Citrullination of H3 and Neutrophil Extracellular Traps are reduced upon activation in Proliferative Diabetic Retinopathy patients

Citrullination of H3 and Neutrophil Extracellular Traps are reduced upon activation in Proliferative Diabetic Retinopathy patients | 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 Citrullination of H3 and Neutrophil Extracellular Traps are reduced upon activation in Proliferative Diabetic Retinopathy patients Beatriz Buentello-Volante, Fátima Sofía Magaña-Guerrero, Norma Angélica Magaña-Guerrero, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7935515/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 Proliferative diabetic retinopathy (PDR) is the most advanced stage of diabetic retinopathy (DR). Although spontaneous NET release has been reported in DR, the capacity of neutrophils from PDR patients to undergo inducible NET formation remains unexplored. This study aimed to evaluate inducible NET formation and underlying regulatory mechanisms in PDR. Methods We performed a case–control study including 9 non-diabetic controls, 17 patients with type 2 diabetes (T2D) without DR, and 15 patients with PDR. Peripheral neutrophils were isolated and stimulated with ionomycin to assess NET formation and histone H3 citrullination (H3Cit). PADI4 promoter methylation and mRNA expression were analyzed, together with the surface expression of activation markers CD11b and CD66b. An ex vivo high-glucose model was performed to examine the impact of persistent hyperglycemia on NET induction. Results Neutrophils from PDR exhibited significantly reduced inducible NET formation and impaired H3 citrullination compared with controls and T2D patients. The NETs Index (stimulated/non-stimulated ratio) was markedly lower in the PDR group. Despite increased PADI4 mRNA expression in PDR neutrophils, promoter methylation remained unchanged, as well as cell activation markers. Notably, persistent hyperglycemia in healthy neutrophils reproduced the impaired NET formation and reduced H3 citrullination observed in PDR. Conclusions Neutrophils from PDR patients display a selective defect in inducible NET formation and histone citrullination, likely driven by chronic hyperglycemia and metabolic stress rather than epigenetic silencing of PADI4. These findings support the concept of neutrophil functional exhaustion in PDR and identify PAD4 as a potential therapeutic target in advanced diabetic retinopathy Proliferative Diabetic Retinopathy Neutrophil Extracellular Traps (NETs) Histone H3 Citrullination PADI4 Immune Dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Diabetic retinopathy (DR), a microvascular complication of type-2 diabetes (T2D), represents the principal global cause of preventable blindness. Its most advanced manifestation, proliferative diabetic retinopathy (PDR), is pathologically characterized by retinal neovascularization, subsequent fibrosis, and extensive ischemia. These pathological changes collectively contribute to significant visual impairment and, if left untreated, result in irreversible blindness [ 1 ]. While the pathophysiology of DR has classically focused on hyperglycemia-induced vascular damage, emerging evidence accentuates a significant role for immune dysregulation and chronic inflammation in disease progression [ 2 ], particularly involving innate immune cells such as neutrophils [ 3 , 4 ]. Neutrophils contribute to host defense through multiple mechanisms, including degranulation, phagocytosis, and the formation of neutrophil extracellular traps (NETs), among many others. NETs are composed of a web-like meshwork of chromatin and antimicrobial proteins expelled during a specialized form of cell death known as NETosis [ 5 ]. While NETs serve as potent antimicrobial structures, their aberrant release has been linked to microvascular injury [ 6 ], endothelial dysfunction [ 7 ], and sterile inflammation[ 8 ], all of which are relevant to diabetic complications. Recent studies have shown elevated NET markers, including citrullinated histone H3 (H3Cit) and neutrophil elastase (NE), in the circulation and retinal tissues of patients with PDR [ 9 , 10 ]. Peptidylarginine deiminase 4 (PAD4) encoded by the PADI4 gene, is a critical enzyme that participates in the formation of NETs. PAD4 catalyzes the citrullination of histone H3, a modification critical for chromatin decondensation and subsequent NET release [ 11 ]. Regulation of PAD4 is calcium-dependent and tightly linked to the metabolic and inflammatory state of the cell. Hyperglycemia and oxidative stress have been shown to modulate PAD4 activity, either by impairing calcium homeostasis [ 12 ] or altering intracellular signaling cascades [ 13 ]. However, whether PAD4 activity is impaired in neutrophils from individuals with PDR, and how this contributes to defective NET formation, remains poorly understood. Moreover, the transcriptional and epigenetic regulation of PADI4 under diabetic conditions is an area of active investigation. DNA methylation of the PADI4 promoter has been implicated in autoimmune diseases [ 14 ], but its role in diabetes or DR is unknown. Exploring whether PAD4 dysregulation stems from transcriptional repression, epigenetic silencing, or metabolic suppression is essential to understanding the functional capacity of neutrophils in diabetes. Despite reports of increased spontaneous NET release in diabetes and its complications [ 15 ], little is known about inducible NET formation capacity in PDR neutrophils, a defining characteristic of robust immune responsiveness. It remains unclear whether these cells are primed for pro-inflammatory activity or display signs of exhaustion and unresponsiveness due to prolonged stimulation, a state recently described as innate immune tolerance [ 16 ]. This is further complicated by the observation that surface activation markers such as CD11b and CD66b, involved in adhesion and degranulation, may not correlate with functional NET formation, which can be either diminished or exaggerated independently of their expression [ 17 ]. This study investigated the regulation of NET formation in peripheral isolated neutrophils upon activation by comparing responses among non-diabetic controls, subjects with T2D without DR, and patients with PDR. We evaluated multiple parameters, including NET release and histone H3Cit following ionomycin-induced activation, PADI4 promoter methylation and gene expression, the surface expression of activation markers CD11b and CD66b, and the effect of persistent hyperglycemic exposure on NET release using an ex vivo model. Methods This was a case–control study conducted at the Institute of Ophthalmology “Conde de Valenciana” Foundation, derived from a previously reported cohort with slight modifications. In accordance with the Declaration of Helsinki, the study protocol was approved by the local Institutional Review Board for Human Ethics (CEI-2020/01/01), and written informed consent was obtained from all participants prior to enrollment. Clinical trial number: not applicable. Control individuals had no diagnosis of type 2 diabetes (T2D) or systemic pathologies, including autoimmune or renal disease, hypertension, or active infections. Patients with T2D were defined according to American Diabetes Association criteria, included both sexes and were older than 18 years. Exclusion criteria comprised other inflammatory diseases, acute or chronic infections, immunocompromised status, and inability to undergo retinal examination. Unsuitable biological samples were