associated myocardial injury, saturated palmitic acid direct binds to TLR4 accessory protein MD2 and activates downstream inflammatory responses ( 38 ). Considering that ROS generation is fundamental for NET formation, we conclude that SFAs promote NETs production through TLR4-MD2/ROS signaling pathway ( 53 ). Further studies need to determine whether other abnormal lipid metabolites are engaged in obesity-promoted NETs generation. Our results reconfirmed the known role for NETs in promoting IL-17/Th17 responses ( 54 , 55 ). With well-documented proinflammatory functions, neutrophils externalize IL-17A-decorated NETs in many diseases such as psoriasis, ankylosing spondylitis, pulmonary fibrosis, asthma, Alzheimer’s disease, and acute myocardial infarction ( 29 , 56 – 60 ). Previous studies have also illustrated the indirect amplification of Th17 responses by NETs through macrophages, dendritic cells, and monocytes ( 22 , 61 – 63 ). In addition, NETs directly prime T cells by lowering their activation threshold and promotes Th17 differentiation, which is mediated through a TLR2/MyD88-dependent pathway ( 24 , 64 , 65 ). Interestingly, in our study, other than the augmentation of Th17 responses by NETs, we propose that NETs induce innate IL-17 production from γδT cells, which even seems to play a more important role in obesity-exacerbated psoriasis than Th17-derived IL-17. Furthermore, IL-17A induces recruitment of neutrophils and enhances NETs generation ( 66 – 68 ). Thus, a corollary to these findings implies that NETs formation has a positive feedback function on the IL-17A driven dynamic inflammation, which further elicits NETs formation and promotes immunopathology in obesity-exacerbated psoriasis. However, the putative mechanism about interactions between NETs and IL-17A-producing T cells remains to be investigated. Clinically, a weight-based approach to secukinumab and ustekinumab has been proposed, indicating that higher doses of biologics may be required for psoriasis patients with greater bodyweight to optimize their efficacy ( 11 , 13 ). As many obese psoriasis patients show poor responses to immunotherapy and carry an intolerable psychological and economic burden, it is vital to identify biomarkers for the evaluation of therapeutic efficacy and to establish new strategies for combined immunotherapy ( 11 , 12 , 69 ). However, little is known about the precise mechanism of decreased responses resulting from lipid metabolism disorder. Our research identifies SFAs as a major hindrance for anti-IL17A treatment in psoriatic mice with severe symptoms and remarkable local pathological changes. Incredibly, NETs inhibitor combined with anti-IL17A treatment could improve the decreased response in the light of ameliorated symptoms and pathological changes. Thus, our results suggest an intriguing possibility that NETs inhibitors combined with anti-IL17A could be a promising option for psoriasis patients with lipid metabolism disorders. However, although we identified SFAs as the major stimulus for augmented skin inflammation, the effects of unsaturated fatty acids were not investigated and their clear functional differences were not clarified. And further clinical investigation is warranted to learn whether NETs inhibition combined with anti-IL17A treatment can reverse the severe condition of obese psoriasis patients. In summary, we highlight the importance of SFAs-induced NETs for dermatologic conditions with overnutrition status. Mechanistically, TLR4-MD2/ROS signaling pathway is critical for SFAs-induced NETs generation. In addition, our research suggests a feed-forward loop in which NETs trigger IL-17 immunity, evolving neutrophils from terminal effectors to complex mediators. Thus, our study reveals that breaking the vicious cycle with NETs inhibitors could be a novel and promising candidate for psoriasis patients with obesity, especially those who show poor responses to various biological agents clinically. Materials and methods Patient samples Serum and skin samples were collected from outpatients with psoriasis vulgaris in department of dermatology at Wuhan Union Hospital between August 2022 and August 2023. Human studies were approved by the Ethics Committee for Clinical Trials at Huazhong University of Science and Technology and all patients signed informed consent. All patients were evaluated for baseline characteristics including age, gender, height, weight, BMI, diagnosis, PASI, body surface area (BSA), dermatology life quality index (DLQI). Mice research Female C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Starting at 3 weeks of age, female mice were fed a CD or HFD for 16 weeks. An HFD provides 60% of its energy in the form of fat (Cat # 112252, Dyets Inc.), whereas an CD provides 10% of its energy in the form of fat. For psoriatic dermatitis mouse model, 62.5 mg of 5% IMQ cream was applied on the back skin. Clinical scores and skin thickness were assessed as described. To inhibit NETs formation, mice were intravenously injected daily with 50 mg/kg/d of DNase I (DNase I; Roche) resuspended in PBS starting 2 d prior to the IMQ treatment and during the whole duration of the IMQ treatment. For PA or anti-IL17A treatment, mice were intravenously injected daily with 5 µmol/d of PA or 30 µg/d of anti-IL17A starting 2 d prior to the IMQ treatment and during the whole duration of the IMQ treatment. All mice were maintained in the animal facilities under specific pathogen-free conditions. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology. Neutrophil isolation and ex vivo NETs formation Bone marrow cells was acquired from mice as described. Neutrophils were isolated by density gradient centrifugation in Histopaque 1077 (Sigma-Aldrich) and Histopaque 1119 (Sigma-Aldrich). Freshly isolated neutrophils were seeded on 96-well round bottom culture plates (1.0 × 10 5 cells) and poly-D-lysine coated coverslips in 12-well round bottom culture plates (1.0 × 10 6 cells). After a 1-hour incubation period at 37℃, the PA, TAK-242, DPI, and H 2 O 2 were added for corresponding incubation in different experiments. LPS or PMA were used as positive controls, whereas DNase Ⅰ served as a negative control. Cell-impermeable DNA dye SYTOX Red (Thermo Fisher Scientific) were added to the incubation system in some experiments. Neutrophils were then cultured for 4 h at 37° in a 5% CO 2 atmosphere, after which cells in 96-well plates were collected for flow cytometry and coverslips were used for scanning electron microscopy or immunofluorescent staining. Detection of NETs in serum dsDNA was measured in the serum using Quant-iT PicoGreen double-stranded DNA reagent (Invitrogen) according to the manufacturer’s protocol. A capture ELISA assay was used to detect myeloperoxidase MPO-DNA complexes in the serum as described. Briefly, a MPO ELISA kit (Hycult) was used according to the manufacturer’s directions. After blocking in 2% BSA, 40 µl of serum was added per well in combination with the peroxidase-labeled anti-DNA monoclonal antibody (Cell Death ELISAPLUS; Roche) according to the manufacturer´s instructions. After 4 h of incubation at room temperature on a shaking device (300 rpm), the samples were washed three times with 200 µl PBS per well and the peroxidase substrate (ABTS) of the kit (Cell Death ELISAPLUS; Roche) was added. The absorbance at 405 nm wavelength was measured after 40 min incubation at 37°C in the dark. Histology and immunohistochemistry Mouse back skin and skin samples from psoriasis patients were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 mm sections. H&E staining was performed to evaluate inflammatory infiltration and epidermal thickness. Immunohistochemistry staining was applied with the following antibodies respectively: anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam), anti- myeloperoxidase antibody (Abcam), anti-IL-17A antibody (Abcam), anti-Ki67 antibody (Abcam), anti-Ly6G antibody (Abcam), anti-TLR4 antibody (Abcam), anti-TLR4/MD2 antibody (Biolegend), anti‑Mouse TCR Gamma + TCR Delta (LifeSpan), anti-CD4 antibody (Abcam). The primary antibody was omitted in negative controls. Gas chromatography mass spectrometry The GC analysis was performed on trace 1300 gas chromatograph (Thermo Fisher Scientific, USA). The GC was fitted with a capillary column Thermo TG-FAME (50 m*0.25 mm ID*0.20 µm) and helium was used as the carrier gas at 0.63 mL/min. Injection was made in split mode at 8:1 with an injection volume of 1 µL and an injector temperature of 250℃. The temperature of the ion source and MS transfer line were 300℃ and 280℃, respectively. The column temperature was programmed to increase from an initial temperature of 80℃, which was maintained for 1 min, followed by an increase to 160℃ at 20℃/min, which was maintained for 1.5 min, and increase to 196℃ at 3℃/min, which was maintained for 8.5 min, and finally to 250℃ at 20℃/min and kept at this temperature for 3 min. Mass spectrometric detection of metabolites was performed on TSQ 9000 (Thermo Fisher Scientific, USA) with electron impact ionization mode. Single ion monitoring (SIM) mode was used with the electron energy of 70 eV. RNA sequencing and transcriptomics analysis Total RNA of neutrophils isolated from CD-fed and HFD-fed mouse bone marrow was extracted with TRIzol (Invitrogen). The cDNA library construction, library purification, and RNA sequencing (RNA-seq) were conducted on a DNBSEQ platform with PE150 (read length) at Huada Gene Technology Co., Ltd. (Shenzhen, China) following standard protocols. Expression level of gene was calculated by RSEM (v1.3.1). Differential expression analysis was performed using the DESeq2 with P value ≤ 0.05. Heatmaps were analyzed and plotted using the R programming language (version 4.2.3; pheatmap package and ComplexHeatmap package). Immunofluorescence Neutrophils were fixed in 4% paraformaldehyde in PBS for 30 min and permeabilized in 0.5% Triton X-100 in PBS for 3 min; after blocking with 2% BSA, they were incubated with the specific primary antibodies for citH3 (Abcam), MPO (Abcam), or anti-TLR4/MD2 antibody (Biolegend). Secondary antibody conjugated with FITC or Cy3 were used. The samples were observed under a confocal microscope (Dragonfly/CR-DFLY-201-40). Scanning electron microscopy Neutrophils were fixed with 2.5% glutaraldehyde for 1 h and then washed three times with PBS. Cells were post-fixed with 1% OsO 4 for 1 h and then further washed three times with PBS. Samples were dehydrated, by using graded ethanol concentrations, from 30% to absolute ethanol, for 10 min each, plus two additional immersions in absolute ethanol, for 10 min each. The glass coverslips were dried to the critical point and covered with gold. The samples were analyzed in a scanning electron microscope (Hitachi/SU8100). ROS detection Neutrophil ROS levels were detected using the fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. Neutrophils were incubated for 45 min in serum-free media containing 10 mM DCFH-DA. The conversion of DCFH-DA to the fluorescent product 2',7'-dichlorofluorescein (DCF) was measured using flow cytometry with wavelength of excitation at 488 nm and emission at 525 nm. ROS in skin of mice and patients was observed using the fluorescence probe dihydroethidium (DHE). Sections of skin were incubated for 30 min in PBS containing 10 µM DHE and were analyzed under fluorescence microscope (Nikon Eclipse Ti-SR) with wavelength of excitation at 535 nm and emission at 610 nm. Flow cytometry Nonspecific antibody binding was blocked with an anti-CD16/32 antibody (Biolegend). Cells were washed and stained with surface antibodies. For intracellular cytokines staining, cells were collected and restimulated with PMA (100 ng/ml) and ionomycin (500 ng/ml) in the presence of Golgi-plug (BD) for 5 h. Cells were then fixed and permeabilized with Cytofix/Cytoperm Kit (BD) and stained with the appropriate antibodies. Intracellular staining for transcription factor was performed using the Foxp3 Permeabilization/Fixation kit (eBioscience), followed by staining with appropriate antibodies. For citH3 staining, cells were incubated with a primary anti-citH3 antibody (Abcam) and labeled with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Invitrogen). LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen Life Technology) was used to gate out dead cells. The following antibodies were used: anti-mouse CD45 (Biolegend), anti-mouse/human CD11b (Biolegend), anti-mouse Ly6G (Biolegend), anti-mouse CD3ε (Biolegend), anti-mouse CD4 (Biolegend), anti-mouse CD8a (Biolegend), anti-mouse TCRγδ (Biolegend), anti-mouse IL-17A (Biolegend), anti-mouse IL-17F (Biolegend), anti-mouse RORγt (BD), anti-mouse Foxp3 (BD), anti-mouse IFNγ (Biolegend), anti-mouse IL-4 (Biolegend), anti-mouse IL-13 (Biolegend), anti-mouse IL-22 (eBioscience), anti-mouse TLR4/MD2 complex (Biolegend). Cells were measured with a ID7000™ Spectral Cell Analyzer (Sony Biotechnology) and analyzed using FlowJo software (TreeStar). Statistical analysis Data were processed using GraphPad Prism 9.0. Flow cytometry analysis was performed with FlowJo_V10. Experimental