Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

Suprewicz, Łukasz; Fiedoruk, Krzysztof; Skłodowski, Karol; Hutt, Evan; Zakrzewska, Magdalena; Walewska, Alicja; Deptuła, Piotr; Lesiak, Agata; Okła, Sławomir; Galie, Peter A.; Patteson, Alison E.; Janmey, Paul A.; Bucki, Robert

doi:10.1186/s12964-025-02062-w

Research
Open access
Published: 05 February 2025

Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

Łukasz Suprewicz¹,
Krzysztof Fiedoruk¹,
Karol Skłodowski¹,
Evan Hutt²,
Magdalena Zakrzewska¹,
Alicja Walewska³,
Piotr Deptuła⁴,
Agata Lesiak⁵,
Sławomir Okła⁵,
Peter A. Galie²,
Alison E. Patteson⁶,
Paul A. Janmey⁷ &
…
Robert Bucki¹

Cell Communication and Signaling volume 23, Article number: 64 (2025) Cite this article

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Abstract

Background

Vimentin is a type III intermediate filament protein that plays an important role in cytoskeletal mechanics. It is now known that vimentin also has distinct functions outside the cell. Recent studies show the controlled release of vimentin into the extracellular environment, where it functions as a signaling molecule. Such observations are expanding our current knowledge of vimentin as a structural cellular component towards additional roles as an active participant in cell signaling.

Methods

Our study investigates the immunological roles of extracellular vimentin (eVim) and its citrullinated form (CitVim) as a damage-associated molecular pattern (DAMP) engaging the Toll-like receptor 4 (TLR4) of human neutrophils. We used in vitro assays to study neutrophil migration through endothelial cell monolayers and activation markers such as NADPH oxidase subunit 2 (NOX2/gp91phox). The comparison of eVim with CitVim and its effect on human neutrophils was extended to the induction of extracellular traps (NETs) and phagocytosis of pathogens.

Results

Both eVim and CitVim interact with and trigger TLR4, leading to increased neutrophil migration and adhesion. CitVim stimulated the enhanced migratory ability of neutrophils, activation of NF-κB, and induction of NET formation mainly mediated through reactive oxygen species (ROS)-dependent and TLR4-dependent pathways. In contrast, neutrophils exposed to non-citrullinated vimentin exhibited higher efficiency in favoring pathogen phagocytosis, such as Escherichia coli and Candida albicans, compared to CitVim.

Conclusions

Our study identifies new functions of eVim in its native and modified forms as an extracellular matrix DAMP and highlights its importance in the modulation of immune system functions. The differential effects of eVim and CitVim on neutrophil functions highlight their potential as new molecular targets for therapeutic strategies aimed at regulation of neutrophil activity in different pathological conditions. This, in turn, opens new windows of therapeutic intervention in inflammatory and immunological diseases characterized by immune system dysfunction, in which eVim and CitVim play a key role.

Introduction

Neutrophils are the most abundant leukocytes in human circulation and play a vital role in the inflammatory process [1]. Neutrophils migrate to the site of inflammation and release antimicrobial agents, pro-inflammatory cytokines, and a wide range of other factors [2]. Neutrophil activation can be triggered by various external stimuli, including proteins classified as damage-associated molecular patterns (DAMPs). DAMPs are endogenous molecules that accumulate during cellular injury or stress and act as ‘alarmins’ or ‘danger’ signals in response to infectious and non-infectious pathologies [3, 4]. DAMPs stimulate neutrophils by binding pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These receptors, often activated by aberrantly expressed or bioactive fragments of extracellular matrix components (ECM), serve as critical mediators in the DAMP-induced activation of neutrophils [5]. The role of DAMPs in neutrophil activation represents a delicate balance. While they are critical in defending against pathogens, their excessive or dysregulated activation can lead to detrimental consequences [6,7,8].