excluded. All participants underwent mydriatic fundus photography, independently assessed by a retina specialist masked to patient data, and classified according to the Diabetic Retinopathy Disease Severity Scale and the International Clinical Diabetic Retinopathy Disease Severity Scale. Patients with T2D were categorised as without DR or with proliferative diabetic retinopathy (PDR). Clinical information included demographic data, duration of diabetes, HbA1c, renal function, urinalysis, and comprehensive haematological analysis. Estimated glomerular filtration rate (eGFR) was calculated, as well as serum osmolality and the triglyceride–glucose (TyG) index. Peripheral blood was collected after fasting and neutrophils were isolated by density gradient centrifugation followed by erythrocyte lysis. Purity and viability were confirmed by flow cytometry, with preparations reaching > 98% CD11b + CD15 + cells. For in vitro experiments, neutrophils were seeded on coated coverslips, allowed to adhere, and incubated under either non-stimulated (NS) or stimulated (S) conditions. Stimulation was performed using ionomycin and calcium chloride. After incubation, NETs were fixed, processed for immunofluorescence, and stained with antibodies against neutrophil elastase (NE) or citrullinated histone H3 (H3Cit), along with DNA counterstaining. Negative controls omitted primary antibodies. Images were acquired using an ApoTome II microscope. To assess the effect of hyperglycaemia, neutrophils from healthy donors were incubated under normoglycaemic or high-glucose conditions, followed by stimulation or left unstimulated. NETs were fixed and visualised as described above. NET area was quantified from random images across independent experiments using a machine learning algorithm adapted from a published protocol. Only fields with ≥ 80 nuclei were analysed. Segmentation and quantification were performed with ZEN Intellesis software, excluding background and nuclei. A NETs Index was calculated for each participant as the ratio of stimulated to non-stimulated conditions (S/NS). Neutrophil protein lysates were prepared under experimental conditions and subjected to SDS-PAGE followed by transfer to membranes. After blocking, membranes were incubated with antibodies against H3Cit, with total histone H3 as loading control. Detection was performed with chemiluminescence and quantified with dedicated imaging software. Genomic DNA from neutrophils was bisulfite-converted and analysed by quantitative methylation-specific PCR with high-resolution melting. Standard curves from fully methylated and unmethylated controls were used to estimate methylation status. Negative controls were included in all runs. Total RNA was isolated and quantified. PADI4 expression was measured by real-time RT-qPCR, normalised to β2 microglobulin (B2M), and expressed as fold change relative to controls using the ΔΔCt method. Results were log2-transformed prior to analysis. Whole blood was incubated under NS and S conditions, stained with fluorochrome-conjugated monoclonal antibodies against CD11b and CD66b, and processed by standard procedures. Data acquisition was performed on a flow cytometer, collecting 100,000 events in the polymorphonuclear region. Neutrophils were gated by scatter properties, and expression levels were reported as mean fluorescence intensity (MFI). Normality of variables was assessed with Shapiro–Wilk and Kolmogorov–Smirnov tests. Group comparisons were performed using Kruskal–Wallis tests, with p < 0.05 considered significant. Linear regressions and Spearman correlations were used to evaluate associations. Analyses and graphical outputs were generated with GraphPad Prism software. Further methodological details, including reagent sources, concentrations, incubation conditions, primers, and protocols, are provided in the Supplementary Methods . Results A total of 41 subjects were included: 9 controls, 17 with type 2 diabetes (T2D) without diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). As shown in Table 1 , groups differed significantly in demographic, clinical, and metabolic parameters, including age, diabetes duration, systolic blood pressure, fasting glucose, HbA1c, renal function markers (urea, creatinine, eGFR, BUN), lipid profile (HDL-C, triglycerides, atherogenic index), TyG, uric acid, bilirubin, and serum osmolality. Notably, diabetes duration and renal function parameters also differed between T2D and PDR groups. NETs release patterns varied across groups (Fig. 1 ). In controls, ionomycin stimulation markedly increased NETs formation, whereas T2D neutrophils showed only a modest, non-significant rise. PDR neutrophils released NETs spontaneously under basal conditions but exhibited a paradoxical suppression upon stimulation, resulting in a significantly lower NETs Index compared with controls. Histone H3 citrullination increased upon stimulation in controls and T2D but not in PDR, where basal H3Cit levels were elevated yet unresponsive to stimulation (Fig. 2 ). NETs Index correlated negatively with age, diabetes duration, systolic blood pressure, fasting glucose, HbA1c, renal dysfunction (urea, creatinine, BUN), TyG, uric acid, and serum osmolality, while positive correlations were found with eGFR and HDL-C ( Table 2 ). These associations link impaired inducible NET formation with poor glycemic control and renal impairment. Epigenetic and transcriptional analyses (Fig. 3 ) showed no significant group differences in PADI4 promoter methylation, but PADI4 mRNA was significantly upregulated in PDR vs controls. By contrast, surface activation markers CD11b and CD66b increased upon stimulation in all groups without intergroup differences, indicating preserved neutrophil activation capacity. Finally, glucose modulation experiments demonstrated that persistent hyperglycemia (24 h) attenuated ionomycin-induced NETs release and H3 citrullination (Fig. 4 ). Acute high glucose (1 h) reduced NETs release but did not impair H3 citrullination, as confirmed by Western blot analysis (Fig. 5 ). Discussion This study uncovers a complex pattern of neutrophil dysregulation in patients with PDR, characterized by impaired inducible NET formation, altered citrullination dynamics, and preserved surface activation capacity. Our integrated approach—encompassing immunofluorescence microscopy, Western blotting, epigenetic profiling, gene expression, and flow cytometry—provides robust evidence that chronic hyperglycemia disrupts PAD4-dependent neutrophil responses through mechanisms that are independent of PADI4 promoter methylation, but potentially driven by metabolic modulation of enzyme activity. We report that NET formation is significantly attenuated upon stimulation in neutrophils derived from PDR patients. Unlike neutrophils obtained from control subjects, which showed robust NET release following ionomycin/calcium activation, neutrophils from PDR patients exhibited a paradoxical decrease in NET production upon stimulation, despite showing elevated baseline NET presence. This blunted response, quantified through a reduced NETs Index (S/NS ratio), reveals a previously unrecognized defect in inducible NETs during advanced diabetic complications. The aberrant increase in spontaneous NET release at rest observed in different stages of DR [ 15 ], including PDR, together with the reduced capacity for further activation seen in diabetes [ 18 ], suggests a pre-activated neutrophil phenotype [ 19 ]. This reflects a state of functional exhaustion in proliferative PDR (manuscript under