Pentagalloyl glucose Suppresses MSU Crystal–Induced Gout Inflammation and Arachidonic Acid Production In Vitro and In Vivo

Pentagalloyl glucose Suppresses MSU Crystal–Induced Gout Inflammation and Arachidonic Acid Production In Vitro and In Vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pentagalloyl glucose Suppresses MSU Crystal–Induced Gout Inflammation and Arachidonic Acid Production In Vitro and In Vivo Sadiq Umar, Yu Lu, Sugasini Dhavamani, Poorna CR Yalagala, Matez S Wietecha, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8904164/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Gout is an acute inflammatory arthritis triggered by monosodium urate (MSU) crystal deposition and activation of innate immune responses. In addition to inflammasome signaling, emerging evidence suggests that metabolic reprogramming of arachidonic acid (AA) pathways amplifies inflammatory responses during gout flares. However, the contribution of upstream fatty acid desaturation processes that regulate endogenous AA availability remains poorly defined. 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) is a naturally occurring polyphenol with reported anti-inflammatory activity, but its effects on MSU-induced fatty acid metabolism and gouty inflammation have not been well established. Methods Publicly available bulk and single-cell transcriptomic datasets from human and mouse gout studies were analyzed to assess dysregulation of AA-associated pathways. MSU-induced inflammatory responses were examined in mouse bone marrow–derived macrophages and in a murine MSU-induced gout model. Macrophages were treated with PGG prior to MSU stimulation, and inflammatory cytokine production, phagocytosis, and expression of fatty acid desaturases were assessed. Lipidomic analysis of macrophages and plasma was performed using gas chromatography–mass spectrometry (GC–MS) to quantify arachidonic acid and related fatty acids. In vivo disease severity, cytokine expression, and anti-inflammatory markers were evaluated following PGG treatment. Results Analysis of public datasets revealed consistent dysregulation of arachidonic acid–associated inflammatory pathways during gout flares. In macrophages, MSU stimulation increased expression of fatty acid desaturases FADS1 and FADS2 and promoted accumulation of arachidonic acid, concomitant with robust production of pro-inflammatory cytokines. PGG treatment significantly suppressed MSU-induced FADS1, FADS2 and arachidonic acid levels, and attenuated pro-inflammatory cytokine production. PGG also markedly impaired macrophage phagocytosis of MSU crystals. In vivo, PGG treatment significantly reduced clinical disease severity in an MSU-induced gout model, suppressed fatty acid desaturation and arachidonic acid accumulation in plasma, decreased pro-inflammatory cytokine expression, and enhanced anti-inflammatory markers. Conclusion These findings identify fatty acid desaturation as an important metabolic contributor to gouty inflammation and demonstrate that PGG suppresses MSU-induced inflammation by limiting endogenous arachidonic acid availability, reducing inflammatory amplification, and impairing MSU crystal phagocytosis. Targeting upstream fatty acid metabolism represents a potential therapeutic strategy for modulating acute gout flares beyond conventional anti-inflammatory approaches. Rheumatology Immunology macrophages MSU-gout model Pentagalloyl glucose inflammation arachidonic acid pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Gout is a prevalent and increasingly common form of inflammatory arthritis, with a rising global incidence in both developed and developing countries 1 – 4 . The initiation of gouty inflammation occurs when resident joint macrophages phagocytose monosodium urate (MSU) crystals. Crystal uptake activates innate immune signaling pathways, notably through Toll-like receptors (TLR2 and TLR4), leading to NF-κB activation NF-κB 5–10 and assembly of the NLRP3 inflammasome 8 , 11 – 13 . This cascade promotes the release of pro-inflammatory cytokines and drives the recruitment of neutrophils to the inflamed joint. Activated neutrophils further amplify inflammation by releasing reactive oxygen species, proteases, cytokines, chemokines, and lipid mediators 6 , 14 – 22 . MSU crystals potently stimulate arachidonic acid (AA) metabolism, resulting in robust prostanoid production—particularly prostaglandin E₂ (PGE₂)—through enhanced cytosolic phospholipase A₂ (cPLA₂) activity and cyclooxygenase-2 (COX-2) expression in immune cells and osteoblasts. Elevated levels of prostanoids within gouty synovial fluid are strongly associated with the cardinal clinical features of acute gout flares, including severe pain, swelling, and erythema 23 , 24 , 25 . Despite the established role of AA-derived lipid mediators in inflammatory diseases, their specific contribution to gout pathogenesis remains incompletely understood. AA metabolism generates a diverse array of bioactive lipids, including prostaglandins, thromboxanes, and leukotrienes, which are potent regulators of immune cell activation and resolution 26 , 27 . Recent studies have demonstrated that MSU crystals upregulate PTGS2 (COX-2) expression in peripheral monocytes from patients with advanced gout, linking AA metabolism to systemic inflammatory burden 28 . In addition, MSU-induced leukotriene production by neutrophils and elevated synovial fluid levels of leukotriene B₄ (LTB₄) further support a pathogenic role for lipid mediators in gout, exceeding those observed in other inflammatory arthritides such as rheumatoid arthritis 29 . Current treatment of acute gout relies largely on NSAIDs, colchicine, and corticosteroids, which effectively suppress symptoms but are often limited by systemic toxicity and contraindications in patients with renal, cardiovascular, or metabolic comorbidities 4 , 30 , 31 . NSAIDs primarily inhibit prostaglandin synthesis via cyclooxygenase blockade, while colchicine reduces inflammation by impairing microtubule-dependent neutrophil recruitment; however, neither strategy directly regulates upstream arachidonic acid metabolism or coordinated lipid mediator signaling during MSU-driven inflammation. 1,2,3,4,6-Penta- O -galloyl-β-D-glucose (PGG) is a naturally occurring polyphenolic compound that has been reported to exhibit broad anti-inflammatory, anti-oxidative, and immunomodulatory activities across multiple experimental systems. Previous studies have demonstrated that PGG suppresses inflammatory mediator production in macrophages, inhibits oxidative stress, and attenuates tissue injury in models of inflammatory and metabolic disease. Mechanistically, PGG has been shown to interfere with upstream innate immune signaling and lipid metabolic pathways rather than acting solely on terminal cytokine effectors, suggesting a capacity to modulate inflammatory amplification at an early stage. Despite these observations, the therapeutic potential of PGG in acute gouty inflammation and its impact on monosodium urate (MSU)–driven arachidonic acid metabolism remain poorly defined. In the present study, we aimed to delineate the anti-inflammatory mechanisms of PGG in macrophages and to evaluate its therapeutic efficacy in a murine MSU-induced gout model, with particular emphasis on its regulation of arachidonic acid–derived prostanoid production and downstream inflammatory responses. Methods Gene expression analysis from gout patients and mouse model Bulk RNA sequencing dataset GSE242872 submitted by chengyu yin et al 32 for gout model at 8 and 24 hr and GSE191054 Human macrophages activated with MSU 33 and single-cell RNA sequencing dataset GSE211783 submitted by Hanjie Yu et al. 34 to evaluate the expression of arachidonic