results are expressed as mean ± SEM. Comparisons were performed using the unpaired student’s t test and one-way analysis of variance. For all analyses, a two-tailed P value < 0.05 was considered statistically significant. Declarations Acknowledgements: This work was supported by the National Natural Science Foundation of China (82130089, 82103730, 82304022, and 32070894); Key R&D Program of Hubei Province (YFXM2021000203); National Key Research and Development Program of China (2020YFA0804400); Program of HUST Academic Frontier Youth Team (2018QYTD10). We thank all members of Medical sub-center of Analytical and Testing Center, Huazhong University of Science and Technology for technical assistance. We thank all members of Institutional Animal Care and Use Committee, Huazhong University of Science and Technology for showing loving care for life. Author contributions Y.X. and J.L. designed the project, performed experiments, analyzed data, and wrote the manuscript. S.Y., X.X., Q.D., H.D., W.N., B.J., L.Z., Z.C., X.Z. and Y.X. conducted experiments. J.Y. and Y.L. interpreted data. R.H. and J.T. designed the project, oversaw the studies, provided intellectual support, and edited the manuscript. All authors edited and approved the manuscript. Conflict of interest: The authors have declared that no conflict of interest exists. Data availability: The data underlying this article will be shared on reasonable request to the corresponding author. Ethics statement: All studies involving mice were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. All human clinical protocols were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from the patients before participation in the study. Funding sources: This work was supported by the National Natural Science Foundation of China (82130089, 82103730, 82304022, and 32070894); Key R&D Program of Hubei Province (YFXM2021000203); National Key Research and Development Program of China (2020YFA0804400); Program of HUST Academic Frontier Youth Team (2018QYTD10). References Griffiths CEM, Armstrong AW, Gudjonsson JE, Barker J. Psoriasis. Lancet. 2021;397(10281):1301–15. Armstrong AW, Read C. Pathophysiology, Clinical Presentation, and Treatment of Psoriasis: A Review. JAMA. 2020;323(19):1945–60. Yu J, Zhao Q, Wang X, Zhou H, Hu J, Gu L, et al. Pathogenesis, multi-omics research, and clinical treatment of psoriasis. J Autoimmun. 2022;133:102916. Bu J, Ding R, Zhou L, Chen X, Shen E. Epidemiology of Psoriasis and Comorbid Diseases: A Narrative Review. Front Immunol. 2022;13:880201. Shah K, Mellars L, Changolkar A, Feldman SR. Real-world burden of comorbidities in US patients with psoriasis. J Am Acad Dermatol. 2017;77(2):287–92 e4. Takeshita J, Grewal S, Langan SM, Mehta NN, Ogdie A, Van Voorhees AS, et al. Psoriasis and comorbid diseases: Epidemiology. J Am Acad Dermatol. 2017;76(3):377–90. Snekvik I, Smith CH, Nilsen TIL, Langan SM, Modalsli EH, Romundstad PR, et al. Obesity, Waist Circumference, Weight Change, and Risk of Incident Psoriasis: Prospective Data from the HUNT Study. J Invest Dermatol. 2017;137(12):2484–90. Norden A, Rekhtman S, Strunk A, Garg A. Risk of psoriasis according to body mass index: A retrospective cohort analysis. J Am Acad Dermatol. 2022;86(5):1020–6. Paroutoglou K, Papadavid E, Christodoulatos GS, Dalamaga M. Deciphering the Association Between Psoriasis and Obesity: Current Evidence and Treatment Considerations. Curr Obes Rep. 2020;9(3):165–78. Wu MY, Yu CL, Yang SJ, Chi CC. Change in body weight and body mass index in psoriasis patients receiving biologics: A systematic review and network meta-analysis. J Am Acad Dermatol. 2020;82(1):101–9. Papp KA, Langley RG, Sigurgeirsson B, Abe M, Baker DR, Konno P, et al. Efficacy and safety of secukinumab in the treatment of moderate-to-severe plaque psoriasis: a randomized, double-blind, placebo-controlled phase II dose-ranging study. Br J Dermatol. 2013;168(2):412–21. Wu NL, Hsu CJ, Sun FJ, Tsai TF. Efficacy and safety of secukinumab in Taiwanese patients with moderate to severe plaque psoriasis: Subanalysis from ERASURE phase III study. J Dermatol. 2017;44(10):1129–37. Lebwohl M, Yeilding N, Szapary P, Wang Y, Li S, Zhu Y, et al. Impact of weight on the efficacy and safety of ustekinumab in patients with moderate to severe psoriasis: rationale for dosing recommendations. Journal of the American Academy of Dermatology. 2010;63(4):571–9. Herbert D, Franz S, Popkova Y, Anderegg U, Schiller J, Schwede K, et al. High-Fat Diet Exacerbates Early Psoriatic Skin Inflammation Independent of Obesity: Saturated Fatty Acids as Key Players. J Invest Dermatol. 2018;138(9):1999–2009. Sivasami P, Elkins C, Diaz-Saldana PP, Goss K, Peng A, Hamersky Mt, et al. Obesity-induced dysregulation of skin-resident PPARgamma(+) Treg cells promotes IL-17A-mediated psoriatic inflammation. Immunity. 2023;56(8):1844–61 e6. Castanheira FVS, Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation. Blood. 2019;133(20):2178–85. Thiam HR, Wong SL, Wagner DD, Waterman CM. Cellular Mechanisms of NETosis. Annu Rev Cell Dev Biol. 2020;36:191–218. McDowell SAC, Luo RBE, Arabzadeh A, Doré S, Bennett NC, Breton V, et al. Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration. Nature Cancer. 2021;2(5):545–62. Brotfain E, Hadad N, Shapira Y, Avinoah E, Zlotnik A, Raichel L, et al. Neutrophil functions in morbidly obese subjects. Clin Exp Immunol. 2015;181(1):156–63. van der Windt DJ, Sud V, Zhang H, Varley PR, Goswami J, Yazdani HO, et al. Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis. Hepatology. 2018;68(4):1347–60. Wang H, Zhang H, Wang Y, Brown ZJ, Xia Y, Huang Z, et al. Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis. J Hepatol. 2021;75(6):1271–83. Warnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis. Science (New York, NY). 2015;349(6245):316–20. Wolberink EA, Peppelman M, van de Kerkhof PC, van Erp PE, Gerritsen MJ. Establishing the dynamics of neutrophil accumulation in vivo by reflectance confocal microscopy. Exp Dermatol. 2014;23(3):184–8. Rodriguez-Rosales YA, Langereis JD, Gorris MAJ, van den Reek J, Fasse E, Netea MG, et al. Immunomodulatory aged neutrophils are augmented in blood and skin of psoriasis patients. J Allergy Clin Immunol. 2021;148(4):1030–40. Hu SC, Yu HS, Yen FL, Lin CL, Chen GS, Lan CC. Neutrophil extracellular trap formation is increased in psoriasis and induces human beta-defensin-2 production in epidermal keratinocytes. Sci Rep. 2016;6:31119. Li B, Li G, Yang X, Song Z, Wang Y, Zhang Z. NETosis in Psoriatic Arthritis: Serum MPO-DNA Complex Level Correlates With Its Disease Activity. Front Immunol. 2022;13:911347. Wojcik P, Garley M, Wronski A, Jablonska E, Skrzydlewska E. Cannabidiol Modifies the Formation of NETs in Neutrophils of Psoriatic Patients. Int J Mol Sci. 2020;21(18). Czerwinska J, Owczarczyk-Saczonek A. The Role of the Neutrophilic Network in the Pathogenesis of Psoriasis. Int J Mol Sci. 2022;23(3). Lin AM, Rubin CJ, Khandpur R, Wang JY, Riblett M, Yalavarthi S, et al. Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J Immunol. 2011;187(1):490–500. Shao S, Fang H, Dang E, Xue K, Zhang J, Li B, et al. Neutrophil Extracellular Traps Promote Inflammatory Responses in Psoriasis via Activating Epidermal TLR4/IL-36R Crosstalk. Front Immunol. 2019;10:746. Alkhouri N, Dixon LJ, Feldstein AE. Lipotoxicity in nonalcoholic fatty liver disease: not all lipids are created equal. Expert Rev Gastroenterol Hepatol. 2009;3(4):445–51. Lian CY, Zhai ZZ, Li ZF, Wang L. High fat diet-triggered non-alcoholic fatty liver disease: A review of proposed mechanisms. Chem Biol Interact. 2020;330:109199. Shi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin Invest. 2006;116(11):3015–25. Mangum KD, Gallagher KA. Obesity confers macrophage memory. Science (New York, NY). 2023;379(6627):28–9. Nicholas DA, Zhang K, Hung C, Glasgow S, Aruni AW, Unternaehrer J, et al. Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1beta. PLoS One. 2017;12(5):e0176793. Maloney E, Sweet IR, Hockenbery DM, Pham M, Rizzo NO, Tateya S, et al. Activation of NF-kappaB by palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation. Arterioscler Thromb Vasc Biol. 2009;29(9):1370–5. Kim SY, Jeong JM, Kim SJ, Seo W, Kim MH, Choi WM, et al. Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4-MD2 complex. Nat Commun. 2017;8(1):2247. Wang Y, Qian Y, Fang Q, Zhong P, Li W, Wang L, et al. Saturated palmitic acid induces myocardial inflammatory injuries through direct binding to TLR4 accessory protein MD2. Nat Commun. 2017;8:13997. Watanabe S, Iwata Y, Fukushima H, Saito K, Tanaka Y, Hasegawa Y, et al. Neutrophil extracellular traps are induced in a psoriasis model of interleukin-36 receptor antagonist-deficient mice. Sci Rep. 2020;10(1):20149. Hurabielle C, Link VM, Bouladoux N, Han SJ, Merrill ED, Lightfoot YL, et al. Immunity to commensal skin fungi promotes psoriasiform skin inflammation. Proc Natl Acad Sci U S A. 2020;117(28):16465–74. Cao T, Yuan X, Fang H, Chen J, Xue K, Li Z, et al. Neutrophil extracellular traps promote keratinocyte inflammation via AIM2 inflammasome and AIM2-XIAP in psoriasis. Exp Dermatol. 2022. Skrzeczynska-Moncznik J, Wlodarczyk A, Zabieglo K, Kapinska-Mrowiecka M, Marewicz E, Dubin A, et al. Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis. J Immunol. 2012;189(4):1611–7. Jiang M, Fang H, Shao S, Dang E, Zhang J, Qiao P, et al. Keratinocyte exosomes activate neutrophils and enhance skin inflammation in psoriasis. FASEB J. 2019;33(12):13241–53. Herster F, Bittner Z, Archer NK, Dickhofer S, Eisel D, Eigenbrod T, et al. Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis. Nat Commun. 2020;11(1):105. Moorthy AN, Tan KB, Wang S, Narasaraju T, Chow VT. Effect of High-Fat Diet on the Formation of Pulmonary Neutrophil Extracellular Traps during Influenza Pneumonia in BALB/c Mice. Front Immunol. 2016;7:289. Wang H, Wang Q, Venugopal J, Wang J, Kleiman K, Guo C, et al. Obesity-induced Endothelial Dysfunction is Prevented by Neutrophil Extracellular Trap Inhibition. Sci Rep. 2018;8(1):4881. Fernandez-Garcia V, Gonzalez-Ramos S, Avendano-Ortiz J, Martin-Sanz P, Gomez-Coronado D, Delgado C, et al. High-fat diet activates splenic NOD1 and enhances neutrophil recruitment and neutrophil extracellular traps release in the spleen of ApoE-deficient mice. Cell Mol Life Sci. 2022;79(8):396. Poli V, Zanoni I. Neutrophil intrinsic and extrinsic regulation of NETosis in health and disease. Trends Microbiol. 2022. Amara N, Cooper MP, Voronkova MA, Webb BA, Lynch EM, Kollman JM, et al. Selective activation of PFKL suppresses the phagocytic oxidative burst. Cell. 2021;184(17):4480-94 e15. Amini P, Stojkov D, Felser A, Jackson CB, Courage C, Schaller A, et al. Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production. Nat Commun. 2018;9(1):2958. Fritsche KL. The science of fatty acids and inflammation. Adv Nutr. 2015;6(3):293S-301S. Hwang DH, Kim JA, Lee JY. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid. Eur J Pharmacol. 2016;785:24–35. Wigerblad G, Kaplan MJ. Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases. Nat Rev Immunol. 2022:1–15. Kim TS, Silva LM, Theofilou VI, Greenwell-Wild T, Li L, Williams DW, et al. Neutrophil extracellular traps and extracellular histones potentiate IL-17 inflammation in periodontitis. J Exp Med. 2023;220(9). Miranda FJB, Rocha BC, Pereira MCA, Pereira LMN, de Souza EHM, Marino AP, et al. Toxoplasma gondii-Induced Neutrophil Extracellular Traps Amplify the Innate and Adaptive Response. mBio. 2021;12(5):e0130721. Papagoras C, Chrysanthopoulou A, Mitsios A, Ntinopoulou M, Tsironidou V, Batsali AK, et al. IL-17A expressed on neutrophil extracellular traps promotes mesenchymal stem cell differentiation toward bone-forming cells in ankylosing spondylitis. Eur J Immunol. 2021;51(4):930–42. Chrysanthopoulou A, Mitroulis I, Apostolidou E, Arelaki S, Mikroulis D, Konstantinidis T, et al. Neutrophil extracellular traps promote differentiation and function of fibroblasts. J Pathol. 2014;233(3):294–307. Zenaro E, Pietronigro E, Della Bianca V, Piacentino G, Marongiu L, Budui S, et al. Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Nat Med. 2015;21(8):880–6. de Boer OJ, Li X, Teeling P, Mackaay C, Ploegmakers HJ, van der Loos CM, et al. Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction. Thromb Haemost. 2013;109(2):290–7. Ntinopoulou M, Cassimos D, Roupakia E, Kolettas E, Panopoulou M, Mantadakis E, et al. Ιnterleukin-17A-Enriched Neutrophil Extracellular Traps Promote Immunofibrotic Aspects of Childhood Asthma Exacerbation. Biomedicines. 2023;11(8). Chen W, Chen H, Yang Z-T, Mao E-Q, Chen Y, Chen E-Z. Free fatty acids-induced neutrophil extracellular traps lead to dendritic cells activation and T cell differentiation in acute lung injury. Aging (Albany NY). 2021;13(24):26148–60. Krishnamoorthy N, Douda DN, Brüggemann TR, Ricklefs I, Duvall MG, Abdulnour R-EE, et al. Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma. Sci Immunol. 2018;3(26). Lambert S, Hambro CA, Johnston A, Stuart PE, Tsoi LC, Nair RP, et al. Neutrophil Extracellular Traps Induce Human Th17 Cells: Effect of Psoriasis-Associated TRAF3IP2 Genotype. J Invest Dermatol. 