Vimentin (Vim), an intermediate filament protein, is predominantly expressed in mesenchymal cells and forms part of the cytoskeleton, maintaining cellular integrity and facilitating migration [9, 10]. New work is highlighting, however, that these intracellular vimentin filaments can be disassembled into subunits that are released into the extracellular environment, to form extracellular vimentin (eVim), which can then attach to the cell surface or the ECM [11,12,13]. Increased expression of eVim and its post-translationally modified (PTM) form, citrullinated vimentin (CitVim), has been observed in various acute inflammatory conditions such as sepsis and chronic conditions like pulmonary fibrosis or rheumatoid arthritis (RA) [14,15,16]. Citrullination is a modification mediated by peptidyl arginine deiminase (PAD) enzymes [17] that convert the amino acid arginine into citrulline, leading to protein structure and function changes [18]. Neutrophils, for example, activate PADs to disassemble their vimentin cytoskeleton, as they also citrullinate histones to release their DNA to form neutrophil extracellular traps (NETs) [19]. Citrullination occurs in the joints of individuals with RA and results in the production of autoantibodies against citrullinated proteins, including CitVim, which can trigger an autoimmune response, leading to chronic inflammation [20]. However, the specific roles of eVim and CitVim in neutrophil-mediated inflammation and their potential as therapeutic targets remain largely unexplored.

Here, we present evidence that eVim activates neutrophils, and citrullination enhances the stimulatory effect of eVim. We conducted a series of biochemical assays, including atomic force microscopy and inhibition studies to demonstrate the interaction between eVim and CitVim with TLR4, providing insights into the molecular mechanisms involved. Through a combination of cellular assays, microscopy, 3D microfluidic models, and gene expression profiling, we assessed the influence of eVim and CitVim on neutrophil activities, including adhesion, migration, phagocytosis, NET formation, and cytokine secretion. The presence of CitVim significantly enhances specific neutrophil activities over non-citrullinated vimentin, but eVim is superior in boosting phagocytic efficacy.

Results

Extracellular vimentin on endothelial cell surfaces and enhanced adhesion and activation of human neutrophils

We assessed eVim presence at the surface of human umbilical vein endothelial cells (HUVECs) (Fig. 1). Immunofluorescence revealed that in HUVECs eVim, unlike its intracellular filamentous form, appeared as surface dots (Fig. 1a). Such a pattern is consistent with previous reports [21, 22]. To rule out antibody-induced membrane permeability changes, we performed control experiments with HUVECs using an anti-β-actin antibody, confirming no intracellular actin stress fiber visualization after vital staining (Fig. S1a). As shown in Fig. S1b, HUVECs lack detectable CitVim on their surface, but exogenous CitVim can bind the cell surface when added to the cell culture medium (Fig. 1b). Furthermore, CitVim from neutrophil-derived NETs also bound to the endothelial cell surface (Fig. 1b). We tested whether eVim or CitVim impacts neutrophil adhesion to cells using a confluent monolayer of vimentin -/- mouse embryonic fibroblasts (mEFs) preincubated with eVim or CitVim (Fig. 1c). Analysis of cell surface attachment of eVim and CitVim to vim -/- mEFs, revealed that eVim and CitVim adhered to the surface of the vim -/- mEFs, displaying a punctate pattern (Fig. S2a, b). We observed a dose-dependent increase in adherent neutrophils after the addition of eVim (Fig. 1d), with an approximately 6-fold increase in adhesion at an eVim concentration of 20 µg/mL. Addition of CitVim had an even greater effect on neutrophil adhesion, with an approximately 9-fold increase at a CitVim concentration of 20 µg/mL.