pathway. Myeloid cells Mouse bone marrow derived macrophages (mBMMs) were isolated from 8-week C57BL/6J mice. Mouse bone marrow cells were cultured with mouse M-CSF (20 ng/ml) for 3 days to obtain myeloid cells differentiated in vitro as MΦs (10% FBS/DMEM). On day 4, MΦs were pretreated for 18 hr with DMSO (PBS), PGG (5 µM, Sigma # G7548, dose is based on our previous studies) 35 , 36 in serum free RPMI. Thereafter cells were stimulated with MSU 9 , 31 (100 µg/ml; Sigma #U2875) for 24 hr. for running ELISA (Protein) and qRT-PCR (mRNA) analysis. Real-time RT-PCR RNA isolated using Trizol and was reverse transcribed to cDNA using the RevertAid RT Reverse Transcription Kit (Thermo Scientific). SYBR green gene expression master mix (Bio-Rad) to perform qRT-PCR. Data was normalized with GAPDH and are presented as fold changes in RNA levels compared to control treatment, calculated following the 2 − ΔΔCt method. ELISA for cytokine analyses Conditioned media from the macrophage, pretreated with PGG (5 µM) overnight followed by stimulation with MSU for 24 hr was collected and cytokine levels of IL-1β, IL-6, TNF-α and IL-18 were measured using DuoSet ELISA (enzyme-linked immunosorbent assay) kits (R&D Systems, MN). In vitro phagocytosis assay The phagocytic activity of macrophages was assessed using the Vybrant™ Phagocytosis Assay Kit (Life Technologies™). Briefly, macrophages (1×10 4 ) were seeded in a 96-well flat-bottom plate, pretreated with PGG overnight, and stimulated with MSU for 2 hours. The culture medium was then replaced with 100 µL of the prepared fluorescent Bioparticle suspension, followed by incubation at 37°C for 2 hours. After incubation, the Bioparticle suspension was removed, and the cells were washed twice with PBS. Subsequently, 100 µL of prepared Trypan Blue suspension was added, incubated for 1 minute, and the fluorescence intensity was measured using a plate reader with ~ 480 nm excitation and ~ 520 nm emission, following the manufacturer's instructions. Lipid Extraction and Fatty Acid Analysis by GC/MS : Blood was collected by cardiac puncture into heparinized syringes, and plasma was isolated by centrifugation at 1,500 × g for 15 min at 4°C. Total lipids were extracted from plasma using a modified version of a previously published method 37 . Briefly, 100 µL of plasma or macrophages lysate (invitro) were mixed with 800 µL of 50% methanol in water containing 0.01 N HCl, followed by the addition of 2 mL chloroform. Samples were vortexed for 30 s, 1 mL water was added, and samples were vortexed again for 30 s before centrifugation. The lower chloroform phase was collected, dried under nitrogen, and used for fatty acid analysis. Lipid extracts were converted to fatty acid methyl esters (FAMEs) as previously described 37 . Dried lipids were resuspended in 0.5 mL toluene containing 25 µg of 22:3 free fatty acid as an internal standard and 250 µg butylated hydroxytoluene. Methanolic HCl (0.3 mL of 8% HCl in methanol) was added, and samples were heated at 100°C for 1 h under nitrogen. The reaction was neutralized with 1.0 mL of 0.33 N NaOH, and FAMEs were extracted twice with 3 mL hexane. Combined hexane extracts were dried under nitrogen, reconstituted in 30 µL hexane, and 1 µL was injected into the GC/MS system. Fatty acid analysis was performed using a Shimadzu QP2010SE GC/MS equipped with a Supelco Omegawax capillary column (30 m × 0.25 mm × 0.25 µm), with data acquired over a total ion current range of m/z 50–400. Murine Model of Gout All animal studies were approved by UIC Animal Care and Use Committee (protocol # 2024-042). After 7 days of acclimatization, 8-10-weeks-old male C57BL/6J mice (Jackson Laboratory) were divided into three groups; a) Control b) Monosodium Uric acid-MSU (gout Model) c) MSU + PGG, (n = 5). In the treatment group, PGG (25 mg/kg, daily oral gavage) was administered from day 0. C57BL/6 mice at 8–10 week are susceptible to the development of gouty arthritis when injected with MSU crystal (0.5 mg) suspended in 25 µl endotoxin free PBS or PBS control will be injected into footpad of mice anaesthetized with 2.5-4% isoflurane. This model is one of the most synchronized and reliable rodent models of gout and produces the least distress 9 , 13 , 38 – 42 . The ∆ ankle circumferences of both the hind ankles from each animal were averaged and monitored for clinical signs of inflammation. Statistical Analysis For comparison between multiple groups, one-way ANOVA followed by Tukey's or Šídák’s multiple comparison test was done using Graph Pad Prism10 software. Values of p < 0.05 were considered significant. Results Altered arachidonic acid–associated metabolic pathways are linked to gout Arachidonic acid (AA) metabolism has emerged as an important amplifier of crystal-induced inflammation. To explore the involvement of AA-associated pathways in gout, we analyzed publicly available bulk and single-cell transcriptomic datasets derived from MSU-induced mouse models and human gout samples. Across datasets, genes involved in prostanoid and leukotriene pathways (e.g., PTGES, ALOX5, and LTA4H) were consistently dysregulated during gout flares compared with remission or control conditions, highlighting a conserved activation of AA-derived inflammatory programs (Fig. 1 ). These analyses support the concept that AA metabolism contributes to gout pathogenesis across species and disease stages and provide a rationale for targeting upstream metabolic processes that regulate AA availability during MSU-driven inflammation 34 . PGG suppresses MSU-induced fatty acid desaturation and arachidonic acid accumulation in macrophages Given the central role of AA as a substrate for inflammatory lipid mediators, we next investigated whether PGG modulates endogenous AA biosynthesis in macrophages. MSU stimulation significantly increased the expression of FADS2 and FADS1, the Δ6- and Δ5-desaturases that catalyze the conversion of linoleic acid to AA. Consistent with enhanced fatty acid desaturation, GC–MS analysis revealed a marked accumulation of AA in MSU-stimulated macrophages. PGG treatment significantly attenuated MSU-induced FADS1, FADS2 and reduced AA levels (Fig. 2 ). These results indicate that PGG suppresses MSU-induced fatty acid desaturation, thereby limiting intracellular AA availability. PGG attenuates MSU-induced inflammatory cytokine production Increased AA availability amplifies inflammatory responses by fueling the generation of bioactive lipid mediators that potentiate cytokine production. Concomitant with FADS1/2 inhibition, PGG robustly attenuated MSU-induced cytokine production. MSU exposure induced pronounced secretion of IL-1β, IL-6, IL18 and TNF-α, while PGG treatment significantly reduced these cytokines, demonstrating broad suppression of the inflammatory response (Fig. 3A-D). At the transcriptional level, MSU stimulation strongly upregulated IL-1β, IL6, IL18 and TNF-α expression, all of which were significantly downregulated following PGG treatment, consistent with reduced inflammatory activation (Fig. 3E-H). Together, these findings indicate that PGG reduced AA accumulation concomitant with reduced cytokine production in macrophages. PGG treatment disrupts MSU-induced phagocytosis Phagocytosis represents a critical early event in gout pathogenesis. Macrophage uptake of monosodium urate (MSU) crystals initiates innate immune activation and downstream inflammatory processes. Our results show that PGG treatment significantly reduced MSU crystal phagocytosis in macrophage (Fig. 4 ), indicating that PGG directly interferes with cellular mechanisms required for MSU uptake. PGG reduces gout severity by modulating fatty acid metabolism and inflammatory responses in vivo To evaluate the therapeutic