2019;139(6):1245–53. Tillack K, Breiden P, Martin R, Sospedra M. T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses. J Immunol. 2012;188(7):3150–9. Wilson AS, Randall KL, Pettitt JA, Ellyard JI, Blumenthal A, Enders A, et al. Neutrophil extracellular traps and their histones promote Th17 cell differentiation directly via TLR2. Nat Commun. 2022;13(1):528. Vural S, Kerl K, Ertop Dogan P, Vollmer S, Puchta U, He M, et al. Lesional activation of T(c) 17 cells in Behcet disease and psoriasis supports HLA class I-mediated autoimmune responses. Br J Dermatol. 2021;185(6):1209–20. Zhang Y, Chandra V, Riquelme Sanchez E, Dutta P, Quesada PR, Rakoski A, et al. Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer. J Exp Med. 2020;217(12). Leppkes M, Maueröder C, Hirth S, Nowecki S, Günther C, Billmeier U, et al. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis. Nature Communications. 2016;7(1). Korman NJ. Management of psoriasis as a systemic disease: what is the evidence? Br J Dermatol. 2020;182(4):840–8. Additional Declarations (Not answered) Supplementary Files Supplementalmaterial20240818.docx Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Cellular & Molecular Immunology → Version 1 posted Editorial decision: revise 16 Oct, 2024 Review # 2 received at journal 04 Oct, 2024 Review # 1 received at journal 15 Sep, 2024 Reviewer # 2 agreed at journal 12 Sep, 2024 Reviewer # 1 agreed at journal 12 Sep, 2024 Reviewers invited by journal 22 Aug, 2024 Editor assigned by journal 20 Aug, 2024 Submission checks completed at journal 19 Aug, 2024 First submitted to journal 19 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Xiong","email":"","orcid":"","institution":"Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xiong","suffix":""},{"id":343679375,"identity":"9cf6927a-a02c-4341-945d-aa8ec086d080","order_by":14,"name":"Yan Li","email":"","orcid":"","institution":"Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""},{"id":343679376,"identity":"98966be8-7f5c-4b1b-b888-a423866ed69c","order_by":15,"name":"Ran He","email":"","orcid":"","institution":"Third Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-08-19 17:50:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4940299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4940299/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41423-025-01278-7","type":"published","date":"2025-04-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66663956,"identity":"3fb0ba73-5db5-423e-995f-8c9cca05cd9a","added_by":"auto","created_at":"2024-10-15 09:14:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3871383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystemic NETs and localized cutaneous NETs increase in obese psoriasis subjects.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative immunofluorescence staining of citH3, MPO and DAPI in skin lesions of psoriasis patients (scale bar = 50 μm). (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eQuantitation of dsDNA and semi-quantitation of MPO-DNA complex in serum of psoriasis patients. (\u003cstrong\u003eC\u003c/strong\u003e) Correlation of clinical severity against dsDNA and MPO-DNA complex (n = 78). (\u003cstrong\u003eD\u003c/strong\u003e) Representative immunohistochemistry staining of citH3 and immunofluorescence staining of citH3, MPO and DAPI in mouse skin (scale bar = 100 μm). (\u003cstrong\u003eE\u003c/strong\u003e) Quantitation of dsDNA and semi-quantitation of MPO-DNA complex in mouse serum. (\u003cstrong\u003eF\u003c/strong\u003e) Flow cytometric analysis of citH3 in neutrophils from mouse dLN. (\u003cstrong\u003eG\u003c/strong\u003e) Representative immunofluorescence staining and scanning electron microscope of \u003cem\u003eex vivo\u003c/em\u003e NETs formation from BM-derived neutrophils (scale bar = 10 μm). (\u003cstrong\u003eH\u003c/strong\u003e) Flow cytometric analysis of \u003cem\u003eex vivo\u003c/em\u003e NETs formation from BM-derived neutrophils. Results are from 3 independent experiments with 3 to 5 mice per group. Error bars show means ± SEM. ns, not significant. *\u003cem\u003eP\u003c/em\u003e \u0026lt; .05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; .01, ***\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/c39859a1d297ba4a04b8f123.png"},{"id":66662629,"identity":"bcbb16fb-6dd5-4c41-8ef3-5a2d004de0d7","added_by":"auto","created_at":"2024-10-15 09:06:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3892433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of NETs attenuates obesity-exacerbated psoriasis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic overview of the experimental protocol. (\u003cstrong\u003eB\u003c/strong\u003e) Quantitation of dsDNA and semi-quantitation of MPO-DNA complex in mouse serum. (\u003cstrong\u003eC\u003c/strong\u003e) Representative immunohistochemistry staining of citH3 and immunofluorescence staining of citH3, MPO and DAPI in mouse skin (scale bar = 100 μm). (\u003cstrong\u003eD\u003c/strong\u003e) Flow cytometric analysis of citH3 in neutrophils from mouse dLN. (\u003cstrong\u003eE\u003c/strong\u003e) Changes of clinical severity. (\u003cstrong\u003eF\u003c/strong\u003e) Representative clinical photographs. Representative images of H\u0026amp;E staining and summarized bar graphs of epidermal thickness (scale bar = 100 μm). Results are from 3 independent experiments with 3 to 5 mice per group. Error bars show means ± SEM. ns, not significant. *\u003cem\u003eP\u003c/em\u003e \u0026lt; .05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; .01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; .001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/fe71e4f52eaea9c9f9d63b66.png"},{"id":66662626,"identity":"7abd1776-be10-4eb8-a80d-ea0c66ba1e5d","added_by":"auto","created_at":"2024-10-15 09:06:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2503496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncrease of SFAs promotes NETs generation in obesity-exacerbated psoriasis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Quantitation of total free fatty acids in serum of psoriasis patients. (\u003cstrong\u003eB\u003c/strong\u003e) Correlation of clinical severity against free fatty acids (n = 78). (\u003cstrong\u003eC\u003c/strong\u003e) Correlation of dsDNA and MPO-DNA complex against free fatty acids (n = 78). (\u003cstrong\u003eD\u003c/strong\u003e) Quantitation of total free fatty acids in mouse serum (n = 5). (\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePCA score plot of free fatty acids in mouse serum (n = 4). (\u003cstrong\u003eF\u003c/strong\u003e) Summarized bar graphs of C16:0, C18:0, C18:1N7, C18:1N9C, C18:1N12, C18:2N6, C19:1N9T, C20:4N6, C22:1N9 and C22:6N3 in mouse serum (n = 4). (\u003cstrong\u003eG \u003c/strong\u003eand\u003cstrong\u003e H\u003c/strong\u003e) Flow cytometric analysis of \u003cem\u003eex vivo\u003c/em\u003e NETs formation from BM-derived neutrophils.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eI\u003c/strong\u003e) Representative immunofluorescence staining and scanning electron microscope of \u003cem\u003eex vivo\u003c/em\u003e NETs formation from BM-derived neutrophils (scale bar = 10 μm). Results are from 3 independent experiments with 3 to 5 mice per group. Error bars show means ± SEM. ns, not significant. *\u003cem\u003eP\u003c/em\u003e \u0026lt; .05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; .01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; .001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/db0a157f6cf20ebdac3ba91f.png"},{"id":66663957,"identity":"fdb3b1e9-b0c5-4d72-a98c-d706a9adde0b","added_by":"auto","created_at":"2024-10-15 09:14:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2936346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTLR4-MD2/ROS signaling pathway regulates SFA-induced NETs production in obesity-exacerbated psoriasis. (A) \u003c/strong\u003eVolcano plot showing the number of differentially expressed genes (DEGs) between neutrophils isolated from CD-fed and HFD-fed mouse bone marrow (n = 3). \u003cstrong\u003e(B) \u003c/strong\u003eHeatmap of DEGs between neutrophils isolated from CD-fed and HFD-fed mouse bone marrow (n = 3). (\u003cstrong\u003eC\u003c/strong\u003e) Flow cytometric analysis of TLR4-MD2 complex in neutrophils from mouse skin. (\u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Flow cytometric analysis of TLR4-MD2 complex and DCFH-DA in BM-derived neutrophils \u003cem\u003eex vivo\u003c/em\u003e. (\u003cstrong\u003eH\u003c/strong\u003e) Flow cytometric analysis of \u003cem\u003eex vivo\u003c/em\u003eNETs formation from BM-derived neutrophils. (\u003cstrong\u003eI\u003c/strong\u003e) Representative immunofluorescence staining of citH3, DHE, TLR4 and DAPI in skin lesions of psoriasis patients (scale bar = 50 μm). (\u003cstrong\u003eJ\u003c/strong\u003e) Representative immunofluorescence staining of citH3, DHE, TLR4 and DAPI in mouse skin (scale bar = 100 μm). Results are from 3 independent experiments with 3 to 5 mice per group. Error bars show means ± SEM. ns, not significant. *\u003cem\u003eP\u003c/em\u003e \u0026lt; .05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; .01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; .001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/83f0d26dc29371aa4c5cc7f4.png"},{"id":66662627,"identity":"93ea7f42-93ce-42ce-929e-4a83555e940d","added_by":"auto","created_at":"2024-10-15 09:06:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3052555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased NETs in obesity-exacerbated psoriasis promote γδT17-mediated responses.\u003c/strong\u003e (\u003cstrong\u003eA)\u003c/strong\u003e Representative immunofluorescence staining of citH3, IL-17A, TCRγδ, CD4 and DAPI in mouse skin. Bottom plots show zoomed-in images of the region enclosed by rectangles in the top images. (scale bar on the top graphs = 100 μm, scale bar on the bottom graphs = 20 μm). (\u003cstrong\u003eB \u003c/strong\u003eand\u003cstrong\u003e C\u003c/strong\u003e) Flow cytometric analysis of the frequencies of γδT and CD4\u003csup\u003e+\u003c/sup\u003e T cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from mouse dLN. (\u003cstrong\u003eD\u003c/strong\u003e) Flow cytometric analysis of the frequencies of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e γδT cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN. (\u003cstrong\u003eE\u003c/strong\u003e) Flow cytometric analysis of the frequencies of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN. (\u003cstrong\u003eF\u003c/strong\u003e) Flow cytometric analysis of the frequencies of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e γδT cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN. (\u003cstrong\u003eG\u003c/strong\u003e) Flow cytometric analysis of the frequencies of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN. Results are from 3 independent experiments with 3 to 5 mice per group. Error bars show means ± SEM. ns, not significant. *\u003cem\u003eP\u003c/em\u003e \u0026lt; .05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; .01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; .001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/def630fa396619e4c5e3b7ae.png"},{"id":66662632,"identity":"24a783a6-3aef-4f43-b85c-4322c449f616","added_by":"auto","created_at":"2024-10-15 09:06:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2815757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting of NETs ameliorates PA-induced insensitivity to anti-IL17A therapy in psoriasis-like mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic overview of the experimental protocol. (\u003cstrong\u003eB\u003c/strong\u003e) Changes of clinical severity. (\u003cstrong\u003eC\u003c/strong\u003e) Representative clinical photographs. Representative images of H\u0026amp;E staining and summarized bar graphs of epidermal thickness (scale bar = 100 μm). Representative immunofluorescence staining of citH3, MPO, TLR4 and DAPI in mouse skin (scale bar = 100 μm). (\u003cstrong\u003eD\u003c/strong\u003e) Flow cytometric analysis of the frequencies of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e γδT cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from mouse dLN. (\u003cstrong\u003eE\u003c/strong\u003e) Flow cytometric analysis of the frequencies of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells gated on CD3\u003csup\u003e+\u003c/sup\u003e T cells from mouse dLN. Results are from 3 independent experiments with 3 to 5 mice per group. Error bars show means ± SEM. ns, not significant. *\u003cem\u003eP\u003c/em\u003e \u0026lt; .05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; .01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; .001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/afc4bbfa72693027597642b9.png"},{"id":66662631,"identity":"96419262-9753-4eaf-b35d-0c53eb9a0d35","added_by":"auto","created_at":"2024-10-15 09:06:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":476858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSFAs-induced NETs contribute to exacerbation and biologic therapy resistance in obesity-related psoriasis.