revision), where neutrophils remain chronically activated yet fail to mount additional responses[ 20 ]. Such phenotypes have been documented in other chronic inflammatory settings, including sepsis and systemic autoimmune diseases [ 16 ], and may reflect persistent exposure to low-grade systemic inflammation or circulating damage-associated molecular patterns (DAMPs) in advanced diabetes [ 21 ]. Citrullination of histone H3, a post-translational modification catalysed by PAD4, is a key chromatin-remodelling event essential for NET release and has been directly implicated in the pathogenesis of diabetes [ 12 ]. In the present study, neutrophils from control subjects showed the expected pattern of increased H3Cit following ionomycin/calcium stimulation. In contrast, neutrophils from PDR subjects exhibited higher H3Cit levels at baseline, but a reduction following stimulation, mirroring the NETs release pattern. This dissociation strongly suggests that the citrullination machinery in PDR neutrophils is dysregulated, with impaired responsiveness to calcium-dependent activation. These findings are consistent with the notion that chronic hyperglycemia may lead to calcium channel dysregulation [ 22 , 23 ], which could impair PAD4 enzyme function. Interestingly, neutrophils from T2D individuals displayed only mild alterations in both NET formation and H3Cit expression, suggesting that this dysregulation worsens with disease progression and is particularly prominent in the PDR stage. The data point toward a staged immune dysfunction in DR, where immune imbalance becomes more severe in tandem with microvascular damage [ 24 ]. One of the outstanding findings of this study is the discordance between PADI4 gene promoter methylation and gene expression. In contrast to previous studies reporting an inverse correlation between PADI4 promoter methylation and anti-PAD4/ACPA antibody levels, as well as disease activity scores in rheumatoid arthritis [ 14 ], our findings show that although PADI4 promoter methylation levels did not differ significantly among control, T2D, and PDR groups, PADI4 mRNA expression was significantly elevated in neutrophils from PDR subjects, suggesting that epigenetic silencing via DNA methylation is not the mechanism that regulates PADI4 expression, at least in this context. The observed upregulation may instead be driven by inflammatory transcription factors [ 25 , 26 ] or hypoxia-inducible elements [ 27 ], hallmarks of the diabetes microenvironment. Yet, despite this transcriptional increase, PAD4-dependent histone citrullination remained defective in neutrophils from PDR subjects, pointing to post-transcriptional or functional inhibition of PAD4 activity, possibly through the activation of PKCα instead of PKCζ [ 28 ]. These findings highlight an important concept: gene expression levels may not necessarily predict enzymatic or cellular function, particularly under conditions of chronic stress such as PDR. This underscores the need for functional validation of transcriptomic data in immune studies of diabetes and its complications. To determine whether the observed defects in NET release and citrullination were a reflection of a global impairment in neutrophil activation, we assessed the expression of classical surface activation markers CD11b and CD66b. These molecules reflect integrin activation and degranulation, respectively. Across all groups, including PDR, these markers significantly increased upon stimulation, indicating that early neutrophil activation steps remain functionally intact. This suggests that the defect is specific to the nuclear events downstream of surface activation, further supporting the hypothesis of selective PAD4 pathway dysfunction in diabetes [ 29 ]. The preservation of these pathways also indicates that neutrophils from PDR patients retain their capacity to adhere and migrate, which may contribute to tissue infiltration and damage [ 30 ] despite impaired NET release. To experimentally model the influence of glycemic status, we exposed neutrophils obtained from healthy individuals to hyperglycemic conditions (25 mM glucose) for 24 h. This led to a significant reduction in ionomycin-induced NET formation and H3 citrullination, effectively recapitulating the PDR phenotype. These findings suggest that persistent hyperglycemia directly impairs PAD4-dependent NET formation, likely through oxidative stress, epigenetic modulation, or metabolic reprogramming [ 31 ]. This glucose-driven impairment may represent a reversible metabolic checkpoint in PAD4 activity and NET release, offering potential therapeutic targets. Pharmacologic modulation of PAD4 activity [ 32 ], or correction of hyperglycemic states, could restore NET competence and limit secondary infections or impaired wound healing observed in diabetes [ 29 ]. Finally, our correlation analyses revealed significant inverse associations between NETs release and key clinical parameters including age, duration of diabetes, blood pressure, HbA1c, urea, creatinine, BUN, and uric acid, along with a positive correlation with eGFR and HDL-C. These associations suggest that NET dysfunction is strongly linked with poor glycemic control and declining renal function [ 33 ], both of which are known risk factors for PDR progression. This highlights the potential value of NETs Index as a biomarker of immune competence and systemic metabolic balance in diabetes. It also supports the broader concept that chronic and systemic inflammation derangement leads to innate immune exhaustion [ 34 ], with significant implications for host defense and microvascular inflammation seen in D2T complications such as DR. This study provides novel insights into the immunometabolic dysfunction of neutrophils in the context of PDR. By integrating functional, transcriptional, and epigenetic analyses, we demonstrate that defective NET formation in PDR is not a consequence of classical activation failure or gene silencing, but instead stems from a complex interplay between hyperglycemia and downstream PAD4-mediated signaling. These findings add evidence suggesting that innate immune tolerance [ 16 ] contributes to the progression of diabetic microvascular complications. From a clinical standpoint, our results suggest that NETs Index and H3Cit levels may serve as functional biomarkers [ 15 ] for immune competence and systemic metabolic stress in patients with diabetes. Furthermore, they raise the possibility that targeted modulation of PAD4 activity through metabolic control or pharmacologic intervention could restore neutrophil function and potentially mitigate secondary infections or exacerbated inflammatory responses in PDR [ 32 ]. Understanding and reversing immune exhaustion in diabetes may therefore represent a paradigm shift in managing diabetic complications beyond glycemic control alone. Conclusion Collectively, our data reveal that PDR is associated with a profound, selective defect in NET formation and histone citrullination, despite intact early neutrophil activation pathways. This dysregulation is not due to epigenetic silencing of PADI4, but may be driven by persistent hyperglycemia and inflammatory stress that impair PAD4 function. These findings advance our understanding of immune dysfunction in diabetic retinopathy and suggest new avenues for therapeutic intervention targeting neutrophil plasticity and PAD4 activity. Future studies should investigate the reversibility of this phenotype and assess its impact on susceptibility to infection and retinal vascular damage in diabetic individuals. Declarations The authors declare that they have no competing interests The present research was funded by SECTEI 159/2023, PAPIIT-DGAPA-UNAM IN210224 and Conde de Valenciana Foundation. Author Contribution All authors has made a significant contribution to the conception, design, execution, or interpretation of the study, and have approved the final version of the manuscript prior to submission.All authors agree to be accountable for all aspects of the work and ensure the accuracy or integrity of any part of the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement We want to thank Dr. Mohamed Ali Pereyra Morales for his invaluable technical support. References Cheung N, Mitchell P, Wong TY (2010) Diabetic retinopathy. Lancet 376(9735):124–136 Pan WW, Lin F, Fort PE (2021) The innate immune system in diabetic retinopathy. 