efficacy of PGG in vivo, an MSU-induced gout model was established in mice. MSU administration resulted in a marked increase in clinical disease severity, as reflected by significantly elevated gout scores compared with controls. PGG treatment significantly attenuated MSU-induced disease severity, leading to a pronounced reduction in clinical scoring over the course of the experiment (Fig. 5 B). At the protein level, MSU challenge induced robust production of IL-1β, IL-6, and TNF-α in joint tissues, whereas PGG treatment significantly suppressed these pro-inflammatory cytokines. In contrast, IL-10 levels were significantly increased in PGG-treated mice, indicating a shift toward an anti-inflammatory environment (Fig. 5 C-F). Consistent with these findings, transcriptional analysis revealed that MSU stimulation markedly upregulated IL-1β, IL-6 and TNF-α expression, all of which were significantly reduced following PGG treatment (Fig. 5 G-J). Conversely, Arg1 expression was significantly enhanced in PGG-treated mice, supporting the induction of an anti-inflammatory phenotype. To further assess whether PGG modulates fatty acid metabolism in vivo, expression of key fatty acid desaturases was examined in plasma from MSU-induced gout mice. MSU challenge significantly increased FADS1, FADS2 and arachidonic acid, consistent with enhanced arachidonic acid biosynthetic activity during gouty inflammation. Notably, PGG treatment markedly suppressed MSU-induced FADS1, FADS2, and AA indicating inhibition of fatty acid desaturation pathways in vivo (Fig. 6 A-C). Together, these findings indicate that PGG suppresses MSU-induced gouty inflammation in vivo by inhibiting fatty acid metabolism, reducing pro-inflammatory mediators, and promoting an anti-inflammatory gene expression profile. Discussion Gout is increasingly recognized as a metabolically driven inflammatory disease in which monosodium urate (MSU) crystals engage innate immune cells and reprogram lipid metabolism to amplify inflammatory responses. In the present study, we demonstrate that PGG exerts potent anti-gout activity both in vitro and in vivo by targeting fatty acid desaturation pathways, suppressing MSU crystal phagocytosis, and shifting macrophage responses toward an anti-inflammatory phenotype. A key finding of this work is the identification of fatty acid desaturases FADS1 and FADS2 as regulated targets during gouty inflammation. These enzymes catalyze critical steps in the biosynthesis of arachidonic acid (AA), a central substrate for pro-inflammatory lipid mediators. Our in vitro data show that MSU stimulation induces FADS1 and FADS2 expressions in macrophages, consistent with metabolic priming toward enhanced AA availability. Importantly, PGG significantly suppressed FADS1 and FADS2 expression, indicating that PGG interferes with upstream lipid metabolic reprogramming rather than solely blocking downstream inflammatory outputs. These findings extend prior observations that AA metabolism contributes to gout pathogenesis and position fatty acid desaturation as a previously underappreciated regulatory node in MSU-driven inflammation. Beyond metabolic regulation, our data highlight phagocytosis as a critical functional target of PGG. Macrophage uptake of MSU crystals is an essential initiating event in gout, triggering intracellular signaling cascades and perpetuating tissue inflammation 43 – 47 . We observed that PGG markedly reduced MSU crystal phagocytosis in macrophages, suggesting that modulation of membrane lipid composition or cytoskeletal dynamics may underlie its inhibitory effects. Given that fatty acid composition directly influences membrane fluidity and phagocytic capacity, suppression of FADS1/FADS2-driven lipid remodeling provides a plausible mechanistic link between altered metabolism and reduced MSU uptake. The in vivo relevance of these findings was confirmed in an MSU-induced mouse model of gout. PGG treatment significantly reduced clinical disease severity, demonstrating robust therapeutic efficacy. PGG suppressed MSU-induced expression of Fads1 and Fads2 in serum, validating that fatty acid metabolic regulation occurs in vivo and is not restricted to cell culture systems. Concomitantly, PGG reduced pro-inflammatory mediators at both the protein and transcriptional levels, including IL-1β, IL-6, TNF-α, while enhancing IL-10 and Arg1 expression. This coordinated molecular shift supports a model in which PGG not only dampens inflammatory activation but also actively promotes resolution-associated macrophage programs. Notably, the increase in IL-10 and Arg1 suggests that PGG favors a reparative immune environment rather than inducing broad immunosuppression. This is particularly relevant in gout, where excessive inflammation coexists with cycles of spontaneous resolution. By limiting fatty acid–driven amplification loops and promoting anti-inflammatory gene expression, PGG may help restore immune balance within the inflamed joint. Collectively, these findings support a multilevel mechanism of action for PGG in gout: (i) inhibition of FADS1/FADS2-mediated fatty acid desaturation, (ii) attenuation of MSU crystal phagocytosis, and (iii) suppressing MSU induced the inflammatory responses in macrophage. This integrated mechanism distinguishes PGG from conventional anti-inflammatory strategies that primarily target single cytokines and underscores the therapeutic potential of metabolic modulation in crystal-induced inflammatory diseases. In summary, our study identifies fatty acid desaturation as a critical contributor to gout pathogenesis and establishes PGG as a metabolically active anti-inflammatory agent capable of suppressing MSU-induced inflammation in vitro and in vivo. These findings provide a strong rationale for further development of PGG or related metabolic modulators as disease-modifying therapies for gout. Limitations. The experimental systems used in this study acute MSU-driven inflammation but do not fully capture the complexity of chronic hyperuricemia or recurrent gout flares observed clinically. Although PGG clearly modulates inflammatory signaling and AA metabolism, the precise molecular targets responsible for these effects remain to be elucidated. Declarations FUNDING: This work was supported by awards from the National Institutes of Health NIH R01DE027404 and R01DE030495 grants. CONFLICT/COMPETING INTEREST: The authors have declared that no commercial or financial conflict of interest exists. CODE AVAILABILITY : Not applicable. AUTHOR CONTRIBUTIONS: All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Ravindran and Umar had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. SU, SR Acquisition of data. SU, YL, PY, SD, MW, SR Analysis and interpretation of data. SU, PY, SD, SR Acknowledgement: The schematic figures were created with BioRender.com. References FitzGerald, J. D.; Dalbeth, N.; Mikuls, T.; Brignardello-Petersen, R.; Guyatt, G.; Abeles, A. M.; Gelber, A. C.; Harrold, L. 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Modulation of IRAK4 as a therapeutic strategy against monosodium urate- and xanthine-induced inflammation in macrophages and HepG2 cells. Front Immunol 2025 , 16 , 1744393. DOI: 10.3389/fimmu.2025.1744393 From NLM Medline. Yin, C.; Liu, B.; Dong, Z.; Shi, S.; Peng, C.; Pan, Y.; Bi, X.; Nie, H.; Zhang, Y.; Tai, Y.; et al. CXCL5 activates CXCR2 in nociceptive sensory neurons to drive joint pain and inflammation in experimental gouty arthritis. Nat Commun 2024 , 15 (1), 3263. DOI: 10.1038/s41467-024-47640-7 From NLM Medline. Cobo, I.; Cheng, A.; Murillo-Saich, J.; Coras, R.; Torres, A.; Abe, Y.; Lana, A. J.; Schlachetzki, J.; Liu-Bryan, R.; Terkeltaub, R.; et al. Monosodium urate crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response. Cell Rep 2022 , 38 (10), 110489. DOI: 10.1016/j.celrep.2022.110489 From NLM Medline. Yu, H.; Xue, W.; Yu, H.; Song, Y.; Liu, X.; Qin, L.; Wang, S.; Bao, H.; Gu, H.; Chen, G.; et al. Single-cell transcriptomics reveals variations in monocytes and Tregs between gout flare and remission. JCI Insight 2024 , 9 (3). DOI: 10.1172/jci.insight.179067 From NLM Medline. Umar, S.; Singh, A. K.; Chourasia, M.; Rasmussen, S. M.; Ruth, J. H.; Ahmed, S. Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model. Front Immunol 2022 , 13 , 928436. DOI: 10.3389/fimmu.2022.928436 From NLM Medline. Panipinto, P. M.; Yue, G. E.; Prasad, B.; Ahmed, S. Pentagalloyl glucose inhibits monosodium urate-induced inflammation and NLRP3 inflammasome formation via TAK1. Am J Physiol Cell Physiol 2025 , 329 (2), C500-C512. DOI: 10.1152/ajpcell.00673.2024 From NLM Medline. Sugasini, D.; Thomas, R.; Yalagala, P. C. R.; Tai, L. M.; Subbaiah, P. V. Dietary docosahexaenoic acid (DHA) as lysophosphatidylcholine, but not as free acid, enriches brain DHA and improves memory in adult mice. Sci Rep 2017 , 7 (1), 11263. DOI: 10.1038/s41598-017-11766-0 From NLM Medline. Joosten, L. A.; Crisan, T. O.; Azam, T.; Cleophas, M. C.; Koenders, M. I.; van de Veerdonk, F. L.; Netea, M. G.; Kim, S.; Dinarello, C. A. Alpha-1-anti-trypsin-Fc fusion protein ameliorates gouty arthritis by reducing release and extracellular processing of IL-1beta and by the induction of endogenous IL-1Ra. Ann Rheum Dis 2016 , 75 (6), 1219-1227. DOI: 10.1136/annrheumdis-2014-206966. Crisan, T. O.; Cleophas, M. C. P.; Novakovic, B.; Erler, K.; van de Veerdonk, F. L.; Stunnenberg, H. G.; Netea, M. G.; Dinarello, C. A.; Joosten, L. A. B. Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway. Proc Natl Acad Sci U S A 2017 , 114 (21), 5485-5490. DOI: 1620910114 [pii] 10.1073/pnas.1620910114. Cheng, J. J.; Ma, X. D.; Ai, G. X.; Yu, Q. X.; Chen, X. Y.; Yan, F.; Li, Y. C.; Xie, J. H.; Su, Z. R.; Xie, Q. F. Palmatine Protects Against MSU-Induced Gouty Arthritis via Regulating the NF-kappaB/NLRP3 and Nrf2 Pathways. Drug Des Devel Ther 2022 , 16 , 2119-2132. DOI: 10.2147/DDDT.S356307. Lan, Z.; Chen, L.; Feng, J.; Xie, Z.; Liu, Z.; Wang, F.; Liu, P.; Yue, X.; Du, L.; Zhao, Y.; et al. Mechanosensitive TRPV4 is required for crystal-induced inflammation. Ann Rheum Dis 2021 , 80 (12), 1604-1614. DOI: 10.1136/annrheumdis-2021-220295. Zhang, Q. B.; Qing, Y. F.; Yin, C. C.; Zhou, L.; Liu, X. S.; Mi, Q. S.; Zhou, J. G. Mice with miR-146a deficiency develop severe gouty arthritis via dysregulation of TRAF 6, IRAK 1 and NALP3 inflammasome. Arthritis Res Ther 2018 , 20 (1), 45. DOI: 10.1186/s13075-018-1546-7. de Almeida, L.; Devi, S.; Indramohan, M.; Huang, Q. Q.; Ratsimandresy, R. A.; Pope, R. M.; Dorfleutner, A.; Stehlik, C. POP1 inhibits MSU-induced inflammasome activation and ameliorates gout. Front Immunol 2022 , 13 , 912069. DOI: 10.3389/fimmu.2022.912069 From NLM Medline. Xu, H.; Zhang, B.; Chen, Y.; Zeng, F.; Wang, W.; Chen, Z.; Cao, L.; Shi, J.; Chen, J.; Zhu, X.; et al. Type II collagen facilitates gouty arthritis by regulating MSU crystallisation and inflammatory cell recruitment. Ann Rheum Dis 2023 , 82 (3), 416-427. DOI: 10.1136/ard-2022-222764 From NLM Medline. Baggio, C.; Sfriso, P.; Cignarella, A.; Galozzi, P.; Scanu, A.; Mastrotto, F.; Favero, M.; Ramonda, R.; Oliviero, F. Phagocytosis and inflammation in crystal-induced arthritis: a synovial fluid and in vitro study. Clin Exp Rheumatol 2021 , 39 (3), 494-500. DOI: 10.55563/clinexprheumatol/jcmrd0 From NLM Medline. Bousoik, E.; Qadri, M.; Elsaid, K. A. CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout. Sci Rep 2020 , 10 (1), 5748. DOI: 10.1038/s41598-020-62727-z From NLM Medline. Piao, M. H.; Wang, H.; Jiang, Y. J.; Wu, Y. L.; Nan, J. X.; Lian, L. H. Taxifolin blocks monosodium urate crystal-induced gouty inflammation by regulating phagocytosis and autophagy. Inflammopharmacology 2022 , 30 (4), 1335-1349. DOI: 10.1007/s10787-022-01014-x From NLM Medline. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8904164","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592952311,"identity":"afca2ff9-aa8c-4499-887d-8b91acb39ccb","order_by":0,"name":"Sadiq Umar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYNCCCgYeECVBgpYzJGthbIPQxGmRn5Gd+Lly3mEZ/gbmg7d5iNFicCN3s+TZbYd5JA6wJVsTp0Uid4NkI1CLAQOPmTRRWuRn5G7+2TgHpIX/G3FaGG7kbpNsbADbwkacFoMzb7dZNhxL55E4zGZsOYcoh7Xnbr7ZUGNtz9/e/PDGG6IcBgfMpCkfBaNgFIyCUYAPAAAXuCu/Vb1DvgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4733-5751","institution":"University of Illinois","correspondingAuthor":true,"prefix":"","firstName":"Sadiq","middleName":"","lastName":"Umar","suffix":""},{"id":592952737,"identity":"98ec9856-2fe7-4e82-b1c4-5d86ee9feb6e","order_by":1,"name":"Yu Lu","email":"","orcid":"","institution":"University of Illinois","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lu","suffix":""},{"id":592952738,"identity":"00223fc9-913d-4890-b875-a6056a67b447","order_by":2,"name":"Sugasini Dhavamani","email":"","orcid":"","institution":"University of Illinois","correspondingAuthor":false,"prefix":"","firstName":"Sugasini","middleName":"","lastName":"Dhavamani","suffix":""},{"id":592952739,"identity":"0751da4f-557b-4fd1-aac3-341805a874e6","order_by":3,"name":"Poorna CR Yalagala","email":"","orcid":"","institution":"University of Illinois","correspondingAuthor":false,"prefix":"","firstName":"Poorna","middleName":"CR","lastName":"Yalagala","suffix":""},{"id":592952740,"identity":"798bdf22-609a-420e-a811-b19ebffba185","order_by":4,"name":"Matez S Wietecha","email":"","orcid":"","institution":"University of Illinois","correspondingAuthor":false,"prefix":"","firstName":"Matez","middleName":"S","lastName":"Wietecha","suffix":""},{"id":592952741,"identity":"23bf34b8-64ae-4b7b-b581-c4b3fcdeac79","order_by":5,"name":"Sriram Ravindran","email":"","orcid":"","institution":"University of Illinois","correspondingAuthor":false,"prefix":"","firstName":"Sriram","middleName":"","lastName":"Ravindran","suffix":""}],"badges":[],"createdAt":"2026-02-17 20:32:07","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8904164/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8904164/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102990189,"identity":"b3e8bb32-3936-4473-ac3e-3eaa49569d92","added_by":"auto","created_at":"2026-02-19 11:21:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2160694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArachidonic acid is associated with the severity of gout. (A) \u003c/strong\u003eSchematic diagram for analysis of public data in mouse and human\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB-D\u003c/strong\u003e) Expression levels of Arachidonic acid related genes in gout.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/c67e10d67a824d7ff15c9552.png"},{"id":102990188,"identity":"d12e7885-19e2-43db-a474-7b202c8c4f20","added_by":"auto","created_at":"2026-02-19 11:21:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":437439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGC–MS profiling of Fatty acid in macrophages. \u003c/strong\u003eTotal lipids were extracted, and fatty acids were derivatized to fatty acid methyl esters (FAMEs) and quantified by gas chromatography–mass spectrometry (GC–MS). Fatty acids associated with FADS2 (Δ6-desaturase) activity, including linoleic acid–derived intermediates, and FADS1 (Δ5-desaturase) products, and arachidonic acid.