To eliminate the potential influence of other proteins in our experimental settings, we fabricated 30 kPa polyacrylamide hydrogels functionalized with collagen (Col), eVim, or CitVim (Fig. S3). This method effectively isolated the impact of eVim and CitVim on neutrophil adhesion by eliminating other surface-bound proteins, clarifying their specific roles [23]. Both forms of Vim supported neutrophil adhesion, but more neutrophils adhered to the CitVim-coated hydrogels compared to eVim-coated hydrogels (Fig. 2a). The number of cells on the Col-coated surface was similar to that on the Vim-coated surface (Fig. 2b), but the average cell area on Vim-coated hydrogels was greater (Fig. 2c). Neutrophils on CitVim-coated hydrogels showed the largest cell area. Additionally, we observed neutrophils tend to form aggregates on both eVim and CitVim substrates with the highest number of cell clusters on CitVim-coated hydrogels (Fig. 2d). Moreover, we observed a significant increase in the NADPH oxidase subunit 2, NOX2/gp91phox (Fig. 2e), an indicator of the production of reactive oxygen species (ROS) and neutrophil activation, in neutrophils on both eVim and CitVim-coated hydrogels [24]. These data suggest that eVim and CitVim, as a coating on hydrogels, enhance neutrophil activation, indicating a potential role of surface vimentin in the inflammatory response.

Extracellular vimentin promotes transendothelial migration of human neutrophils

Since transendothelial migration is a crucial step in neutrophil extravasation at sites of infection or inflammation [25], we investigated the effects of vimentin on neutrophil migration in both transwell assays (Fig. 3a - c) and a more physiologically relevant three-dimensional (3D) microfluidic endothelial model (Fig. 3d - h). In the Transwell assay, more neutrophils adhered to eVim- or CitVim-coated membranes compared to membranes coated with collagen (Fig. 3b). This increased adhesion correlated with the number of cells that migrated through the pores of the membrane to the lower chamber (Fig. 3c). In the microfluidic model, a cylindrical void was created within a collagen hydrogel and seeded with HUVECs to form a confluent monolayer, simulating the blood vessel lining (Fig. 3d). Subsequently, we introduced neutrophils stimulated with eVim, CitVim, or lipopolysaccharide (LPS) into the vessel, as well as unstimulated neutrophils as a control (Fig. 3e). LPS treatment of neutrophils was used for comparison with the effect of a potent neutrophil activator [26]. eVim augments neutrophil adhesion within the blood vessels, and CitVim increases this effect (Fig. 3f). The number of neutrophils escaping the vessel lumen and the distance they traveled within the collagen hydrogel increased in the presence of eVim and was greater upon CitVim addition (Fig. 3g, h) compared to the control and LPS conditions. In comparison, non-stimulated neutrophils (CT) adhered poorly to the endothelial wall, and their extravasation rate was negligible.

Extracellular vimentin enhances phagocytosis and NETosis

Next, we investigated the impact of eVim and CitVim on neutrophil phagocytosis of fungal and bacterial cells (C. albicans and E. coli,respectively) (Fig. 4a). We either simultaneously introduced eVim or CitVim along with the pathogens to the neutrophils or preincubated the neutrophils with eVim (pre-eVim) or CitVim (pre-CitVim) to assess the requirement for neutrophil priming. Neutrophils alone responded to both fungal and bacterial pathogens, as evident from the number of neutrophils engulfing these cells. eVim and CitVim increased the number of neutrophils engaged in phagocytosis, except for neutrophils preincubated with CitVim and then exposed to E. coli (Fig. S4). Further analysis revealed a significant increase in the phagocytosis index (number of ingested pathogens per 100 neutrophils) when eVim or pre-eVim was present, but the effect of CitVim was lower (Fig. 4b, d). Similarly, eVim stimulated more intracellular pathogen killing than CitVim (Fig. 4c, e). We also explored the impact on phagocytosis-related gene expression by exposing neutrophils to eVim or CitVim. eVim triggered more gene expression changes than CitVim (Fig. 4f), with these genes being interrelated and mainly associated with phagocytosis (Fig. S5 and S6).