\u003c/strong\u003e Elevated SFAs in obese individuals promote release of NETs through TLR4–MD2/ROS signaling pathway, which further amplify type 17 immune responses, especially γδT17-mediated immune responses. SFAs drive the abrogation of anti-IL17A therapeutic effect in psoriasis with lipid metabolism disorder and NETs inhibition combined with anti-IL17A treatment could reverse this effect.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/77d38ed801db4c1dbcbaa6a2.png"},{"id":79736342,"identity":"079adb76-ab31-4b20-96bf-071dad637d14","added_by":"auto","created_at":"2025-04-02 07:09:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20219647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/7fc90065-06d2-4e97-a5b5-ab45b84dac54.pdf"},{"id":66662633,"identity":"998c367c-cfb3-4393-858e-f6595af05b92","added_by":"auto","created_at":"2024-10-15 09:06:43","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":23179861,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial20240818.docx","url":"https://assets-eu.researchsquare.com/files/rs-4940299/v1/72ca9f29727f45d38003981a.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Saturated fatty acids-induced neutrophil extracellular traps contribute to exacerbation and biologic therapy resistance in obesity-related psoriasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePsoriasis is a common chronic inflammatory skin disease that is induced by hereditary and environmental factors, affecting approximately 125\u0026nbsp;million people worldwide (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Patients with psoriasis often have systemic metabolic disorders, including obesity, cardiometabolic diseases, metabolic syndrome, type 2 diabetes, and dyslipidemia, which can lead to aggravation of patients' condition and deterioration of their life quality (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). It has been reported that obesity doubles the risk of psoriasis and higher body mass index (BMI) is independently associated with the development of psoriasis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Besides, several clinical studies have shown that psoriasis patients with obesity have poor responses to various biological agents (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Secukinumab has been reported to exhibit better psoriasis area and severity index (PASI) 75 responses in psoriasis patients weighing\u0026thinsp;\u0026lt;\u0026thinsp;90 kg than heavier patients after 12 weeks of treatment and obesity-related factors could adversely affect its efficacy (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). And ustekinumab has been found to show worse PASI 75 and PASI 90 responses in psoriasis patients weighing\u0026thinsp;\u0026gt;\u0026thinsp;100 kg (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition, studies have indicated the exacerbation of psoriatic dermatitis in obese mice (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). As the product of neutral fat metabolism, saturated fatty acids (SFAs) have been found to be major risk factors for the amplification of psoriasis inflammation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, the particular mechanisms of lipid metabolic disorder exacerbating psoriasis is not well understood.\u003c/p\u003e \u003cp\u003eNeutrophil extracellular traps (NETs) are fibrous weblike structures which protrude from the membranes of activated neutrophils and are coated with histones, proteases, and granular cytosolic proteins (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Accumulative evidence has revealed the important roles of NETs in inflammatory diseases and metabolic disorders. The capabilities of neutrophils to migrate and produce NETs are elevated in obese subjects (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In non-alcoholic steatohepatitis, free fatty acids (FFAs) stimulate NETs formation, which facilitate the crosstalk between innate and adaptive immunity by promoting Treg activity through metabolic reprogramming and promote development of hepatocellular carcinoma (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Cholesterol crystals trigger neutrophils to release NETs in cardiometabolic diseases, which prime macrophages and Th17 cells to produce cytokines that amplify immune cell recruitment in atherosclerotic plaques (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). As a hallmark of psoriatic skin lesions, neutrophils infiltration appears early in developing lesions (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Studies show that NETs are prominent within psoriasis lesions and in peripheral blood, and the positive correlation between the amount of NETotic cells in the peripheral blood and psoriasis disease severity has also been reported (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Thus, it would be interesting to clarify the functional link between NETs and the aggravation of psoriasis induced by metabolic disorders.\u003c/p\u003e \u003cp\u003eIn this study, we identified the increase of systemic NETs and localized cutaneous NETs in both obese psoriasis patients and imiquimod (IMQ)-induced psoriasis-like mice, which was positively correlated with disease severity. And NETs inhibitor alleviated psoriatic dermatitis in obese mice. We also present experimental evidence that the increased SFAs promoted the release of NETs through TLR4-MD2/ROS pathway. Moreover, NETs amplified IL-17 inflammation in obesity-exacerbated psoriasis, especially γδT17-mediated immune responses. Interestingly, we demonstrated that SFAs induced decreased response to anti-IL17A treatment in psoriasis-like mice, whereas inhibition of NETs improved the beneficial effects of anti-IL17A in psoriasis-like mice with lipid metabolism disorder. Thus, our research indicates that NETs play a critical role in obesity-exacerbated psoriasis and brings new insights for the treatment of psoriasis patients with lipid metabolism disorders clinically.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNETs elevated systemically and skin locally in obesity-exacerbated psoriasis\u003c/h2\u003e \u003cp\u003eWe compared disease severity in lean psoriasis patients (BMI\u0026thinsp;\u0026lt;\u0026thinsp;24) (n\u0026thinsp;=\u0026thinsp;47) and obese psoriasis patients (BMI\u0026thinsp;\u0026ge;\u0026thinsp;28) (n\u0026thinsp;=\u0026thinsp;31). A significantly increased severity of psoriasis was observed in obese patients (Supplemental Fig.\u0026nbsp;1A) and there was a positive association between PASI and BMI (Supplemental Fig.\u0026nbsp;1B). In addition, we observed more patients showing poor responses to biological agents in BMI\u0026thinsp;\u0026ge;\u0026thinsp;28 group than that in BMI\u0026thinsp;\u0026lt;\u0026thinsp;24 group. Histologic analysis showed increased epidermal hyperplasia and neutrophils infiltration in the skin lesions of obese psoriasis patients (Supplemental Fig.\u0026nbsp;1C). Mice were fed with either a control diet (CD) or high fat diet (HFD) for 16 weeks and HFD consumption led to significant weight gain in mice (Supplemental Fig.\u0026nbsp;2A). Then daily application of IMQ for 6 days was conducted to develop psoriasis-like dermatitis, with Vaseline treatment in sham group (Supplemental Fig.\u0026nbsp;2B). As expected, HFD/IMQ-treated mice exhibited significantly more severe cutaneous manifestations and local pathological changes than CD/IMQ-treated mice (Supplemental Fig.\u0026nbsp;2, C-E). And HFD/IMQ-treatment led to significantly increased proportion of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, IL-22\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e T cells and decreased proportion of Foxp3\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells in skin-draining lymph node (dLN) (Supplemental Fig.\u0026nbsp;2F).\u003c/p\u003e \u003cp\u003ePsoriatic lesions are characterized by neutrophil infiltration, the activation of which leads to release of web-like structures named NETs (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Previous reports have shown that NETs exist in skin lesions of both psoriasis patients and IMQ-induced psoriasis-like mice (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). To identify NETs within psoriasis lesions, immunofluorescent co-staining for MPO and NETs marker, citrullinated histone H3 (citH3), was performed. We found that NETs were present in abundance within obesity-exacerbated inflamed skin of psoriasis patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Sera from psoriasis patients with BMI\u0026thinsp;\u0026lt;\u0026thinsp;24 and BMI\u0026thinsp;\u0026ge;\u0026thinsp;28 were analyzed for myeloperoxidase (MPO) -DNA complexes and double-stranded DNA (dsDNA). Concentrations of serum NETs in obese psoriasis patients were significantly higher than that in lean psoriasis patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and the abundance of serum NETs was positively correlated with disease severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Consistently, NETs markers were significantly increased within psoriasis-like lesions and in serum of HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, D and E). In addition, flow cytometry analysis of dLN single-cell suspensions revealed the significant increase of neutrophil recruitment and NETs formation in HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Supplemental Fig.\u0026nbsp;2F and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Notably, concentrations of serum NETs in HFD/Vas-treated mice were significantly increased compared with those in CD/Vas-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). And HFD alone promoted neutrophil infiltration and NETs generation in mouse skin and dLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, D and F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the effect of obesity on NETs production, neutrophils from CD-fed and HFD-fed mouse bone marrow were isolated. NETs extension was evaluated by confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). Neutrophils from HFD-fed mouse exhibited an increased capacity of releasing more web-like structure to extracellular space compared with those from CD-fed mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The enhanced spontaneous NETs of HFD-fed mouse neutrophils in the absence of specific stimulation \u003cem\u003eex vivo\u003c/em\u003e suggested that NETs have already proceeded in obesity subjects. The result was further validated with flow cytometry analysis. Neutrophils from HFD-fed mouse bone marrow displayed significantly increased mean fluorescence intensity (MFI) of SYTOX Red and citH3 and increased proportion of citH3\u003csup\u003e+\u003c/sup\u003eSYTOX Red\u003csup\u003e+\u003c/sup\u003e populations with or without LPS stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Taken together, these data implicate that obesity leads to exacerbated psoriasis and increased NETs may contribute to obesity-exacerbated psoriasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTargeting NETs alleviated obesity-exacerbated psoriasis in mice\u003c/h2\u003e \u003cp\u003eTo further verify our hypothesis that enhanced NETs generation may contribute to aggravated inflammation caused by obesity, CD-fed or HFD-fed mice were daily treated with DNase Ⅰ, a nuclease that dismantles NETs \u003cem\u003ein vivo\u003c/em\u003e, or equivalent vehicle starting 3 d prior to the imiquimod treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). DNase Ⅰ treatment resulted in the expected reduction of NETs markers in skin, serum, and dLN, demonstrating the efficacy of DNase Ⅰ at eliminating NETs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, B-D). DNase Ⅰ treatment had no significant impact on the pathogenesis of IMQ alone according to clinical severity and histologic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, E and F). However, NETs inhibition significantly reduced inflammation induced by HFD prior to IMQ based on the ameliorated symptoms, reduced epidermal thickening and downregulated infiltration of neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, E and F, Supplemental Fig.\u0026nbsp;3A). Flow cytometry analysis further revealed that DNase Ⅰ treatment significantly reduced leukocytes infiltration in skin and neutrophil accumulation in dLN of HFD/IMQ-treated mice compared with CD/IMQ-treated mice ((Supplemental Fig.