(n=3) The data are shown as mean ± SEM *** represents p\u0026lt;0.001. Significant differences were determined by one-way ANOVA following Šídák’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/6c7f3e6e1f3a3819408cf3f9.png"},{"id":102990192,"identity":"da2e6f2c-d3b8-4dd6-ae1b-46108574a615","added_by":"auto","created_at":"2026-02-19 11:21:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":687999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGG suppresses MSU-induced pro-inflammatory cytokine production and gene expression in macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMonocytes were isolated from mouse bone marrow and differentiated into macrophages. On day 4, cells were pretreated overnight with PGG (5 µM) and subsequently stimulated with MSU crystals (100 µg/mL) for 24 h. (A–D) Cells were harvested for RNA isolation, and mRNA expression of IL-1β, TNF-α, IL-6, and IL-18 was assessed by real-time RT-PCR. (E–H) Conditioned media were collected and analyzed by ELISA to quantify the secretion of IL-1β, TNF-α, IL-6, and IL-18. Data are presented as mean ± SEM (n = 4). Statistical significance was determined by one-way ANOVA followed by Šídák’s multiple-comparison test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/75c6f7894a17b81f3479aec2.png"},{"id":102990191,"identity":"bb2f2cf7-9b8f-4275-9176-6d950cf51571","added_by":"auto","created_at":"2026-02-19 11:21:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":138537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGG impairs MSU induced phagocytosis. \u003c/strong\u003eMacrophages were seeded into 96 well plate and incubated overnight with PGG (5 µM) and stimulated with MSU (100 µg/ml) for 2 hr. and absorbance was taken following manufacturer instructions. n=3. The data are shown as mean ± SEM, *** represents p\u0026lt;0.001 and **** denotes p\u0026lt;0.0001. Significant differences were determined by one-way ANOVA following Šídák’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/29d48084ba6b3743950798fe.png"},{"id":102990193,"identity":"20c40934-eef9-4e10-b972-20076ed11fa6","added_by":"auto","created_at":"2026-02-19 11:21:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1575565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePGG attenuates MSU-induced gouty inflammation by suppressing scoring and inflammatory cytokines in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Mice were subjected to an MSU-induced gout model and treated with PGG as indicated. (B) Clinical gout severity scores over the experimental period show significant attenuation of MSU-induced disease following PGG treatment. (C-F) Protein levels of IL-1β, IL-6, and TNF-α in joint tissues were markedly increased after MSU challenge and significantly reduced by PGG, whereas IL-10 levels were enhanced. (G-J) qPCR analysis of joint tissues demonstrates that MSU stimulation upregulated IL-1β, IL6 and TNF-α expression, all of which were significantly suppressed by PGG treatment, while Arg1 expression was significantly increased. Data are presented as mean ± SEM (n = 4). Statistical significance was determined by one-way ANOVA followed by Šídák’s multiple-comparison test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/bf2c937de4cfd77adc442dd5.png"},{"id":102990194,"identity":"09b5580e-d4a4-4e53-a8ea-66e518a2e5c5","added_by":"auto","created_at":"2026-02-19 11:21:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":314674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo plasma GC–MS profiling of Fatty acid. \u003c/strong\u003eTotal lipids were extracted from mouse plasma and converted to fatty acid methyl esters (FAMEs) for quantification by gas chromatography–mass spectrometry (GC–MS). Fatty acids reflecting FADS2 (Δ6-desaturase) activity, including linoleic acid–derived intermediates, and FADS1 (Δ5-desaturase) products, including arachidonic acid, were quantified. (A-C). Data are presented as mean ± SEM (n = 5). Statistical significance was determined by one-way ANOVA followed by Šídák’s multiple-comparison test. *p \u0026lt; 0.05, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure62.png","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/3903f75ae79740a6bbcede90.png"},{"id":103050086,"identity":"488f686f-eb80-480e-97c0-5c613ddaacdb","added_by":"auto","created_at":"2026-02-20 07:48:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6382054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8904164/v1/83214273-92f4-4114-9c33-abb7a800655b.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003ePentagalloyl glucose Suppresses MSU Crystal–Induced Gout Inflammation and Arachidonic Acid Production In Vitro and In Vivo\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGout is a prevalent and increasingly common form of inflammatory arthritis, with a rising global incidence in both developed and developing countries\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The initiation of gouty inflammation occurs when resident joint macrophages phagocytose monosodium urate (MSU) crystals. Crystal uptake activates innate immune signaling pathways, notably through Toll-like receptors (TLR2 and TLR4), leading to NF-κB activation NF-κB \u003csup\u003e5\u0026ndash;10\u003c/sup\u003e and assembly of the NLRP3 inflammasome\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This cascade promotes the release of pro-inflammatory cytokines and drives the recruitment of neutrophils to the inflamed joint. Activated neutrophils further amplify inflammation by releasing reactive oxygen species, proteases, cytokines, chemokines, and lipid mediators\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMSU crystals potently stimulate arachidonic acid (AA) metabolism, resulting in robust prostanoid production\u0026mdash;particularly prostaglandin E₂ (PGE₂)\u0026mdash;through enhanced cytosolic phospholipase A₂ (cPLA₂) activity and cyclooxygenase-2 (COX-2) expression in immune cells and osteoblasts. Elevated levels of prostanoids within gouty synovial fluid are strongly associated with the cardinal clinical features of acute gout flares, including severe pain, swelling, and erythema\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Despite the established role of AA-derived lipid mediators in inflammatory diseases, their specific contribution to gout pathogenesis remains incompletely understood. AA metabolism generates a diverse array of bioactive lipids, including prostaglandins, thromboxanes, and leukotrienes, which are potent regulators of immune cell activation and resolution\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Recent studies have demonstrated that MSU crystals upregulate PTGS2 (COX-2) expression in peripheral monocytes from patients with advanced gout, linking AA metabolism to systemic inflammatory burden\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In addition, MSU-induced leukotriene production by neutrophils and elevated synovial fluid levels of leukotriene B₄ (LTB₄) further support a pathogenic role for lipid mediators in gout, exceeding those observed in other inflammatory arthritides such as rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent treatment of acute gout relies largely on NSAIDs, colchicine, and corticosteroids, which effectively suppress symptoms but are often limited by systemic toxicity and contraindications in patients with renal, cardiovascular, or metabolic comorbidities\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. NSAIDs primarily inhibit prostaglandin synthesis via cyclooxygenase blockade, while colchicine reduces inflammation by impairing microtubule-dependent neutrophil recruitment; however, neither strategy directly regulates upstream arachidonic acid metabolism or coordinated lipid mediator signaling during MSU-driven inflammation.