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Eur J Pharmacol 916:174623 Gupta A, Singh K, Fatima S et al (2023) Correction: Gupta Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease. Nutrients ;15(11) Lin R, Zhang Y, Pradhan K, Li L (2020) TICAM2-related pathway mediates neutrophil exhaustion. Sci Rep 10(1):14397 Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.docx Table12.docx 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. 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10:14:11","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87720,"visible":true,"origin":"","legend":"","description":"","filename":"12c7d54e22ee4ee78b85351f899a9d8d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/163a7bd6671f7580c1d7addd.xml"},{"id":96357470,"identity":"2af7ab3e-8756-4ebe-84a2-1903756ce1ba","added_by":"auto","created_at":"2025-11-20 08:25:41","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98135,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/63e3ea848ff9751652b6dec8.html"},{"id":96357447,"identity":"95e66769-6d3f-428a-bea2-439646db79f6","added_by":"auto","created_at":"2025-11-20 08:25:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":599967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNETs release was inhibited in neutrophils from PDR subjects after stimulation\u003c/strong\u003e. Peripheral neutrophils from control, T2D (T2D without diabetic retinopathy) and PDR (proliferative diabetic retinopathy) subjects were stimulated or not with ionomycin and NETs release were determined. Representative micrographs of non-stimulated (NS) (upper panels) and stimulated (S) neutrophils (lower panels) on different groups (A). Total NETs release areas of non-stimulated (-) versus stimulated (+) conditions of each group were compared (B). NETs Index comparison between groups was performed (C). PI, propidium iodide; NE, neutrophil elastase. Bars represent mean (±SE). ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. Scale bar= 20 mm.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/8f5c03f4af6801d497a25227.jpg"},{"id":96357452,"identity":"a33c4537-e153-4173-9cb7-0a0e8648b3c6","added_by":"auto","created_at":"2025-11-20 08:25:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCitrullination of H3 is decreased in PDR condition\u003c/strong\u003e. Representative Western blots showing the expression of citrullinated histone H3 (H3Cit) and total histone H3 (H3) in peripheral neutrophils under non-stimulated (NS) and stimulated (S, ionomycin-treated) conditions from control subjects, individuals with T2D (T2D, without diabetic retinopathy), and patients with proliferative diabetic retinopathy (PDR). Western blot representative images of citrullinated histone H3 (H3Cit) and total histone H3 (H3) in neutrophils from control, T2D and PDR subjects under NS and S conditions. S condition notably upregulated citrullination of H3 in control subjects and T2D, while it was not markedly modified in PDR subjects (A). H3Cit relative expression comparison between control, T2D and PDR subjects under S and NS conditions (B). S/NS ratio comparison between control, T2D and PDR subjects. Bars represent mean (±SE). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/d60dc59e2bf4f365499fc4a9.jpg"},{"id":96367465,"identity":"16673e52-bcf0-4efb-9133-d3f98564f18f","added_by":"auto","created_at":"2025-11-20 10:12:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpigenetic regulation and functional activation of neutrophils in control, T2D, and PDR subjects. \u003c/strong\u003ePercentage of DNA methylation at the PADI4 promoter region (Met %) in peripheral blood neutrophils from control, T2D, and PDR subjects. Methylation was assessed by bisulfite conversion and quantified as the mean percentage of methylated CpG sites. Although slight variations were observed among groups, no statistically significant differences were detected, indicating that PADI4 promoter methylation is stable across conditions \u003cstrong\u003e(A). \u003c/strong\u003eRelative PADI4 mRNA expression in peripheral blood neutrophils, calculated using the comparative ΔΔCT method and expressed as log₂(2^–ΔΔCT). Box-and-whisker plots show expression in control, T2D, and PDR groups. A significant increase in PADI4 expression was observed in the PDR group compared to controls (p \u0026lt; 0.05), suggesting transcriptional upregulation independent of promoter methylation \u003cstrong\u003e(B)\u003c/strong\u003e. Surface expression of neutrophil activation markers CD66b (left panel) and CD11b (right panel) measured by flow cytometry in peripheral neutrophils under non-stimulated (NS) and stimulated (S) conditions. Expression is shown as mean fluorescence intensity (MFI, mean ± SEM). Stimulation led to a significant increase in both markers across all groups (p \u0026lt; 0.05, NS vs. S), indicating preserved activation capacity. No significant differences were observed between subject groups under either condition, suggesting that while neutrophil activation upon stimulation is preserved, baseline and induced activation marker levels are comparable across groups \u003cstrong\u003e(C)\u003c/strong\u003e. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/cd063327de8c50ec286d2471.jpg"},{"id":96367470,"identity":"ece78632-b424-4d82-9511-cae8f7e87304","added_by":"auto","created_at":"2025-11-20 10:12:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":322379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNET formation and histone H3 citrullination are reduced under persistent high-glucose conditions.\u003c/strong\u003e Peripheral blood neutrophils from healthy donors were incubated for 24 h in normoglycemic (Glc 5.5 mM) or high-glucose (Glc 25 mM) medium and subsequently stimulated with ionomycin (+) or vehicle (–) for 1 h. Representative immunofluorescence images (top) show PI (red) and citrullinated histone H3 (H3Cit; green) staining, indicating NETs and histone citrullination. Quantification (bottom): Left, percentage of NETs (PI-positive, RFI) significantly increased after ionomycin, with a stronger effect at 5.5 mM glucose. Right, H3Cit signal (RFI) was markedly induced by ionomycin in normoglycemia but attenuated under high glucose (A). Western blot analysis of H3Cit and total H3 confirmed ionomycin-induced citrullination, with greater levels at 5.5 mM glucose (B). Bar graph shows densitometric quantification of H3Cit normalized to H3. Scale bars = 20 μm. Data are mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/49c6b8336eb16e5f1f49a590.jpg"},{"id":96366795,"identity":"b39dbd6e-06a5-4a2f-b5da-1da9a3befbef","added_by":"auto","created_at":"2025-11-20 10:11:54","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":418780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransient high glucose impairs NET formation but not histone citrullination in neutrophils.