\u0026nbsp;3, B and C). Collectively, our data suggest that NETs contribute to obesity-exacerbated psoriasis and inhibition of NETs remarkably relieve the amplified inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDeposit of SFAs in obesity-exacerbated psoriasis promotes the release of NETs\u003c/h2\u003e \u003cp\u003eSFAs are considered as the link between lipid metabolism and the activation of various inflammatory pathways in obese individuals (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). We next assessed whether the disturbance of fatty acid metabolism was related to obesity-exacerbated psoriasis. The total amount of FFAs in the serum of obese psoriasis patients was significantly increased compared with lean psoriasis patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). And the blood FFAs concentration was positively correlated with disease severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, serum FFAs level of psoriasis patients showed positive correlation with serum NETs concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We also investigated the changes in the amount of FFAs in mouse serum after intake of HFD. After 16 weeks of HFD feeding, the total amount of FFAs in mouse serum was significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Targeted lipidomic analysis was further performed and we found HFD consumption changed the composition of FFAs in mouse serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; Supplemental Fig.\u0026nbsp;4, A and B). SFAs such as palmitic acid (PA) and stearic acid (SA) were among the most enriched FFAs in the serum of HFD-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIndependent of obese phenotype, SFAs act as major risk factors for the amplification of skin inflammation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). To investigate whether SFAs functioned as a stimulus for NETs formation, neutrophils from bone marrow of CD-fed mice at 6\u0026ndash;8 weeks of age were treated with different concentrations of PA and SA for 3 h. We found that PA induced NETs \u003cem\u003eex vivo\u003c/em\u003e in a dose-dependent manner and PA-treated neutrophils exhibited the greatest capacity to generate NETs when the concentration of PA was at 200 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Consistently, SA also induced NETs production \u003cem\u003eex vivo\u003c/em\u003e in a dose-dependent manner, which exhibited the greatest ability for NETs generation at 400 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The results of CLSM and SEM further proved that PA promoted the release of web-like structure from neutrophils with PMA and DNase Ⅰ as positive and negative control respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). In summary, HFD feeding leads to an increase of long-chain SFAs, which promote NETs generation during obesity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSFA promotes NETs generation through TLR4\u0026ndash;MD2/ROS signaling pathway in obesity-exacerbated psoriasis\u003c/h2\u003e \u003cp\u003eTo further investigate the effects of abnormal lipid metabolism on neutrophils, RNA sequencing (RNA-seq) was performed on neutrophils isolated from CD-fed and HFD-fed mouse bone marrow. The results revealed differential gene expression, with 2394 genes upregulated and 1918 genes downregulated (FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in neutrophils from HFD-fed mice compared to CD-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). And neutrophils from HFD-fed mice had increased expression of genes involved in ROS generation (i.e., Cgas, Nox1, and Junb) and NETs production (i.e., Selp, Tlr2 and Nlrp3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMyeloid cell\u0026ndash;mediated inflammation has been linked to TLR4 in obesity and insulin resistance, which can be activated by long-chain SFAs (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Endocytosis of palmitate/TLR4\u0026ndash;MD2 complex generates NADPH oxidase (NOX) -mediated superoxide generation in endothelial cells, cardiomyocytes and hepatic macrophages (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Flow cytometry analysis of skin single-cell suspensions from mice showed significantly increased TLR4\u0026ndash;MD2 complex formation in neutrophils from HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To investigate the role of TLR4\u0026ndash;MD2/ROS signaling in SFA-induced NETs, neutrophils from CD-fed and HFD-fed mouse bone marrow were isolated respectively. Flow cytometry analysis and CLSM revealed that neutrophils from HFD-fed mouse bone marrow displayed significantly increased TLR4\u0026ndash;MD2 complex formation and ROS generation with or without LPS stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, D and E; Supplemental Fig.\u0026nbsp;5, A and B). We then treated neutrophils isolated from bone marrow of CD-fed mice at 6\u0026ndash;8 weeks of age with 200 \u0026micro;M PA or 400 \u0026micro;M SA. Both of PA and SA were able to promote TLR4\u0026ndash;MD2 complex formation and ROS generation as measured by flow cytometry analysis and CLSM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, F and G; Supplemental Fig.\u0026nbsp;5, C, D and E). TAK-242, a TLR4 specific inhibitor, and DPI, a NOX inhibitor, were used to further identify the involvement of TLR4\u0026ndash;MD2/ROS signaling in SFA-induced NETs formation. We found that TAK-242 significantly reduced NOX-derived ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH; Supplemental Fig.\u0026nbsp;5F). Flow cytometry analysis revealed that treatment with TAK-242 and DPI significantly abrogated the increase of NETs formation induced by PA and SA while treatment with TAK-242 combined with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH; Supplemental Fig.\u0026nbsp;5F). Moreover, immunofluorescent co-staining for NETs, TLR4, and ROS was performed. The results showed that not only citH3\u003csup\u003e+\u003c/sup\u003e neutrophils infiltration was increased in skin lesions of obese psoriasis patients and mice but also that these cells showed higher TLR4 expression and more ROS generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, I and J). In total, our results implicate that TLR4\u0026ndash;MD2/ROS signaling pathway is crucial for SFAs-induced NETs generation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eObesity-induced generation of NETs promotes γδT17-mediated inflammation in psoriasis\u003c/h2\u003e \u003cp\u003eImmunological studies have identified IL-17 as one of the key drivers of psoriasis pathogenesis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). As our results showed that IL-17A\u003csup\u003e+\u003c/sup\u003e cells infiltration was increased in psoriatic lesions of obese patients and mice, while NETs inhibition remarkably relieved this phenomenon (Supplemental Fig.\u0026nbsp;6, A-C), we hypothesized that NETs promoted IL-17-mediated inflammation in obesity-exacerbated psoriasis. Immunofluorescent co-staining for MPO, citH3, and IL-17A revealed that the abundance of NETs within obesity-exacerbated inflamed skin of psoriasis patients and psoriasis-like mice was accompanied by increased IL-17A\u003csup\u003e+\u003c/sup\u003e cell infiltration (Supplemental Fig.\u0026nbsp;6, D and E). In addition, NETs inhibition in mouse skin with DNase Ⅰ was paralleled by a reduction of IL-17A\u003csup\u003e+\u003c/sup\u003e cells (Supplemental Fig.\u0026nbsp;6F).\u003c/p\u003e \u003cp\u003eWe next sought to further dissect the main source of NETs-promoted IL-17 in obesity-exacerbated psoriasis. Immunofluorescent co-staining for citH3, IL-17A, TCRγδ, and CD4 confirmed that NETs and dermal γδT17 were often found in close proximity while there was a considerable distance between NETs and CD4\u003csup\u003e+\u003c/sup\u003e T cells, implying potential interactions between NETs and γδT17 in obesity-exacerbated psoriasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, HFD/IMQ-treatment brought about significantly increased proportion of γδT cells in CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and NETs inhibition reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Flow cytometry analysis also revealed significant increased proportion of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e γδT, IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells in CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN of HFD/IMQ-treated mice compared with CD/IMQ-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, E and F). Additionally, treatment with DNase I led to a significant decrease proportion of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e γδT, IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells in CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN of HFD/IMQ-treated mice, while that of CD/IMQ-treated mice remained comparable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, F and G). Thus, NETs promote the induction of IL-17 inflammation in obesity-exacerbated psoriasis, especially γδT17-mediated immune responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNase Ⅰ potentiates therapeutic effect of anti-IL17A in PA-treated psoriasis-like mice\u003c/h2\u003e \u003cp\u003eAs SFA-induced NETs heightened IL-17 inflammation in obesity-exacerbated psoriasis, we supposed that SFAs might induce insensitivity to anti-IL17A therapy for psoriasis and NETs inhibition could reverse its effect. Thus, mice were daily treated with vehicle, anti-IL17A, PA and anti-IL17A, or PA and anti-IL17A combined with DNase Ⅰ 3 d prior to the imiquimod treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The curative effect of anti-IL17A was apparent with significantly relieved cutaneous manifestations in anti-IL17A-treated psoriatic mice compared with psoriatic mice without treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B and C). When interfered with PA at the same time, anti-IL17A alone failed to alleviate skin symptoms, including erythema, thickness, and scaling, in psoriasis-like mice, while DNase Ⅰ combined with anti-IL17A treatment significantly remedied these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B and C). Consistently, histologic analysis of mouse dorsal skin showed that anti-IL17A failed to alleviate local pathological changes when psoriasis-like mice were interfered with PA at the same time, while DNase Ⅰ combined with anti-IL17A treatment significantly reduced epidermal thickness, keratinocyte proliferation, and neutrophil infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Supplemental Fig.\u0026nbsp;7A). In agreement with our hypothesis, we found that PA intervention promoted IL-17A\u003csup\u003e+\u003c/sup\u003e cell and citH3\u003csup\u003e+\u003c/sup\u003e cell infiltration in mouse psoriatic lesions (Supplemental Fig.\u0026nbsp;7A). Moreover, anti-IL17A therapy alone failed to inhibit the production and transcription of IL-17A and IL-17F in mouse dLN when interfered with PA at the same time, while DNase Ⅰ combined with anti-IL17A treatment showed reversal effects with decreased proportion of IL-17A\u003csup\u003e+\u003c/sup\u003e, IL-17F\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e γδT, IL-17A\u003csup\u003e+\u003c/sup\u003e and IL-17F\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells in CD3\u003csup\u003e+\u003c/sup\u003e T cells from mouse dLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, D and E). We also found that PA intervention resulted in increased proportion of γδT cells in CD3\u003csup\u003e+\u003c/sup\u003e T cells from dLN and NETs inhibition reversed this effect (Supplemental Fig.\u0026nbsp;7B). Overall, SFAs lead to decreased response to anti-IL17A treatment in psoriasis and inhibition of NETs potentiates therapeutic effect of anti-IL17A in psoriasis-like mice with lipid metabolism disorder.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eObesity is a major risk factor for psoriasis (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Previous work has indicated that disease severity of psoriasis relates to heightened NETs formation, but the regulatory roles and internal mechanism of NETs in psoriasis still remain elusive (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Herein, we delineated that increased NETs induced by abnormal fatty acid metabolism remodulated skin immune microenvironment and it was essential for obesity-exacerbated psoriasis. Elevated SFAs in obese individuals promoted release of NETs through TLR4\u0026ndash;MD2/ROS signaling pathway, which further amplified type 17 immune responses. Moreover, our results provided evidence that SFAs drove the abrogation of anti-IL17A therapeutic effect in psoriasis with lipid metabolism disorder and that NETs inhibition combined with anti-IL17A treatment could reverse this phenomenon. Therefore, we document that NETs inhibition may serve as a potential therapeutic avenue in defined psoriatic settings with lipid metabolism disorder clinically (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe existence of NETs in skin lesions and blood of both psoriasis patients and imiquimod-induced psoriasis-like mice has been addressed (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Here, we found that systemic NETs and localized cutaneous NETs in both obese psoriasis patients and IMQ-induced psoriasis-like mice were significantly higher than lean subjects. Inhibition of NETs significantly alleviated the inflammation in obesity-aggravated psoriasis but failed to influence the pathogenesis of imiquimod alone. In line with our study, NETs inhibition ameliorates skin inflammation in psoriasis exacerbated by IL-36 receptor antagonist deficiency and fungi-aggravated psoriasis but exhibits no significant impact on mice treated by imiquimod alone (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Moreover, studies have reported that NETs directly promote psoriasis exacerbation by mediating keratinocytes activation, plasmacytoid dendritic cells stimulation, and release of IL-17A (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). However, little is known about the precise mechanism regulating the production of NETs in psoriasis. According to our results, changes in fatty acid metabolism led to the increase of NETs generation. Others have reported that the release of keratinocyte exosomes activates neutrophils and induces NETs formation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Moreover, the self-propagating vicious cycle mediated by RNA and LL37 via TLRs recognition and NETs formation contributes to chronic inflammation in psoriasis (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Thus, it\u0026rsquo;s plausible that NETs formation is vital for the exacerbation of psoriatic inflammation induced by obesity.