To investigate the impact of eVim and its citrullinated form on NET secretion, both types of vimentin were added to neutrophils for 4 h, and NETosis was assessed by staining for neutrophil elastase (NE) (Fig. 5a). Both eVim and CitVim stimulated the release of NETs, but CitVim induced more NETs than eVim (Fig. 5b). We also investigated the protein levels and gene expression of citrullinated histone H3 (CitH3), NADPH Oxidase Subunit 2 (NOX2), TLR4, and NE, which are associated with NET formation (Fig. 5c). At the protein level, CitVim caused more elevation in NOX2 and NE (Fig. 5d - g). At the gene level, CitVim was more effective than eVim in stimulating the expression of all the tested genes associated with NET formation (Fig. 5h - k).

Taken together, these results show that eVim, in both its native and citrullinated forms, enhances neutrophil phagocytosis and NETosis. eVim generally promotes phagocytosis more effectively than CitVim, which is especially evident in eVim-preincubated neutrophils. For NETosis, CitVim induces a more robust response than eVim, elevating protein levels and gene expression associated with NET formation to levels comparable to those induced by the potent neutrophil activator (PMA - phorbol 12-myristate 13-acetate) [27].

Citrullinated vimentin boosts the inflammatory response of human neutrophils, and antibodies targeting vimentin diminish this response

Given the fact that NETs also contain eVim and CitVim, NET formation can potentially result in autocrine and paracrine effects on neutrophils (Fig. 6a). We examined the secretion of cytokines and chemokines in response to different doses of eVim and CitVim (Fig. 6b - i). CitVim strongly stimulates human neutrophils to produce pro-inflammatory cytokines, specifically IL-1β, IL-6, IL-8, and TNF-α. In contrast, eVim only induced IL-8 secretion. CitVim exhibited a potent effect even at a dose as low as 0.1 µg/mL, with a minimal further increase beyond 1 µg/mL. Additionally, we observed a modest increase (up to 2-fold) in IL-10 secretion following CitVim treatment.

CitVim augmentation of the inflammatory response in human neutrophils is further supported by gene expression analysis, which showed numerous elevated genes following CitVim stimulation, indicating its impact on transcription and inflammation (Fig. 6j). These genes are associated with NF-κB activation, consistent with the secretion of pro-inflammatory cytokines. More genes were upregulated by CitVim than by eVim (Fig. S7 and S8). These CitVim-triggered genes were strongly co-expressed and were associated with pathways linked to inflammation, including TLR and TNF-α receptor signaling, as well as cancer and viral disease markers (Fig. S7).

To assess whether inhibition of CitVim can mitigate cytokine secretion (Fig. 7a), various anti-Vim antibodies (Supplementary Table 1) were tested for their ability to prevent the secretion of IL-1β, IL-6, IL-8, and TNF-α (Fig. 7b - e). Neutrophils were treated with antibodies at varying dilutions (ranging from 1:1000 to 1:100) simultaneously with CitVim at 1 µg/mL for 4 h.

The extent of inhibition of inflammatory mediator secretion depended on the specific antibody used. All antibodies generally had an inhibitory effect on IL-1β, IL-6, and TNF-α secretion. For IL-1β and IL-6, the most pronounced effect was observed with Pritumumab and anti-CSV (cell-surface vimentin) antibodies. In contrast, the secretion of TNF-α was most significantly impacted by the anti-CitVim antibody. None of the antibodies inhibited IL-8 secretion, which might suggest a distinct pathway exploited by CitVim to stimulate IL-8 secretion compared to other mediators.

In human neutrophils, eVim and CitVim serve as a ligand for toll-like receptor 4

Our study thus far demonstrates that eVim and CitVim trigger the expression of key immune response regulators: NADPH 2 oxidase, NF-κB, and MAPK3 (Figs. 2e and 5e and i and Fig. 6j). This activation mirrors the typical upregulation observed in TLR4-dependent immune responses [28].