\u003c/strong\u003e Representative immunofluorescence images of neutrophils cultured under low glucose (Glc 5.5 mM) or high glucose (Glc 25 mM) conditions for 1 h, in the absence or presence of ionomycin. Cells were stained with propidium iodide (PI, red) and citrullinated histone H3 (H3Cit, green). Quantification of NETs formation (left graph) and H3Cit fluorescence intensity (middle graph) shows that ionomycin markedly increased both parameters under normoglycemic conditions, whereas the effect was attenuated in high glucose. Western blot analysis of H3Cit and total histone H3 (right, upper panel) confirmed increased histone citrullination in the presence of ionomycin, with densitometric quantification shown below (right, lower graph). Data are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/e62e4ef50d6fb4b13b39b263.jpg"},{"id":108183266,"identity":"a9e4f72f-43cd-4cfc-9fea-2709a05c874a","added_by":"auto","created_at":"2026-04-30 09:00:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2018491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/2ed1eeb1-f3cb-4154-b688-c8e2d1ad0db9.pdf"},{"id":96366705,"identity":"1682b2b1-353a-4474-a384-9be17a12d29e","added_by":"auto","created_at":"2025-11-20 10:11:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":602157,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/6f6392926e073d71183ef5a6.docx"},{"id":96357448,"identity":"f8d3f84a-213b-45ef-b181-29247692eeb3","added_by":"auto","created_at":"2025-11-20 08:25:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17774,"visible":true,"origin":"","legend":"","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-7935515/v1/2396b8666edfadbd1d5191d2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Citrullination of H3 and Neutrophil Extracellular Traps are reduced upon activation in Proliferative Diabetic Retinopathy patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic retinopathy (DR), a microvascular complication of type-2 diabetes (T2D), represents the principal global cause of preventable blindness. Its most advanced manifestation, proliferative diabetic retinopathy (PDR), is pathologically characterized by retinal neovascularization, subsequent fibrosis, and extensive ischemia. These pathological changes collectively contribute to significant visual impairment and, if left untreated, result in irreversible blindness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While the pathophysiology of DR has classically focused on hyperglycemia-induced vascular damage, emerging evidence accentuates a significant role for immune dysregulation and chronic inflammation in disease progression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], particularly involving innate immune cells such as neutrophils [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Neutrophils contribute to host defense through multiple mechanisms, including degranulation, phagocytosis, and the formation of neutrophil extracellular traps (NETs), among many others. NETs are composed of a web-like meshwork of chromatin and antimicrobial proteins expelled during a specialized form of cell death known as NETosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While NETs serve as potent antimicrobial structures, their aberrant release has been linked to microvascular injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], endothelial dysfunction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and sterile inflammation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], all of which are relevant to diabetic complications. Recent studies have shown elevated NET markers, including citrullinated histone H3 (H3Cit) and neutrophil elastase (NE), in the circulation and retinal tissues of patients with PDR [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Peptidylarginine deiminase 4 (PAD4) encoded by the PADI4 gene, is a critical enzyme that participates in the formation of NETs. PAD4 catalyzes the citrullination of histone H3, a modification critical for chromatin decondensation and subsequent NET release [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Regulation of PAD4 is calcium-dependent and tightly linked to the metabolic and inflammatory state of the cell. Hyperglycemia and oxidative stress have been shown to modulate PAD4 activity, either by impairing calcium homeostasis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] or altering intracellular signaling cascades [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, whether PAD4 activity is impaired in neutrophils from individuals with PDR, and how this contributes to defective NET formation, remains poorly understood. Moreover, the transcriptional and epigenetic regulation of PADI4 under diabetic conditions is an area of active investigation. DNA methylation of the PADI4 promoter has been implicated in autoimmune diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], but its role in diabetes or DR is unknown. Exploring whether PAD4 dysregulation stems from transcriptional repression, epigenetic silencing, or metabolic suppression is essential to understanding the functional capacity of neutrophils in diabetes. Despite reports of increased spontaneous NET release in diabetes and its complications [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], little is known about inducible NET formation capacity in PDR neutrophils, a defining characteristic of robust immune responsiveness. It remains unclear whether these cells are primed for pro-inflammatory activity or display signs of exhaustion and unresponsiveness due to prolonged stimulation, a state recently described as innate immune tolerance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This is further complicated by the observation that surface activation markers such as CD11b and CD66b, involved in adhesion and degranulation, may not correlate with functional NET formation, which can be either diminished or exaggerated independently of their expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This study investigated the regulation of NET formation in peripheral isolated neutrophils upon activation by comparing responses among non-diabetic controls, subjects with T2D without DR, and patients with PDR. We evaluated multiple parameters, including NET release and histone H3Cit following ionomycin-induced activation, PADI4 promoter methylation and gene expression, the surface expression of activation markers CD11b and CD66b, and the effect of persistent hyperglycemic exposure on NET release using an ex vivo model.