\u003c/p\u003e \u003cp\u003eAccording to our results, neutrophils from obese subjects are more prone to generate NETs. Other investigators have shown similar results that obesity/lipid metabolism disorder leads to greater NETs formation in influenza pneumonia mouse model, vascular dysfunction mouse model, the STAM mouse model, and ApoE-deficient mice (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). However, the exact mechanism of obesity/lipid metabolism disorder promoting NETs generation awaits further study. Metabolic pathways that regulate glycolysis and energy supply have been proved to be tightly linked to the capacity of neutrophils to produce NETs (\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Moreover, SFAs such as PA and SA are among the most enriched FFAs in both serum and skin from HFD-fed mice and SFAs have been regarded as major risk factors for the amplification of skin inflammation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Thus, we focused on SFAs as a contributor for augmented NETs formation in obesity-exacerbated psoriasis. Our study indicates that NETs have already proceeded in obesity subjects and SFAs promote NETs production in a dose-dependent manner.\u003c/p\u003e \u003cp\u003eMechanistically, our data reveal that SFA-induced TLR4-MD2 complex formation further promote NOX-derived ROS, which leads to NETs generation in obesity-exacerbated psoriasis. SFAs directly stimulate inflammatory gene expression by way of TLR4 signaling, while MUFAs and PUFAs fails to activate TLR4 signaling (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). In hepatic macrophages of an HFD-induced hepatic steatosis mouse model, endocytosis of palmitate/TLR4\u0026ndash;MD2 complex generates NOX2-mediated ROS (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In obesity-associated myocardial injury, saturated palmitic acid direct binds to TLR4 accessory protein MD2 and activates downstream inflammatory responses (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Considering that ROS generation is fundamental for NET formation, we conclude that SFAs promote NETs production through TLR4-MD2/ROS signaling pathway (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Further studies need to determine whether other abnormal lipid metabolites are engaged in obesity-promoted NETs generation.\u003c/p\u003e \u003cp\u003eOur results reconfirmed the known role for NETs in promoting IL-17/Th17 responses (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). With well-documented proinflammatory functions, neutrophils externalize IL-17A-decorated NETs in many diseases such as psoriasis, ankylosing spondylitis, pulmonary fibrosis, asthma, Alzheimer\u0026rsquo;s disease, and acute myocardial infarction (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57 CR58 CR59\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Previous studies have also illustrated the indirect amplification of Th17 responses by NETs through macrophages, dendritic cells, and monocytes (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). In addition, NETs directly prime T cells by lowering their activation threshold and promotes Th17 differentiation, which is mediated through a TLR2/MyD88-dependent pathway (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Interestingly, in our study, other than the augmentation of Th17 responses by NETs, we propose that NETs induce innate IL-17 production from γδT cells, which even seems to play a more important role in obesity-exacerbated psoriasis than Th17-derived IL-17. Furthermore, IL-17A induces recruitment of neutrophils and enhances NETs generation (\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). Thus, a corollary to these findings implies that NETs formation has a positive feedback function on the IL-17A driven dynamic inflammation, which further elicits NETs formation and promotes immunopathology in obesity-exacerbated psoriasis. However, the putative mechanism about interactions between NETs and IL-17A-producing T cells remains to be investigated.\u003c/p\u003e \u003cp\u003eClinically, a weight-based approach to secukinumab and ustekinumab has been proposed, indicating that higher doses of biologics may be required for psoriasis patients with greater bodyweight to optimize their efficacy (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). As many obese psoriasis patients show poor responses to immunotherapy and carry an intolerable psychological and economic burden, it is vital to identify biomarkers for the evaluation of therapeutic efficacy and to establish new strategies for combined immunotherapy (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). However, little is known about the precise mechanism of decreased responses resulting from lipid metabolism disorder. Our research identifies SFAs as a major hindrance for anti-IL17A treatment in psoriatic mice with severe symptoms and remarkable local pathological changes. Incredibly, NETs inhibitor combined with anti-IL17A treatment could improve the decreased response in the light of ameliorated symptoms and pathological changes. Thus, our results suggest an intriguing possibility that NETs inhibitors combined with anti-IL17A could be a promising option for psoriasis patients with lipid metabolism disorders. However, although we identified SFAs as the major stimulus for augmented skin inflammation, the effects of unsaturated fatty acids were not investigated and their clear functional differences were not clarified. And further clinical investigation is warranted to learn whether NETs inhibition combined with anti-IL17A treatment can reverse the severe condition of obese psoriasis patients.\u003c/p\u003e \u003cp\u003eIn summary, we highlight the importance of SFAs-induced NETs for dermatologic conditions with overnutrition status. Mechanistically, TLR4-MD2/ROS signaling pathway is critical for SFAs-induced NETs generation. In addition, our research suggests a feed-forward loop in which NETs trigger IL-17 immunity, evolving neutrophils from terminal effectors to complex mediators. Thus, our study reveals that breaking the vicious cycle with NETs inhibitors could be a novel and promising candidate for psoriasis patients with obesity, especially those who show poor responses to various biological agents clinically.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePatient samples\u003c/h2\u003e \u003cp\u003eSerum and skin samples were collected from outpatients with psoriasis vulgaris in department of dermatology at Wuhan Union Hospital between August 2022 and August 2023. Human studies were approved by the Ethics Committee for Clinical Trials at Huazhong University of Science and Technology and all patients signed informed consent. All patients were evaluated for baseline characteristics including age, gender, height, weight, BMI, diagnosis, PASI, body surface area (BSA), dermatology life quality index (DLQI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMice research\u003c/h2\u003e \u003cp\u003eFemale C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Starting at 3 weeks of age, female mice were fed a CD or HFD for 16 weeks. An HFD provides 60% of its energy in the form of fat (Cat # 112252, Dyets Inc.), whereas an CD provides 10% of its energy in the form of fat. For psoriatic dermatitis mouse model, 62.5 mg of 5% IMQ cream was applied on the back skin. Clinical scores and skin thickness were assessed as described. To inhibit NETs formation, mice were intravenously injected daily with 50 mg/kg/d of DNase I (DNase I; Roche) resuspended in PBS starting 2 d prior to the IMQ treatment and during the whole duration of the IMQ treatment. For PA or anti-IL17A treatment, mice were intravenously injected daily with 5 \u0026micro;mol/d of PA or 30 \u0026micro;g/d of anti-IL17A starting 2 d prior to the IMQ treatment and during the whole duration of the IMQ treatment. All mice were maintained in the animal facilities under specific pathogen-free conditions. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil isolation and\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003eNETs formation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBone marrow cells was acquired from mice as described. Neutrophils were isolated by density gradient centrifugation in Histopaque 1077 (Sigma-Aldrich) and Histopaque 1119 (Sigma-Aldrich). Freshly isolated neutrophils were seeded on 96-well round bottom culture plates (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) and poly-D-lysine coated coverslips in 12-well round bottom culture plates (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells). After a 1-hour incubation period at 37℃, the PA, TAK-242, DPI, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added for corresponding incubation in different experiments. LPS or PMA were used as positive controls, whereas DNase Ⅰ served as a negative control. Cell-impermeable DNA dye SYTOX Red (Thermo Fisher Scientific) were added to the incubation system in some experiments. Neutrophils were then cultured for 4 h at 37\u0026deg; in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, after which cells in 96-well plates were collected for flow cytometry and coverslips were used for scanning electron microscopy or immunofluorescent staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of NETs in serum\u003c/h2\u003e \u003cp\u003edsDNA was measured in the serum using Quant-iT PicoGreen double-stranded DNA reagent (Invitrogen) according to the manufacturer\u0026rsquo;s protocol. A capture ELISA assay was used to detect myeloperoxidase MPO-DNA complexes in the serum as described. Briefly, a MPO ELISA kit (Hycult) was used according to the manufacturer\u0026rsquo;s directions. After blocking in 2% BSA, 40 \u0026micro;l of serum was added per well in combination with the peroxidase-labeled anti-DNA monoclonal antibody (Cell Death ELISAPLUS; Roche) according to the manufacturer\u0026acute;s instructions. After 4 h of incubation at room temperature on a shaking device (300 rpm), the samples were washed three times with 200 \u0026micro;l PBS per well and the peroxidase substrate (ABTS) of the kit (Cell Death ELISAPLUS; Roche) was added. The absorbance at 405 nm wavelength was measured after 40 min incubation at 37\u0026deg;C in the dark.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eMouse back skin and skin samples from psoriasis patients were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 mm sections. H\u0026amp;E staining was performed to evaluate inflammatory infiltration and epidermal thickness. Immunohistochemistry staining was applied with the following antibodies respectively: anti-histone H3 (citrulline R2\u0026thinsp;+\u0026thinsp;R8\u0026thinsp;+\u0026thinsp;R17) antibody (Abcam), anti- myeloperoxidase antibody (Abcam), anti-IL-17A antibody (Abcam), anti-Ki67 antibody (Abcam), anti-Ly6G antibody (Abcam), anti-TLR4 antibody (Abcam), anti-TLR4/MD2 antibody (Biolegend), anti‑Mouse TCR Gamma\u0026thinsp;+\u0026thinsp;TCR Delta (LifeSpan), anti-CD4 antibody (Abcam). The primary antibody was omitted in negative controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGas chromatography mass spectrometry\u003c/h2\u003e \u003cp\u003eThe GC analysis was performed on trace 1300 gas chromatograph (Thermo Fisher Scientific, USA). The GC was fitted with a capillary column Thermo TG-FAME (50 m*0.25 mm ID*0.20 \u0026micro;m) and helium was used as the carrier gas at 0.63 mL/min. Injection was made in split mode at 8:1 with an injection volume of 1 \u0026micro;L and an injector temperature of 250℃. The temperature of the ion source and MS transfer line were 300℃ and 280℃, respectively. The column temperature was programmed to increase from an initial temperature of 80℃, which was maintained for 1 min, followed by an increase to 160℃ at 20℃/min, which was maintained for 1.5 min, and increase to 196℃ at 3℃/min, which was maintained for 8.5 min, and finally to 250℃ at 20℃/min and kept at this temperature for 3 min. Mass spectrometric detection of metabolites was performed on TSQ 9000 (Thermo Fisher Scientific, USA) with electron impact ionization mode. Single ion monitoring (SIM) mode was used with the electron energy of 70 eV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing and transcriptomics analysis\u003c/h2\u003e \u003cp\u003eTotal RNA of neutrophils isolated from CD-fed and HFD-fed mouse bone marrow was extracted with TRIzol (Invitrogen). The cDNA library construction, library purification, and RNA sequencing (RNA-seq) were conducted on a DNBSEQ platform with PE150 (read length) at Huada Gene Technology Co., Ltd. (Shenzhen, China) following standard protocols. Expression level of gene was calculated by RSEM (v1.3.1). Differential expression analysis was performed using the DESeq2 with \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.05. Heatmaps were analyzed and plotted using the R programming language (version 4.2.3; pheatmap package and ComplexHeatmap package).