Using confocal microscopy, we next explored the colocalization of TLR4 with eVim and CitVim on neutrophil surfaces (Fig. 8a, b). External application of eVim to neutrophils led to its adsorption and an increased overlap with TLR4 in fluorescence signal, measured by a colocalization coefficient ranging from 0 (no colocalization) to 1 (complete colocalization). Control (CT) neutrophils displayed negligible surface CitVim. However, when CitVim was externally administered, it adhered to the cell surface, exhibiting a colocalization with TLR4 that was less pronounced than with eVim.

Dot blot binding assays confirmed the interaction between TLR4 and both eVim and CitVim. Initially, we checked the binding of eVim and CitVim on nitrocellulose (NC) membranes (Fig. S10). Then, NC with immobilized eVim and CitVim was incubated with lysates of human neutrophils to check whether TLR4 would bind to the proteins. BSA served as a negative control. TLR4 binds to both eVim and CitVim but not to BSA (Fig. 8c). Density analysis shows that the CitVim– TLR4 interaction occurs at higher affinity than the eVim– TLR4 interaction.

To assess the molecular interactions between TLR4 with eVim and CitVim, we used atomic force microscopy (AFM) and force spectroscopy analysis. We affixed either eVim or CitVim to the end of an AFM tip functionalized with glutaraldehyde (Fig. 8d). Examination of binding involved recording force-distance (FD) curves through repetitive approaches and retractions of the eVim or CitVim-functionalized tip from a TLR4-coated mica surface as a modification of previous studies [29,30,31]. Specific adhesion events were identified in more than 5% of the retraction FD curves, aligning with findings from other studies on different surface receptor systems (Fig. 8e) [29, 30, 32]. Control experiments to validate interaction specificity included (i) deploying an AFM tip functionalized only with glutaraldehyde and (ii) directing the tip towards mica surfaces coated with BSA or lacking TLR4 (Fig. 8e and Fig. S9). The binding probability observed in these control experiments was notably lower, thus affirming the specificity of the eVim/CitVim and TLR4 complexes under our experimental conditions. The rupture forces detected for the AFM probe at an approach/retraction speed of 5 μm/s were comparable for both eVim and CitVim (Fig. 8f), and in the same range as other AFM-based ligand-receptor studies [29, 33].

Next, we employed a functional assay to determine whether blocking TLR4 on the surface of neutrophils inhibits the increased production of ROS caused by eVim and CitVim (Fig. 8g). To do so, we used anti-TLR4 antibody at 1:100 and TAK-242, a TLR4 inhibitor at 1 µM, a concentration that did not affect ROS production in untreated conditions (Fig. S11a). CitVim caused a significant dose-dependent increase in ROS to the level of that induced by LPS, which was potently inhibited when TLR4 was blocked with anti-TLR4 antibodies. eVim also caused increased ROS formation; however, it was weaker than CitVim. Heat denaturation of eVim or CitVim eliminated their stimulation of ROS production (Fig. S11b). The TLR4 inhibitor also partially decreased ROS production induced by CitVim and our positive control with LPS but did not block ROS production induced by PMA, which acts through a TLR4-independent mechanism (Fig. S12).

To determine whether eVim- and CitVim-induced cytokine and chemokine secretion depends on TLR4-mediated NF-κB activation (Fig. 8h), we used human embryonic kidney cells (HEK) with an NF-κB reporter, transiently transfected with TLR4 and Myeloid Differentiation factor 2 (MD2). MD2 facilitates the dimerization of TLR4-MD2 complexes to activate intracellular signaling. TLR4-expressing cells that did not express MD2 served as a control for cells expressing both TLR4 and MD2 proteins. Both eVim and CitVim significantly increased NF-κB reporter fluorescence, with eVim showing a stronger effect (Fig. 8i). LPS was used as a positive control, confirming NF-κB activation. To assess whether CitVim’s effects stem from its citrulline residue per se or its altered oligomeric structure, we included PAD2, fibrinogen, and citrullinated fibrinogen controls (Fig. S13). We observed weak NF-κB activation by fibrinogen, reported previously [34]. The effect of citrullinated fibrinogen or PAD2 itself was smaller. These findings demonstrate that eVim and CitVim stimulate intracellular ROS production in neutrophils via TLR4 interaction and subsequent NF-κB translocation.