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis was a case\u0026ndash;control study conducted at the Institute of Ophthalmology \u0026ldquo;Conde de Valenciana\u0026rdquo; Foundation, derived from a previously reported cohort with slight modifications. In accordance with the Declaration of Helsinki, the study protocol was approved by the local Institutional Review Board for Human Ethics (CEI-2020/01/01), and written informed consent was obtained from all participants prior to enrollment. Clinical trial number: not applicable. Control individuals had no diagnosis of type 2 diabetes (T2D) or systemic pathologies, including autoimmune or renal disease, hypertension, or active infections. Patients with T2D were defined according to American Diabetes Association criteria, included both sexes and were older than 18 years. Exclusion criteria comprised other inflammatory diseases, acute or chronic infections, immunocompromised status, and inability to undergo retinal examination. Unsuitable biological samples were excluded. All participants underwent mydriatic fundus photography, independently assessed by a retina specialist masked to patient data, and classified according to the Diabetic Retinopathy Disease Severity Scale and the International Clinical Diabetic Retinopathy Disease Severity Scale. Patients with T2D were categorised as without DR or with proliferative diabetic retinopathy (PDR). Clinical information included demographic data, duration of diabetes, HbA1c, renal function, urinalysis, and comprehensive haematological analysis. Estimated glomerular filtration rate (eGFR) was calculated, as well as serum osmolality and the triglyceride\u0026ndash;glucose (TyG) index. Peripheral blood was collected after fasting and neutrophils were isolated by density gradient centrifugation followed by erythrocyte lysis. Purity and viability were confirmed by flow cytometry, with preparations reaching\u0026thinsp;\u0026gt;\u0026thinsp;98% CD11b\u0026thinsp;+\u0026thinsp;CD15\u0026thinsp;+\u0026thinsp;cells. For in vitro experiments, neutrophils were seeded on coated coverslips, allowed to adhere, and incubated under either non-stimulated (NS) or stimulated (S) conditions. Stimulation was performed using ionomycin and calcium chloride. After incubation, NETs were fixed, processed for immunofluorescence, and stained with antibodies against neutrophil elastase (NE) or citrullinated histone H3 (H3Cit), along with DNA counterstaining. Negative controls omitted primary antibodies. Images were acquired using an ApoTome II microscope. To assess the effect of hyperglycaemia, neutrophils from healthy donors were incubated under normoglycaemic or high-glucose conditions, followed by stimulation or left unstimulated. NETs were fixed and visualised as described above. NET area was quantified from random images across independent experiments using a machine learning algorithm adapted from a published protocol. Only fields with \u0026ge;\u0026thinsp;80 nuclei were analysed. Segmentation and quantification were performed with ZEN Intellesis software, excluding background and nuclei. A NETs Index was calculated for each participant as the ratio of stimulated to non-stimulated conditions (S/NS). Neutrophil protein lysates were prepared under experimental conditions and subjected to SDS-PAGE followed by transfer to membranes. After blocking, membranes were incubated with antibodies against H3Cit, with total histone H3 as loading control. Detection was performed with chemiluminescence and quantified with dedicated imaging software. Genomic DNA from neutrophils was bisulfite-converted and analysed by quantitative methylation-specific PCR with high-resolution melting. Standard curves from fully methylated and unmethylated controls were used to estimate methylation status. Negative controls were included in all runs. Total RNA was isolated and quantified. PADI4 expression was measured by real-time RT-qPCR, normalised to β2 microglobulin (B2M), and expressed as fold change relative to controls using the ΔΔCt method. Results were log2-transformed prior to analysis. Whole blood was incubated under NS and S conditions, stained with fluorochrome-conjugated monoclonal antibodies against CD11b and CD66b, and processed by standard procedures. Data acquisition was performed on a flow cytometer, collecting 100,000 events in the polymorphonuclear region. Neutrophils were gated by scatter properties, and expression levels were reported as mean fluorescence intensity (MFI). Normality of variables was assessed with Shapiro\u0026ndash;Wilk and Kolmogorov\u0026ndash;Smirnov tests. Group comparisons were performed using Kruskal\u0026ndash;Wallis tests, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant. Linear regressions and Spearman correlations were used to evaluate associations. Analyses and graphical outputs were generated with GraphPad Prism software. Further methodological details, including reagent sources, concentrations, incubation conditions, primers, and protocols, are provided in the \u003cem\u003eSupplementary Methods\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 41 subjects were included: 9 controls, 17 with type 2 diabetes (T2D) without diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). As shown in \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e, groups differed significantly in demographic, clinical, and metabolic parameters, including age, diabetes duration, systolic blood pressure, fasting glucose, HbA1c, renal function markers (urea, creatinine, eGFR, BUN), lipid profile (HDL-C, triglycerides, atherogenic index), TyG, uric acid, bilirubin, and serum osmolality. Notably, diabetes duration and renal function parameters also differed between T2D and PDR groups.\u003c/p\u003e\n\u003cp\u003eNETs release patterns varied across groups (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In controls, ionomycin stimulation markedly increased NETs formation, whereas T2D neutrophils showed only a modest, non-significant rise. PDR neutrophils released NETs spontaneously under basal conditions but exhibited a paradoxical suppression upon stimulation, resulting in a significantly lower NETs Index compared with controls.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eHistone H3 citrullination increased upon stimulation in controls and T2D but not in PDR, where basal H3Cit levels were elevated yet unresponsive to stimulation (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eNETs Index correlated negatively with age, diabetes duration, systolic blood pressure, fasting glucose, HbA1c, renal dysfunction (urea, creatinine, BUN), TyG, uric acid, and serum osmolality, while positive correlations were found with eGFR and HDL-C (\u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e). These associations link impaired inducible NET formation with poor glycemic control and renal impairment.\u003c/p\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003cdiv id=\"Sec20\" class=\"Section4\"\u003e\n \u003cp\u003eEpigenetic and transcriptional analyses (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) showed no significant group differences in PADI4 promoter methylation, but PADI4 mRNA was significantly upregulated in PDR vs controls. By contrast, surface activation markers CD11b and CD66b increased upon stimulation in all groups without intergroup differences, indicating preserved neutrophil activation capacity.