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eNeutrophils were fixed in 4% paraformaldehyde in PBS for 30 min and permeabilized in 0.5% Triton X-100 in PBS for 3 min; after blocking with 2% BSA, they were incubated with the specific primary antibodies for citH3 (Abcam), MPO (Abcam), or anti-TLR4/MD2 antibody (Biolegend). Secondary antibody conjugated with FITC or Cy3 were used. The samples were observed under a confocal microscope (Dragonfly/CR-DFLY-201-40).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy\u003c/h2\u003e \u003cp\u003eNeutrophils were fixed with 2.5% glutaraldehyde for 1 h and then washed three times with PBS. Cells were post-fixed with 1% OsO\u003csub\u003e4\u003c/sub\u003e for 1 h and then further washed three times with PBS. Samples were dehydrated, by using graded ethanol concentrations, from 30% to absolute ethanol, for 10 min each, plus two additional immersions in absolute ethanol, for 10 min each. The glass coverslips were dried to the critical point and covered with gold. The samples were analyzed in a scanning electron microscope (Hitachi/SU8100).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eROS detection\u003c/h2\u003e \u003cp\u003eNeutrophil ROS levels were detected using the fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. Neutrophils were incubated for 45 min in serum-free media containing 10 mM DCFH-DA. The conversion of DCFH-DA to the fluorescent product 2',7'-dichlorofluorescein (DCF) was measured using flow cytometry with wavelength of excitation at 488 nm and emission at 525 nm. ROS in skin of mice and patients was observed using the fluorescence probe dihydroethidium (DHE). Sections of skin were incubated for 30 min in PBS containing 10 \u0026micro;M DHE and were analyzed under fluorescence microscope (Nikon Eclipse Ti-SR) with wavelength of excitation at 535 nm and emission at 610 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eNonspecific antibody binding was blocked with an anti-CD16/32 antibody (Biolegend). Cells were washed and stained with surface antibodies. For intracellular cytokines staining, cells were collected and restimulated with PMA (100 ng/ml) and ionomycin (500 ng/ml) in the presence of Golgi-plug (BD) for 5 h. Cells were then fixed and permeabilized with Cytofix/Cytoperm Kit (BD) and stained with the appropriate antibodies. Intracellular staining for transcription factor was performed using the Foxp3 Permeabilization/Fixation kit (eBioscience), followed by staining with appropriate antibodies. For citH3 staining, cells were incubated with a primary anti-citH3 antibody (Abcam) and labeled with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody (Invitrogen). LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen Life Technology) was used to gate out dead cells. The following antibodies were used: anti-mouse CD45 (Biolegend), anti-mouse/human CD11b (Biolegend), anti-mouse Ly6G (Biolegend), anti-mouse CD3ε (Biolegend), anti-mouse CD4 (Biolegend), anti-mouse CD8a (Biolegend), anti-mouse TCRγδ (Biolegend), anti-mouse IL-17A (Biolegend), anti-mouse IL-17F (Biolegend), anti-mouse RORγt (BD), anti-mouse Foxp3 (BD), anti-mouse IFNγ (Biolegend), anti-mouse IL-4 (Biolegend), anti-mouse IL-13 (Biolegend), anti-mouse IL-22 (eBioscience), anti-mouse TLR4/MD2 complex (Biolegend). Cells were measured with a ID7000\u0026trade; Spectral Cell Analyzer (Sony Biotechnology) and analyzed using FlowJo software (TreeStar).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were processed using GraphPad Prism 9.0. Flow cytometry analysis was performed with FlowJo_V10. Experimental results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Comparisons were performed using the unpaired student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test and one-way analysis of variance. For all analyses, a two-tailed P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (82130089, 82103730, 82304022, and 32070894); Key R\u0026amp;D Program of Hubei Province (YFXM2021000203); National Key Research and Development Program of China (2020YFA0804400); Program of HUST Academic Frontier Youth Team (2018QYTD10). We thank all members of Medical sub-center of Analytical and Testing Center, Huazhong University of Science and Technology for technical assistance. We thank all members of Institutional Animal Care and Use Committee, Huazhong University of Science and Technology for showing loving care for life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.X. and J.L. designed the project, performed experiments, analyzed data, and wrote the manuscript. S.Y., X.X., Q.D., H.D., W.N., B.J., L.Z., Z.C., X.Z. and Y.X. conducted experiments. J.Y. and Y.L. interpreted data. R.H. and J.T. designed the project, oversaw the studies, provided intellectual support, and edited the manuscript. All authors edited and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u0026nbsp;\u003c/strong\u003eAll studies involving mice were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. All human clinical protocols were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from the patients before participation in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources:\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (82130089, 82103730, 82304022, and 32070894); Key R\u0026amp;D Program of Hubei Province (YFXM2021000203); National Key Research and Development Program of China (2020YFA0804400); Program of HUST Academic Frontier Youth Team (2018QYTD10).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGriffiths CEM, Armstrong AW, Gudjonsson JE, Barker J. Psoriasis. Lancet. 2021;397(10281):1301\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong AW, Read C. Pathophysiology, Clinical Presentation, and Treatment of Psoriasis: A Review. JAMA. 2020;323(19):1945\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, Zhao Q, Wang X, Zhou H, Hu J, Gu L, et al. Pathogenesis, multi-omics research, and clinical treatment of psoriasis. J Autoimmun. 2022;133:102916.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBu J, Ding R, Zhou L, Chen X, Shen E. Epidemiology of Psoriasis and Comorbid Diseases: A Narrative Review. Front Immunol. 2022;13:880201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah K, Mellars L, Changolkar A, Feldman SR. Real-world burden of comorbidities in US patients with psoriasis. J Am Acad Dermatol. 2017;77(2):287\u0026ndash;92 e4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeshita J, Grewal S, Langan SM, Mehta NN, Ogdie A, Van Voorhees AS, et al. Psoriasis and comorbid diseases: Epidemiology. J Am Acad Dermatol. 2017;76(3):377\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnekvik I, Smith CH, Nilsen TIL, Langan SM, Modalsli EH, Romundstad PR, et al. Obesity, Waist Circumference, Weight Change, and Risk of Incident Psoriasis: Prospective Data from the HUNT Study. J Invest Dermatol. 2017;137(12):2484\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorden A, Rekhtman S, Strunk A, Garg A. Risk of psoriasis according to body mass index: A retrospective cohort analysis. J Am Acad Dermatol. 2022;86(5):1020\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParoutoglou K, Papadavid E, Christodoulatos GS, Dalamaga M. Deciphering the Association Between Psoriasis and Obesity: Current Evidence and Treatment Considerations. Curr Obes Rep. 2020;9(3):165\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu MY, Yu CL, Yang SJ, Chi CC. Change in body weight and body mass index in psoriasis patients receiving biologics: A systematic review and network meta-analysis. J Am Acad Dermatol. 2020;82(1):101\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapp KA, Langley RG, Sigurgeirsson B, Abe M, Baker DR, Konno P, et al. Efficacy and safety of secukinumab in the treatment of moderate-to-severe plaque psoriasis: a randomized, double-blind, placebo-controlled phase II dose-ranging study. Br J Dermatol. 2013;168(2):412\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu NL, Hsu CJ, Sun FJ, Tsai TF. Efficacy and safety of secukinumab in Taiwanese patients with moderate to severe plaque psoriasis: Subanalysis from ERASURE phase III study. J Dermatol. 2017;44(10):1129\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebwohl M, Yeilding N, Szapary P, Wang Y, Li S, Zhu Y, et al. Impact of weight on the efficacy and safety of ustekinumab in patients with moderate to severe psoriasis: rationale for dosing recommendations. Journal of the American Academy of Dermatology. 2010;63(4):571\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbert D, Franz S, Popkova Y, Anderegg U, Schiller J, Schwede K, et al. High-Fat Diet Exacerbates Early Psoriatic Skin Inflammation Independent of Obesity: Saturated Fatty Acids as Key Players. J Invest Dermatol. 2018;138(9):1999\u0026ndash;2009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSivasami P, Elkins C, Diaz-Saldana PP, Goss K, Peng A, Hamersky Mt, et al. Obesity-induced dysregulation of skin-resident PPARgamma(+) Treg cells promotes IL-17A-mediated psoriatic inflammation. Immunity. 2023;56(8):1844\u0026ndash;61 e6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastanheira FVS, Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation. Blood. 2019;133(20):2178\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThiam HR, Wong SL, Wagner DD, Waterman CM. Cellular Mechanisms of NETosis. Annu Rev Cell Dev Biol. 2020;36:191\u0026ndash;218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcDowell SAC, Luo RBE, Arabzadeh A, Dor\u0026eacute; S, Bennett NC, Breton V, et al. Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration. Nature Cancer. 2021;2(5):545\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrotfain E, Hadad N, Shapira Y, Avinoah E, Zlotnik A, Raichel L, et al. Neutrophil functions in morbidly obese subjects. Clin Exp Immunol. 2015;181(1):156\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Windt DJ, Sud V, Zhang H, Varley PR, Goswami J, Yazdani HO, et al. Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis. Hepatology. 2018;68(4):1347\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Zhang H, Wang Y, Brown ZJ, Xia Y, Huang Z, et al. Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis. J Hepatol. 2021;75(6):1271\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis. Science (New York, NY). 2015;349(6245):316\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolberink EA, Peppelman M, van de Kerkhof PC, van Erp PE, Gerritsen MJ. Establishing the dynamics of neutrophil accumulation in vivo by reflectance confocal microscopy. Exp Dermatol. 2014;23(3):184\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Rosales YA, Langereis JD, Gorris MAJ, van den Reek J, Fasse E, Netea MG, et al. Immunomodulatory aged neutrophils are augmented in blood and skin of psoriasis patients. J Allergy Clin Immunol. 2021;148(4):1030\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu SC, Yu HS, Yen FL, Lin CL, Chen GS, Lan CC. Neutrophil extracellular trap formation is increased in psoriasis and induces human beta-defensin-2 production in epidermal keratinocytes. Sci Rep. 2016;6:31119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, Li G, Yang X, Song Z, Wang Y, Zhang Z. NETosis in Psoriatic Arthritis: Serum MPO-DNA Complex Level Correlates With Its Disease Activity. Front Immunol. 2022;13:911347.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWojcik P, Garley M, Wronski A, Jablonska E, Skrzydlewska E. Cannabidiol Modifies the Formation of NETs in Neutrophils of Psoriatic Patients. Int J Mol Sci. 2020;21(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCzerwinska J, Owczarczyk-Saczonek A. The Role of the Neutrophilic Network in the Pathogenesis of Psoriasis. Int J Mol Sci. 2022;23(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin AM, Rubin CJ, Khandpur R, Wang JY, Riblett M, Yalavarthi S, et al. Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J Immunol. 2011;187(1):490\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao S, Fang H, Dang E, Xue K, Zhang J, Li B, et al. Neutrophil Extracellular Traps Promote Inflammatory Responses in Psoriasis via Activating Epidermal TLR4/IL-36R Crosstalk. Front Immunol. 