Discussion

In this study, we investigated the effects of eVim on human neutrophils to determine the significance of vimentin release to extracellular fluids and the importance of vimentin citrullination for immune responses and possibly immunopathology. We found that CitVim significantly outcompetes its native form (eVim) in the promotion of (i) neutrophil adhesion, (ii) transendothelial migration, (iii) NET formation, (iv) pro-inflammatory response and (v) immune gene expression. In contrast, eVim is more effective than CitVim in inducing neutrophil phagocytic activity. Both forms of vimentin appear to exert these immunomodulatory actions serving as ligands for TLR4.

The presence of Vim on the cell surface results from its release from Vim-expressing cells [11, 21, 35]. Our study uncovers a novel binding interaction between Vim and TLR4, adding a to our understanding of Vim’s functional role. This discovery not only broadens the scope of known interactions but also provides insights into novel mechanistic pathways where Vim and TLR4 interplay could be critical in inflammatory and immune responses.

Both eVim and CitVim can be deposited on the cell surface. However, the distribution of CitVim differs from eVim, possibly due to differences in the binding properties of both vimentins with other cell surface components. TLR4 is unlikely to be the only attachment point for Vim on the neutrophil surface, since previous studies have shown binding of eVim to N-acetylglucosamine, glycosaminoglycans, and several transmembrane proteins [23, 36,37,38]. On hydrogels coated with eVim and CitVim, both forms of vimentin promote neutrophil adhesion, clustering, and aggregation. This phenomenon is consistent with previous reports that neutrophils tend to aggregate in response to external stimulation with inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or PMA, mediated by the β2-integrin ligand CD54 [39]. Furthermore, two independent groups have demonstrated that ICAM-1 plays a pivotal role in neutrophil activation and aggregation, highlighting its involvement in increased adherence and aggregation upon exposure to inflammatory factors also in a murine model of endotoxemia [40, 41].

The capacity of eVim to enhance transendothelial migration suggests that eVim released from damaged or stressed cells into the extracellular space can influence the extent of neutrophil infiltration into tissues and could impact the degree of tissue damage. Our results suggest that both eVim and CitVim act as DAMPs in the modulation of the immune response, particularly by activation of neutrophils. In this light, the release of NETs containing CitVim during neutrophil activation might act as a positive feedback loop that propagates neutrophil activation within sites of inflammation.

Elevated eVim levels are prevalent in severe conditions like sepsis, idiopathic pulmonary fibrosis (IPF), and viral infections, with intensive care unit (ICU) patients showing average serum Vim concentrations around 500 ng/mL with levels nearly triple this amount observed in some patients [14, 42,43,44]. Notably, during inflammation, eVim and CitVim levels at the site often surpass peripheral blood levels, as evidenced by findings of higher CitVim concentrations in lung tissue and bronchoalveolar lavage fluid of IPF patients compared to their bloodstream [14]. Moreover, eVim concentration in COVID-19 patient saliva can reach up to 70 µg/mL [45]. These findings underline the relevance and necessity of the diverse range of eVim and CitVim doses (0.1–20 µg/mL) employed in our study, reflecting the complex and varying nature of eVim and CitVim levels in different disease states.