\u003c/p\u003e \u003cp\u003e1,2,3,4,6-Penta-\u003cem\u003eO\u003c/em\u003e-galloyl-β-D-glucose (PGG) is a naturally occurring polyphenolic compound that has been reported to exhibit broad anti-inflammatory, anti-oxidative, and immunomodulatory activities across multiple experimental systems. Previous studies have demonstrated that PGG suppresses inflammatory mediator production in macrophages, inhibits oxidative stress, and attenuates tissue injury in models of inflammatory and metabolic disease. Mechanistically, PGG has been shown to interfere with upstream innate immune signaling and lipid metabolic pathways rather than acting solely on terminal cytokine effectors, suggesting a capacity to modulate inflammatory amplification at an early stage. Despite these observations, the therapeutic potential of PGG in acute gouty inflammation and its impact on monosodium urate (MSU)\u0026ndash;driven arachidonic acid metabolism remain poorly defined. In the present study, we aimed to delineate the anti-inflammatory mechanisms of PGG in macrophages and to evaluate its therapeutic efficacy in a murine MSU-induced gout model, with particular emphasis on its regulation of arachidonic acid\u0026ndash;derived prostanoid production and downstream inflammatory responses.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cstrong\u003eGene expression analysis from gout patients and mouse model\u003c/strong\u003e \u003cp\u003eBulk RNA sequencing dataset GSE242872 submitted by chengyu yin et al\u003csup\u003e32\u003c/sup\u003e for gout model at 8 and 24 hr and GSE191054 Human macrophages activated with MSU\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and single-cell RNA sequencing dataset GSE211783 submitted by Hanjie Yu et al.\u003csup\u003e34\u003c/sup\u003e to evaluate the expression of arachidonic pathway.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMyeloid cells\u003c/strong\u003e \u003cp\u003eMouse bone marrow derived macrophages (mBMMs) were isolated from 8-week C57BL/6J mice. Mouse bone marrow cells were cultured with mouse M-CSF (20 ng/ml) for 3 days to obtain myeloid cells differentiated \u003cem\u003ein vitro\u003c/em\u003e as MΦs (10% FBS/DMEM). On day 4, MΦs were pretreated for 18 hr with DMSO (PBS), PGG (5 \u0026micro;M, Sigma # G7548, dose is based on our previous studies)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e in serum free RPMI. Thereafter cells were stimulated with MSU\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (100 \u0026micro;g/ml; Sigma #U2875) for 24 hr. for running ELISA (Protein) and qRT-PCR (mRNA) analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eReal-time RT-PCR\u003c/strong\u003e \u003cp\u003eRNA isolated using Trizol and was reverse transcribed to cDNA using the RevertAid RT Reverse Transcription Kit (Thermo Scientific). SYBR green gene expression master mix (Bio-Rad) to perform qRT-PCR. Data was normalized with GAPDH and are presented as fold changes in RNA levels compared to control treatment, calculated following the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eELISA for cytokine analyses\u003c/strong\u003e \u003cp\u003eConditioned media from the macrophage, pretreated with PGG (5 \u0026micro;M) overnight followed by stimulation with MSU for 24 hr was collected and cytokine levels of IL-1β, IL-6, TNF-α and IL-18 were measured using DuoSet ELISA (enzyme-linked immunosorbent assay) kits (R\u0026amp;D Systems, MN).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIn vitro phagocytosis assay\u003c/strong\u003e \u003cp\u003eThe phagocytic activity of macrophages was assessed using the Vybrant\u0026trade; Phagocytosis Assay Kit (Life Technologies\u0026trade;). Briefly, macrophages (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were seeded in a 96-well flat-bottom plate, pretreated with PGG overnight, and stimulated with MSU for 2 hours. The culture medium was then replaced with 100 \u0026micro;L of the prepared fluorescent Bioparticle suspension, followed by incubation at 37\u0026deg;C for 2 hours. After incubation, the Bioparticle suspension was removed, and the cells were washed twice with PBS. Subsequently, 100 \u0026micro;L of prepared Trypan Blue suspension was added, incubated for 1 minute, and the fluorescence intensity was measured using a plate reader with ~\u0026thinsp;480 nm excitation and ~\u0026thinsp;520 nm emission, following the manufacturer's instructions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLipid Extraction and Fatty Acid Analysis by GC/MS\u003c/b\u003e: Blood was collected by cardiac puncture into heparinized syringes, and plasma was isolated by centrifugation at 1,500 \u0026times; g for 15 min at 4\u0026deg;C. Total lipids were extracted from plasma using a modified version of a previously published method\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Briefly, 100 \u0026micro;L of plasma or macrophages lysate (invitro) were mixed with 800 \u0026micro;L of 50% methanol in water containing 0.01 N HCl, followed by the addition of 2 mL chloroform. Samples were vortexed for 30 s, 1 mL water was added, and samples were vortexed again for 30 s before centrifugation. The lower chloroform phase was collected, dried under nitrogen, and used for fatty acid analysis. Lipid extracts were converted to fatty acid methyl esters (FAMEs) as previously described\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Dried lipids were resuspended in 0.5 mL toluene containing 25 \u0026micro;g of 22:3 free fatty acid as an internal standard and 250 \u0026micro;g butylated hydroxytoluene. Methanolic HCl (0.3 mL of 8% HCl in methanol) was added, and samples were heated at 100\u0026deg;C for 1 h under nitrogen. The reaction was neutralized with 1.0 mL of 0.33 N NaOH, and FAMEs were extracted twice with 3 mL hexane. Combined hexane extracts were dried under nitrogen, reconstituted in 30 \u0026micro;L hexane, and 1 \u0026micro;L was injected into the GC/MS system. Fatty acid analysis was performed using a Shimadzu QP2010SE GC/MS equipped with a Supelco Omegawax capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m), with data acquired over a total ion current range of m/z 50\u0026ndash;400.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMurine Model of Gout\u003c/strong\u003e \u003cp\u003e All animal studies were approved by UIC Animal Care and Use Committee (protocol # 2024-042). After 7 days of acclimatization, 8-10-weeks-old male C57BL/6J mice (Jackson Laboratory) were divided into three groups; \u003cb\u003ea)\u003c/b\u003e Control \u003cb\u003eb)\u003c/b\u003e Monosodium Uric acid-MSU (gout Model) \u003cb\u003ec)\u003c/b\u003e MSU\u0026thinsp;+\u0026thinsp;PGG, (n\u0026thinsp;=\u0026thinsp;5). In the treatment group, PGG (25 mg/kg, daily oral gavage) was administered from day 0. C57BL/6 mice at 8\u0026ndash;10 week are susceptible to the development of gouty arthritis when injected with MSU crystal (0.5 mg) suspended in 25 \u0026micro;l endotoxin free PBS or PBS control will be injected into footpad of mice anaesthetized with 2.5-4% isoflurane. This model is one of the most synchronized and reliable rodent models of gout and produces the least distress \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40 CR41\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The ∆ ankle circumferences of both the hind ankles from each animal were averaged and monitored for clinical signs of inflammation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical Analysis\u003c/strong\u003e \u003cp\u003eFor comparison between multiple groups, one-way ANOVA followed by Tukey's or Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s multiple comparison test was done using Graph Pad Prism10 software. Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAltered arachidonic acid\u0026ndash;associated metabolic pathways are linked to gout\u003c/h2\u003e \u003cp\u003eArachidonic acid (AA) metabolism has emerged as an important amplifier of crystal-induced inflammation. To explore the involvement of AA-associated pathways in gout, we analyzed publicly available bulk and single-cell transcriptomic datasets derived from MSU-induced mouse models and human gout samples. Across datasets, genes involved in prostanoid and leukotriene pathways (e.g., PTGES, ALOX5, and LTA4H) were consistently dysregulated during gout flares compared with remission or control conditions, highlighting a conserved activation of AA-derived inflammatory programs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese analyses support the concept that AA metabolism contributes to gout pathogenesis across species and disease stages and provide a rationale for targeting upstream metabolic processes that regulate AA availability during MSU-driven inflammation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePGG suppresses MSU-induced fatty acid desaturation and arachidonic acid accumulation in macrophages\u003c/h3\u003e\n\u003cp\u003eGiven the central role of AA as a substrate for inflammatory lipid mediators, we next investigated whether PGG modulates endogenous AA biosynthesis in macrophages. MSU stimulation significantly increased the expression of FADS2 and FADS1, the Δ6- and Δ5-desaturases that catalyze the conversion of linoleic acid to AA. Consistent with enhanced fatty acid desaturation, GC\u0026ndash;MS analysis revealed a marked accumulation of AA in MSU-stimulated macrophages. PGG treatment significantly attenuated MSU-induced FADS1, FADS2 and reduced AA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results indicate that PGG suppresses MSU-induced fatty acid desaturation, thereby limiting intracellular AA availability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePGG attenuates MSU-induced inflammatory cytokine production\u003c/h3\u003e\n\u003cp\u003eIncreased AA availability amplifies inflammatory responses by fueling the generation of bioactive lipid mediators that potentiate cytokine production. Concomitant with FADS1/2 inhibition, PGG robustly attenuated MSU-induced cytokine production. MSU exposure induced pronounced secretion of IL-1β, IL-6, IL18 and TNF-α, while PGG treatment significantly reduced these cytokines, demonstrating broad suppression of the inflammatory response (Fig.\u0026nbsp;3A-D). At the transcriptional level, MSU stimulation strongly upregulated IL-1β, IL6, IL18 and TNF-α expression, all of which were significantly downregulated following PGG treatment, consistent with reduced inflammatory activation (Fig.\u0026nbsp;3E-H). Together, these findings indicate that PGG reduced AA accumulation concomitant with reduced cytokine production in macrophages.\u003c/p\u003e\n\u003ch3\u003ePGG treatment disrupts MSU-induced phagocytosis\u003c/h3\u003e\n\u003cp\u003ePhagocytosis represents a critical early event in gout pathogenesis. Macrophage uptake of monosodium urate (MSU) crystals initiates innate immune activation and downstream inflammatory processes. Our results show that PGG treatment significantly reduced MSU crystal phagocytosis in macrophage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating that PGG directly interferes with cellular mechanisms required for MSU uptake.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePGG reduces gout severity by modulating fatty acid metabolism and inflammatory responses in vivo\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic efficacy of PGG in vivo, an MSU-induced gout model was established in mice. MSU administration resulted in a marked increase in clinical disease severity, as reflected by significantly elevated gout scores compared with controls. PGG treatment significantly attenuated MSU-induced disease severity, leading to a pronounced reduction in clinical scoring over the course of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the protein level, MSU challenge induced robust production of IL-1β, IL-6, and TNF-α in joint tissues, whereas PGG treatment significantly suppressed these pro-inflammatory cytokines. In contrast, IL-10 levels were significantly increased in PGG-treated mice, indicating a shift toward an anti-inflammatory environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F).\u003c/p\u003e \u003cp\u003eConsistent with these findings, transcriptional analysis revealed that MSU stimulation markedly upregulated IL-1β, IL-6 and TNF-α expression, all of which were significantly reduced following PGG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Conversely, Arg1 expression was significantly enhanced in PGG-treated mice, supporting the induction of an anti-inflammatory phenotype.\u003c/p\u003e \u003cp\u003eTo further assess whether PGG modulates fatty acid metabolism in vivo, expression of key fatty acid desaturases was examined in plasma from MSU-induced gout mice. MSU challenge significantly increased FADS1, FADS2 and arachidonic acid, consistent with enhanced arachidonic acid biosynthetic activity during gouty inflammation. Notably, PGG treatment markedly suppressed MSU-induced FADS1, FADS2, and AA indicating inhibition of fatty acid desaturation pathways in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether, these findings indicate that PGG suppresses MSU-induced gouty inflammation in vivo by inhibiting fatty acid metabolism, reducing pro-inflammatory mediators, and promoting an anti-inflammatory gene expression profile.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGout is increasingly recognized as a metabolically driven inflammatory disease in which monosodium urate (MSU) crystals engage innate immune cells and reprogram lipid metabolism to amplify inflammatory responses. In the present study, we demonstrate that PGG exerts potent anti-gout activity both in vitro and in vivo by targeting fatty acid desaturation pathways, suppressing MSU crystal phagocytosis, and shifting macrophage responses toward an anti-inflammatory phenotype.\u003c/p\u003e \u003cp\u003eA key finding of this work is the identification of fatty acid desaturases FADS1 and FADS2 as regulated targets during gouty inflammation. These enzymes catalyze critical steps in the biosynthesis of arachidonic acid (AA), a central substrate for pro-inflammatory lipid mediators. Our in vitro data show that MSU stimulation induces FADS1 and FADS2 expressions in macrophages, consistent with metabolic priming toward enhanced AA availability. Importantly, PGG significantly suppressed FADS1 and FADS2 expression, indicating that PGG interferes with upstream lipid metabolic reprogramming rather than solely blocking downstream inflammatory outputs. These findings extend prior observations that AA metabolism contributes to gout pathogenesis and position fatty acid desaturation as a previously underappreciated regulatory node in MSU-driven inflammation.\u003c/p\u003e \u003cp\u003eBeyond metabolic regulation, our data highlight phagocytosis as a critical functional target of PGG. Macrophage uptake of MSU crystals is an essential initiating event in gout, triggering intracellular signaling cascades and perpetuating tissue inflammation \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. We observed that PGG markedly reduced MSU crystal phagocytosis in macrophages, suggesting that modulation of membrane lipid composition or cytoskeletal dynamics may underlie its inhibitory effects. Given that fatty acid composition directly influences membrane fluidity and phagocytic capacity, suppression of FADS1/FADS2-driven lipid remodeling provides a plausible mechanistic link between altered metabolism and reduced MSU uptake.