2019;10:746.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlkhouri N, Dixon LJ, Feldstein AE. Lipotoxicity in nonalcoholic fatty liver disease: not all lipids are created equal. Expert Rev Gastroenterol Hepatol. 2009;3(4):445\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLian CY, Zhai ZZ, Li ZF, Wang L. High fat diet-triggered non-alcoholic fatty liver disease: A review of proposed mechanisms. Chem Biol Interact. 2020;330:109199.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin Invest. 2006;116(11):3015\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMangum KD, Gallagher KA. Obesity confers macrophage memory. Science (New York, NY). 2023;379(6627):28\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicholas DA, Zhang K, Hung C, Glasgow S, Aruni AW, Unternaehrer J, et al. Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1beta. PLoS One. 2017;12(5):e0176793.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaloney E, Sweet IR, Hockenbery DM, Pham M, Rizzo NO, Tateya S, et al. Activation of NF-kappaB by palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation. Arterioscler Thromb Vasc Biol. 2009;29(9):1370\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SY, Jeong JM, Kim SJ, Seo W, Kim MH, Choi WM, et al. Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4-MD2 complex. Nat Commun. 2017;8(1):2247.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Qian Y, Fang Q, Zhong P, Li W, Wang L, et al. Saturated palmitic acid induces myocardial inflammatory injuries through direct binding to TLR4 accessory protein MD2. Nat Commun. 2017;8:13997.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe S, Iwata Y, Fukushima H, Saito K, Tanaka Y, Hasegawa Y, et al. Neutrophil extracellular traps are induced in a psoriasis model of interleukin-36 receptor antagonist-deficient mice. Sci Rep. 2020;10(1):20149.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHurabielle C, Link VM, Bouladoux N, Han SJ, Merrill ED, Lightfoot YL, et al. Immunity to commensal skin fungi promotes psoriasiform skin inflammation. Proc Natl Acad Sci U S A. 2020;117(28):16465\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao T, Yuan X, Fang H, Chen J, Xue K, Li Z, et al. Neutrophil extracellular traps promote keratinocyte inflammation via AIM2 inflammasome and AIM2-XIAP in psoriasis. Exp Dermatol. 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkrzeczynska-Moncznik J, Wlodarczyk A, Zabieglo K, Kapinska-Mrowiecka M, Marewicz E, Dubin A, et al. Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis. J Immunol. 2012;189(4):1611\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang M, Fang H, Shao S, Dang E, Zhang J, Qiao P, et al. Keratinocyte exosomes activate neutrophils and enhance skin inflammation in psoriasis. FASEB J. 2019;33(12):13241\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerster F, Bittner Z, Archer NK, Dickhofer S, Eisel D, Eigenbrod T, et al. Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis. Nat Commun. 2020;11(1):105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoorthy AN, Tan KB, Wang S, Narasaraju T, Chow VT. Effect of High-Fat Diet on the Formation of Pulmonary Neutrophil Extracellular Traps during Influenza Pneumonia in BALB/c Mice. Front Immunol. 2016;7:289.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Wang Q, Venugopal J, Wang J, Kleiman K, Guo C, et al. Obesity-induced Endothelial Dysfunction is Prevented by Neutrophil Extracellular Trap Inhibition. Sci Rep. 2018;8(1):4881.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandez-Garcia V, Gonzalez-Ramos S, Avendano-Ortiz J, Martin-Sanz P, Gomez-Coronado D, Delgado C, et al. High-fat diet activates splenic NOD1 and enhances neutrophil recruitment and neutrophil extracellular traps release in the spleen of ApoE-deficient mice. Cell Mol Life Sci. 2022;79(8):396.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoli V, Zanoni I. Neutrophil intrinsic and extrinsic regulation of NETosis in health and disease. Trends Microbiol. 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmara N, Cooper MP, Voronkova MA, Webb BA, Lynch EM, Kollman JM, et al. Selective activation of PFKL suppresses the phagocytic oxidative burst. Cell. 2021;184(17):4480-94 e15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmini P, Stojkov D, Felser A, Jackson CB, Courage C, Schaller A, et al. Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production. Nat Commun. 2018;9(1):2958.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFritsche KL. The science of fatty acids and inflammation. Adv Nutr. 2015;6(3):293S-301S.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang DH, Kim JA, Lee JY. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid. Eur J Pharmacol. 2016;785:24\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWigerblad G, Kaplan MJ. Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases. Nat Rev Immunol. 2022:1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim TS, Silva LM, Theofilou VI, Greenwell-Wild T, Li L, Williams DW, et al. Neutrophil extracellular traps and extracellular histones potentiate IL-17 inflammation in periodontitis. J Exp Med. 2023;220(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiranda FJB, Rocha BC, Pereira MCA, Pereira LMN, de Souza EHM, Marino AP, et al. Toxoplasma gondii-Induced Neutrophil Extracellular Traps Amplify the Innate and Adaptive Response. mBio. 2021;12(5):e0130721.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapagoras C, Chrysanthopoulou A, Mitsios A, Ntinopoulou M, Tsironidou V, Batsali AK, et al. IL-17A expressed on neutrophil extracellular traps promotes mesenchymal stem cell differentiation toward bone-forming cells in ankylosing spondylitis. Eur J Immunol. 2021;51(4):930\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChrysanthopoulou A, Mitroulis I, Apostolidou E, Arelaki S, Mikroulis D, Konstantinidis T, et al. Neutrophil extracellular traps promote differentiation and function of fibroblasts. J Pathol. 2014;233(3):294\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZenaro E, Pietronigro E, Della Bianca V, Piacentino G, Marongiu L, Budui S, et al. Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Nat Med. 2015;21(8):880\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Boer OJ, Li X, Teeling P, Mackaay C, Ploegmakers HJ, van der Loos CM, et al. Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction. Thromb Haemost. 2013;109(2):290\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNtinopoulou M, Cassimos D, Roupakia E, Kolettas E, Panopoulou M, Mantadakis E, et al. Ιnterleukin-17A-Enriched Neutrophil Extracellular Traps Promote Immunofibrotic Aspects of Childhood Asthma Exacerbation. Biomedicines. 2023;11(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Chen H, Yang Z-T, Mao E-Q, Chen Y, Chen E-Z. Free fatty acids-induced neutrophil extracellular traps lead to dendritic cells activation and T cell differentiation in acute lung injury. Aging (Albany NY). 2021;13(24):26148\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrishnamoorthy N, Douda DN, Br\u0026uuml;ggemann TR, Ricklefs I, Duvall MG, Abdulnour R-EE, et al. Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma. Sci Immunol. 2018;3(26).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLambert S, Hambro CA, Johnston A, Stuart PE, Tsoi LC, Nair RP, et al. Neutrophil Extracellular Traps Induce Human Th17 Cells: Effect of Psoriasis-Associated TRAF3IP2 Genotype. J Invest Dermatol. 2019;139(6):1245\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTillack K, Breiden P, Martin R, Sospedra M. T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses. J Immunol. 2012;188(7):3150\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson AS, Randall KL, Pettitt JA, Ellyard JI, Blumenthal A, Enders A, et al. Neutrophil extracellular traps and their histones promote Th17 cell differentiation directly via TLR2. Nat Commun. 2022;13(1):528.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVural S, Kerl K, Ertop Dogan P, Vollmer S, Puchta U, He M, et al. Lesional activation of T(c) 17 cells in Behcet disease and psoriasis supports HLA class I-mediated autoimmune responses. Br J Dermatol. 2021;185(6):1209\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Chandra V, Riquelme Sanchez E, Dutta P, Quesada PR, Rakoski A, et al. Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer. J Exp Med. 2020;217(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeppkes M, Mauer\u0026ouml;der C, Hirth S, Nowecki S, G\u0026uuml;nther C, Billmeier U, et al. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis. Nature Communications. 2016;7(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorman NJ. Management of psoriasis as a systemic disease: what is the evidence? Br J Dermatol. 2020;182(4):840\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-immunology","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cmi","sideBox":"Learn more about [Cellular \u0026 Molecular Immunology](http://www.nature.com/cmi/)","snPcode":"41423","submissionUrl":"https://mts-cmi.nature.com/cgi-bin/main.plex","title":"Cellular \u0026 Molecular Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4940299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4940299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePsoriasis patients with obesity tend to show a serious clinical manifestation and have poor responses to various biological agents in most cases. However, the mechanisms in obesity-exacerbated psoriasis remain enigmatic. In this study, we found that the abundance of systemic and localized cutaneous neutrophil extracellular traps (NETs) in obesity-induced aggravation of psoriasis was positively correlated with disease severity, and inhibition of NETs alleviated psoriatic dermatitis in obese mice. Mechanistically, we found that changes of fatty acid composition in obese subjects resulted in the deposit of saturated fatty acids (SFAs), which promoted the release of NETs via the TLR4-MD2/ROS signaling pathway. We further found that NETs potentiated IL-17 inflammation in obesity-exacerbated psoriasis, especially γδT17-mediated immune responses. Moreover, SFAs induced decreased response to anti-IL17A treatment in psoriasis-like mice, whereas inhibition of NETs improved the beneficial effects of anti-IL17A in psoriasis-like mice with lipid metabolism disorders. Our findings collectively suggest that SFAs-induced NETs play a critical role in the exacerbation of obesity-related psoriasis, and provide potential new strategies for the treatment of refractory psoriasis patients with lipid metabolism disorders clinically.\u003c/p\u003e","manuscriptTitle":"Saturated fatty acids-induced neutrophil extracellular traps contribute to exacerbation and biologic therapy resistance in obesity-related psoriasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-15 09:06:38","doi":"10.21203/rs.3.rs-4940299/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-10-16T11:40:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-10-04T12:17:26+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-15T19:27:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-12T21:15:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-12T06:32:13+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-08-22T11:36:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-20T04:19:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-20T03:38:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular \u0026 Molecular Immunology","date":"2024-08-19T17:45:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-immunology","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cmi","sideBox":"Learn more about [Cellular \u0026 Molecular Immunology](http://www.nature.com/cmi/)","snPcode":"41423","submissionUrl":"https://mts-cmi.nature.com/cgi-bin/main.plex","title":"Cellular \u0026 Molecular Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f68be94c-7132-42eb-936f-8959723ed425","owner":[],"postedDate":"October 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36418169,"name":"Biological sciences/Immunology/Innate immune cells/Granulocytes/Neutrophils"},{"id":36418170,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":36418171,"name":"Biological sciences/Immunology/Inflammation/Chronic inflammation"},{"id":36418172,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":36418173,"name":"Biological sciences/Immunology/Translational immunology"}],"tags":[],"updatedAt":"2025-04-02T07:09:16+00:00","versionOfRecord":{"articleIdentity":"rs-4940299","link":"https://doi.org/10.1038/s41423-025-01278-7","journal":{"identity":"cellular-and-molecular-immunology","isVorOnly":false,"title":"Cellular \u0026 Molecular Immunology"},"publishedOn":"2025-04-01 04:00:00","publishedOnDateReadable":"April 1st, 2025"},"versionCreatedAt":"2024-10-15 09:06:38","video":"","vorDoi":"10.1038/s41423-025-01278-7","vorDoiUrl":"https://doi.org/10.1038/s41423-025-01278-7","workflowStages":[]},"version":"v1","identity":"rs-4940299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4940299","identity":"rs-4940299","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}