\u003c/p\u003e\n \u003cp\u003eFinally, glucose modulation experiments demonstrated that persistent hyperglycemia (24 h) attenuated ionomycin-induced NETs release and H3 citrullination (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Acute high glucose (1 h) reduced NETs release but did not impair H3 citrullination, as confirmed by Western blot analysis (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study uncovers a complex pattern of neutrophil dysregulation in patients with PDR, characterized by impaired inducible NET formation, altered citrullination dynamics, and preserved surface activation capacity. Our integrated approach\u0026mdash;encompassing immunofluorescence microscopy, Western blotting, epigenetic profiling, gene expression, and flow cytometry\u0026mdash;provides robust evidence that chronic hyperglycemia disrupts PAD4-dependent neutrophil responses through mechanisms that are independent of PADI4 promoter methylation, but potentially driven by metabolic modulation of enzyme activity. We report that NET formation is significantly attenuated upon stimulation in neutrophils derived from PDR patients. Unlike neutrophils obtained from control subjects, which showed robust NET release following ionomycin/calcium activation, neutrophils from PDR patients exhibited a paradoxical decrease in NET production upon stimulation, despite showing elevated baseline NET presence. This blunted response, quantified through a reduced NETs Index (S/NS ratio), reveals a previously unrecognized defect in inducible NETs during advanced diabetic complications. The aberrant increase in spontaneous NET release at rest observed in different stages of DR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], including PDR, together with the reduced capacity for further activation seen in diabetes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], suggests a pre-activated neutrophil phenotype [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This reflects a state of functional exhaustion in proliferative PDR (manuscript under revision), where neutrophils remain chronically activated yet fail to mount additional responses[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Such phenotypes have been documented in other chronic inflammatory settings, including sepsis and systemic autoimmune diseases [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and may reflect persistent exposure to low-grade systemic inflammation or circulating damage-associated molecular patterns (DAMPs) in advanced diabetes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Citrullination of histone H3, a post-translational modification catalysed by PAD4, is a key chromatin-remodelling event essential for NET release and has been directly implicated in the pathogenesis of diabetes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the present study, neutrophils from control subjects showed the expected pattern of increased H3Cit following ionomycin/calcium stimulation. In contrast, neutrophils from PDR subjects exhibited higher H3Cit levels at baseline, but a reduction following stimulation, mirroring the NETs release pattern. This dissociation strongly suggests that the citrullination machinery in PDR neutrophils is dysregulated, with impaired responsiveness to calcium-dependent activation. These findings are consistent with the notion that chronic hyperglycemia may lead to calcium channel dysregulation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which could impair PAD4 enzyme function. Interestingly, neutrophils from T2D individuals displayed only mild alterations in both NET formation and H3Cit expression, suggesting that this dysregulation worsens with disease progression and is particularly prominent in the PDR stage. The data point toward a staged immune dysfunction in DR, where immune imbalance becomes more severe in tandem with microvascular damage [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. One of the outstanding findings of this study is the discordance between PADI4 gene promoter methylation and gene expression. In contrast to previous studies reporting an inverse correlation between PADI4 promoter methylation and anti-PAD4/ACPA antibody levels, as well as disease activity scores in rheumatoid arthritis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], our findings show that although PADI4 promoter methylation levels did not differ significantly among control, T2D, and PDR groups, PADI4 mRNA expression was significantly elevated in neutrophils from PDR subjects, suggesting that epigenetic silencing via DNA methylation is not the mechanism that regulates PADI4 expression, at least in this context. The observed upregulation may instead be driven by inflammatory transcription factors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] or hypoxia-inducible elements [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], hallmarks of the diabetes microenvironment. Yet, despite this transcriptional increase, PAD4-dependent histone citrullination remained defective in neutrophils from PDR subjects, pointing to post-transcriptional or functional inhibition of PAD4 activity, possibly through the activation of PKCα instead of PKCζ [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings highlight an important concept: gene expression levels may not necessarily predict enzymatic or cellular function, particularly under conditions of chronic stress such as PDR. This underscores the need for functional validation of transcriptomic data in immune studies of diabetes and its complications. To determine whether the observed defects in NET release and citrullination were a reflection of a global impairment in neutrophil activation, we assessed the expression of classical surface activation markers CD11b and CD66b. These molecules reflect integrin activation and degranulation, respectively. Across all groups, including PDR, these markers significantly increased upon stimulation, indicating that early neutrophil activation steps remain functionally intact. This suggests that the defect is specific to the nuclear events downstream of surface activation, further supporting the hypothesis of selective PAD4 pathway dysfunction in diabetes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The preservation of these pathways also indicates that neutrophils from PDR patients retain their capacity to adhere and migrate, which may contribute to tissue infiltration and damage [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] despite impaired NET release.\u003c/p\u003e\u003cp\u003eTo experimentally model the influence of glycemic status, we exposed neutrophils obtained from healthy individuals to hyperglycemic conditions (25 mM glucose) for 24 h. This led to a significant reduction in ionomycin-induced NET formation and H3 citrullination, effectively recapitulating the PDR phenotype. These findings suggest that persistent hyperglycemia directly impairs PAD4-dependent NET formation, likely through oxidative stress, epigenetic modulation, or metabolic reprogramming [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This glucose-driven impairment may represent a reversible metabolic checkpoint in PAD4 activity and NET release, offering potential therapeutic targets. Pharmacologic modulation of PAD4 activity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], or correction of hyperglycemic states, could restore NET competence and limit secondary infections or impaired wound healing observed in diabetes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Finally, our correlation analyses revealed significant inverse associations between NETs release and key clinical parameters including age, duration of diabetes, blood pressure, HbA1c, urea, creatinine, BUN, and uric acid, along with a positive correlation with eGFR and HDL-C. These associations suggest that NET dysfunction is strongly linked with poor glycemic control and declining renal function [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], both of which are known risk factors for PDR progression. This highlights the potential value of NETs Index as a biomarker of immune competence and systemic metabolic balance in diabetes. It also supports the broader concept that chronic and systemic inflammation derangement leads to innate immune exhaustion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], with significant implications for host defense and microvascular inflammation seen in D2T complications such as DR.