\u003c/p\u003e \u003cp\u003eThe in vivo relevance of these findings was confirmed in an MSU-induced mouse model of gout. PGG treatment significantly reduced clinical disease severity, demonstrating robust therapeutic efficacy. PGG suppressed MSU-induced expression of Fads1 and Fads2 in serum, validating that fatty acid metabolic regulation occurs in vivo and is not restricted to cell culture systems. Concomitantly, PGG reduced pro-inflammatory mediators at both the protein and transcriptional levels, including IL-1β, IL-6, TNF-α, while enhancing IL-10 and Arg1 expression. This coordinated molecular shift supports a model in which PGG not only dampens inflammatory activation but also actively promotes resolution-associated macrophage programs.\u003c/p\u003e \u003cp\u003eNotably, the increase in IL-10 and Arg1 suggests that PGG favors a reparative immune environment rather than inducing broad immunosuppression. This is particularly relevant in gout, where excessive inflammation coexists with cycles of spontaneous resolution. By limiting fatty acid\u0026ndash;driven amplification loops and promoting anti-inflammatory gene expression, PGG may help restore immune balance within the inflamed joint.\u003c/p\u003e \u003cp\u003eCollectively, these findings support a multilevel mechanism of action for PGG in gout: (i) inhibition of FADS1/FADS2-mediated fatty acid desaturation, (ii) attenuation of MSU crystal phagocytosis, and (iii) suppressing MSU induced the inflammatory responses in macrophage. This integrated mechanism distinguishes PGG from conventional anti-inflammatory strategies that primarily target single cytokines and underscores the therapeutic potential of metabolic modulation in crystal-induced inflammatory diseases.\u003c/p\u003e \u003cp\u003eIn summary, our study identifies fatty acid desaturation as a critical contributor to gout pathogenesis and establishes PGG as a metabolically active anti-inflammatory agent capable of suppressing MSU-induced inflammation in vitro and in vivo. These findings provide a strong rationale for further development of PGG or related metabolic modulators as disease-modifying therapies for gout.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimitations.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe experimental systems used in this study acute MSU-driven inflammation but do not fully capture the complexity of chronic hyperuricemia or recurrent gout flares observed clinically. Although PGG clearly modulates inflammatory signaling and AA metabolism, the precise molecular targets responsible for these effects remain to be elucidated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING:\u0026nbsp;\u003c/strong\u003eThis work was supported by awards from the National Institutes of Health NIH R01DE027404 and R01DE030495 grants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT/COMPETING INTEREST:\u003c/strong\u003e The authors have declared that no commercial or financial conflict of interest exists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCODE AVAILABILITY\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS:\u0026nbsp;\u003c/strong\u003eAll authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Ravindran and Umar had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy conception and design.\u003c/strong\u003e SU, SR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcquisition of data.\u0026nbsp;\u003c/strong\u003eSU, YL, PY, SD, MW, SR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis and interpretation of data.\u003c/strong\u003e SU, PY, SD, SR\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e The schematic figures were created with BioRender.com.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFitzGerald, J. D.; Dalbeth, N.; Mikuls, T.; Brignardello-Petersen, R.; Guyatt, G.; Abeles, A. M.; Gelber, A. C.; Harrold, L. 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DOI: 10.1007/s10787-022-01014-x From NLM Medline.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"National Institute of Health","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"macrophages, MSU-gout model, Pentagalloyl glucose, inflammation, arachidonic acid pathway","lastPublishedDoi":"10.21203/rs.3.rs-8904164/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8904164/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGout is an acute inflammatory arthritis triggered by monosodium urate (MSU) crystal deposition and activation of innate immune responses. In addition to inflammasome signaling, emerging evidence suggests that metabolic reprogramming of arachidonic acid (AA) pathways amplifies inflammatory responses during gout flares. However, the contribution of upstream fatty acid desaturation processes that regulate endogenous AA availability remains poorly defined. 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) is a naturally occurring polyphenol with reported anti-inflammatory activity, but its effects on MSU-induced fatty acid metabolism and gouty inflammation have not been well established.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePublicly available bulk and single-cell transcriptomic datasets from human and mouse gout studies were analyzed to assess dysregulation of AA-associated pathways. MSU-induced inflammatory responses were examined in mouse bone marrow–derived macrophages and in a murine MSU-induced gout model. Macrophages were treated with PGG prior to MSU stimulation, and inflammatory cytokine production, phagocytosis, and expression of fatty acid desaturases were assessed. Lipidomic analysis of macrophages and plasma was performed using gas chromatography–mass spectrometry (GC–MS) to quantify arachidonic acid and related fatty acids. In vivo disease severity, cytokine expression, and anti-inflammatory markers were evaluated following PGG treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of public datasets revealed consistent dysregulation of arachidonic acid–associated inflammatory pathways during gout flares. In macrophages, MSU stimulation increased expression of fatty acid desaturases FADS1 and FADS2 and promoted accumulation of arachidonic acid, concomitant with robust production of pro-inflammatory cytokines. PGG treatment significantly suppressed MSU-induced FADS1, FADS2 and arachidonic acid levels, and attenuated pro-inflammatory cytokine production. PGG also markedly impaired macrophage phagocytosis of MSU crystals. In vivo, PGG treatment significantly reduced clinical disease severity in an MSU-induced gout model, suppressed fatty acid desaturation and arachidonic acid accumulation in plasma, decreased pro-inflammatory cytokine expression, and enhanced anti-inflammatory markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings identify fatty acid desaturation as an important metabolic contributor to gouty inflammation and demonstrate that PGG suppresses MSU-induced inflammation by limiting endogenous arachidonic acid availability, reducing inflammatory amplification, and impairing MSU crystal phagocytosis. Targeting upstream fatty acid metabolism represents a potential therapeutic strategy for modulating acute gout flares beyond conventional anti-inflammatory approaches.\u003c/p\u003e","manuscriptTitle":"Pentagalloyl glucose Suppresses MSU Crystal–Induced Gout Inflammation and Arachidonic Acid Production In Vitro and In Vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 11:21:42","doi":"10.21203/rs.3.rs-8904164/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94d839eb-a9eb-4fa6-af40-28c4ffee5cc0","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63094775,"name":"Rheumatology"},{"id":63094776,"name":"Immunology"}],"tags":[],"updatedAt":"2026-02-19T11:21:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 11:21:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8904164","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8904164","identity":"rs-8904164","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}