\u003c/p\u003e\u003cp\u003eThis study provides novel insights into the immunometabolic dysfunction of neutrophils in the context of PDR. By integrating functional, transcriptional, and epigenetic analyses, we demonstrate that defective NET formation in PDR is not a consequence of classical activation failure or gene silencing, but instead stems from a complex interplay between hyperglycemia and downstream PAD4-mediated signaling. These findings add evidence suggesting that innate immune tolerance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] contributes to the progression of diabetic microvascular complications. From a clinical standpoint, our results suggest that NETs Index and H3Cit levels may serve as functional biomarkers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] for immune competence and systemic metabolic stress in patients with diabetes. Furthermore, they raise the possibility that targeted modulation of PAD4 activity through metabolic control or pharmacologic intervention could restore neutrophil function and potentially mitigate secondary infections or exacerbated inflammatory responses in PDR [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Understanding and reversing immune exhaustion in diabetes may therefore represent a paradigm shift in managing diabetic complications beyond glycemic control alone.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCollectively, our data reveal that PDR is associated with a profound, selective defect in NET formation and histone citrullination, despite intact early neutrophil activation pathways. This dysregulation is not due to epigenetic silencing of PADI4, but may be driven by persistent hyperglycemia and inflammatory stress that impair PAD4 function. These findings advance our understanding of immune dysfunction in diabetic retinopathy and suggest new avenues for therapeutic intervention targeting neutrophil plasticity and PAD4 activity. Future studies should investigate the reversibility of this phenotype and assess its impact on susceptibility to infection and retinal vascular damage in diabetic individuals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eThe present research was funded by SECTEI 159/2023, PAPIIT-DGAPA-UNAM IN210224 and Conde de Valenciana Foundation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors has made a significant contribution to the conception, design, execution, or interpretation of the study, and have approved the final version of the manuscript prior to submission.All authors agree to be accountable for all aspects of the work and ensure the accuracy or integrity of any part of the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe want to thank Dr. Mohamed Ali Pereyra Morales for his invaluable technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheung N, Mitchell P, Wong TY (2010) Diabetic retinopathy. 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Mol Endocrinol 21(7):1617\u0026ndash;1629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng Y, Si Y, Wang L et al (2021) The regulation of macrophage polarization by hypoxia-PADI4 coordination in Rheumatoid arthritis. Int Immunopharmacol 99:107988\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKonig MF, Andrade F (2016) A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination. Front Immunol 7:461\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong SL, Demers M, Martinod K et al (2015) Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat Med 21(7):815\u0026ndash;819\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoy SS, Siddiqui K (2019) Molecular and Pathophysiological Mechanisms of Diabetic Retinopathy in Relation to Adhesion Molecules. Curr Diabetes Rev 15(5):363\u0026ndash;371\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoshi MB, Lad A, Bharath Prasad AS, Balakrishnan A, Ramachandra L, Satyamoorthy K (2013) High glucose modulates IL-6 mediated immune homeostasis through impeding neutrophil extracellular trap formation. FEBS Lett 587(14):2241\u0026ndash;2246\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen Y, You Q, Wu Y, Wu J (2022) Inhibition of PAD4-mediated NET formation by cl-amidine prevents diabetes development in nonobese diabetic mice. Eur J Pharmacol 916:174623\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta A, Singh K, Fatima S et al (2023) Correction: Gupta Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease. Nutrients ;15(11)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin R, Zhang Y, Pradhan K, Li L (2020) TICAM2-related pathway mediates neutrophil exhaustion. Sci Rep 10(1):14397\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Proliferative Diabetic Retinopathy, Neutrophil Extracellular Traps (NETs), Histone H3 Citrullination, PADI4, Immune Dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-7935515/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7935515/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eProliferative diabetic retinopathy (PDR) is the most advanced stage of diabetic retinopathy (DR). Although spontaneous NET release has been reported in DR, the capacity of neutrophils from PDR patients to undergo inducible NET formation remains unexplored. This study aimed to evaluate inducible NET formation and underlying regulatory mechanisms in PDR.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe performed a case\u0026ndash;control study including 9 non-diabetic controls, 17 patients with type 2 diabetes (T2D) without DR, and 15 patients with PDR. Peripheral neutrophils were isolated and stimulated with ionomycin to assess NET formation and histone H3 citrullination (H3Cit). PADI4 promoter methylation and mRNA expression were analyzed, together with the surface expression of activation markers CD11b and CD66b. An ex vivo high-glucose model was performed to examine the impact of persistent hyperglycemia on NET induction.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNeutrophils from PDR exhibited significantly reduced inducible NET formation and impaired H3 citrullination compared with controls and T2D patients. The NETs Index (stimulated/non-stimulated ratio) was markedly lower in the PDR group. Despite increased PADI4 mRNA expression in PDR neutrophils, promoter methylation remained unchanged, as well as cell activation markers. Notably, persistent hyperglycemia in healthy neutrophils reproduced the impaired NET formation and reduced H3 citrullination observed in PDR.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eNeutrophils from PDR patients display a selective defect in inducible NET formation and histone citrullination, likely driven by chronic hyperglycemia and metabolic stress rather than epigenetic silencing of PADI4. These findings support the concept of neutrophil functional exhaustion in PDR and identify PAD4 as a potential therapeutic target in advanced diabetic retinopathy\u003c/p\u003e","manuscriptTitle":"Citrullination of H3 and Neutrophil Extracellular Traps are reduced upon activation in Proliferative Diabetic Retinopathy patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 08:25:36","doi":"10.21203/rs.3.rs-7935515/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":"125f3b00-893e-48aa-969e-9cc46cf98574","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T23:39:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-20 08:25:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7935515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7935515","identity":"rs-7935515","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}