The current understanding of eVim’s role in the host inflammatory response includes several different cellular or molecular effects and different conclusions about whether eVim systemically is pro- or anti-inflammatory. For example, eVim modulates oxLDL-induced secretion of pro-inflammatory cytokines in macrophages and alters cytokine profiles in LPS-activated dendritic cells, affecting T cell differentiation [46, 47]. Additionally, its involvement in diapedesis, influencing the distribution and expression of adhesion molecules in lymphocytes and endothelial cells, highlights its multifaceted role [48]. eVim’s stimulatory effect on leukemic macrophage migration and phagocytosis aligns with our findings on human neutrophils [49]. Secretion of vimentin by tumor endothelial cells can lead to immunosuppression, and vaccination to produce anti-vimentin antibodies shows efficacy in prolonging survival in dogs with bladder cancer [50]. eVim’s capability as a substrate for attachment and motility of various cell types underscores its diverse, context-dependent interactions [23]. Collectively, these findings highlight the pivotal role of eVim in orchestrating key elements of the immune response, revealing its potential as a critical target in therapeutic interventions aimed at modulating inflammatory processes.

One aspect of eVim that might determine its effect in vivo is the state of vimentin citrullination. Cytoskeletal vimentin needs to be disassembled before it can be secreted, and this disassembly appears to involve either citrullination or phosphorylation. In neutrophils, citrullination appears to be required, whereas, in macrophages, phosphorylation appears to be dominant [19, 51, 52]. It is not yet clear whether the effect of citrullinating vimentin is to reduce the filaments to small soluble oligomers, or whether the change from arginine to citrulline creates new binding sites on the cell or ECM. The impact of citrullination on protein function is complex and varies significantly. For example, citrullination diminishes the osteoprotective effect of fibrinogen, suggesting a detrimental impact in this context [53]. In contrast, citrullination of histone H4 leads to a decrease in calcium influx compared to its native form, resulting in reduced NET formation [54]. Citrullinated collagen, as opposed to its native form, does not decrease the severity of autoimmune arthritis [55]. These examples highlight that citrullination can lead to diverse functional outcomes, ranging from loss of beneficial properties to a reduction in potentially harmful activities, depending on the specific protein undergoing modification.

CitVim specifically binds to the human leukocyte antigen class II histocompatibility antigen, DRB1 beta chain (HLA-DRB1) in rheumatoid arthritis (RA) patients, enhancing cytokine production, notably INF-γ [56]. CitVim increases proliferation and cytokine secretion in RA and osteoarthritis patient-derived fibroblast-like synoviocytes (FLS), contrasting with the inhibitory effects of non-modified Vim [57]. Additionally, CitVim induces TGF-β1, CTGF, and IL-8 secretion in lung fibroblasts and increases bone resorption in a mouse periodontitis model [14, 58]. Our research aligns with these findings, revealing that vimentin-targeting antibodies inhibit CitVim-mediated pro-inflammatory cytokine secretion but not IL-8. This is partially corroborated by data showing that NF-κB inhibition reduces specific cytokines in RA FLS, suggesting that anti-vimentin antibodies may specifically inhibit NF-κB pathways [59].

Recent research suggests that while dimerization is generally necessary for TLR4 activation, TLR4 signaling can, under certain conditions, occur through alternative mechanisms involving conformational changes or interactions with accessory proteins [60]. For instance, docking studies have shown that the spike protein of wild-type SARS-CoV-2 and its Omicron variant exhibit similar binding to the human TLR4/MD-2 complex but differ in dimerization potency, analogous to differences observed between eVim and CitVim in our study [61]. This implies alternative binding modalities for danger signals to TLR4 and/or MD2. For the larger eVim structure, it is plausible that multiple high-affinity configurations collectively may induce TLR4/MD2 dimerization, leading to a higher dimerization probability compared to the smaller CitVim protein. Additionally, TLR4 signaling can be turned on after events at the cell surface or in endosomes, regulated by different pathways [62]. CitVim could preferentially enhance endosomal TLR4 signaling (TRIF-dependent), which may involve less receptor dimerization than surface signaling [63, 64]. We also cannot exclude the involvement of accessory proteins interacting with TLR4 to mediate CitVim-induced NF-κB activation. CitVim might act as a partial agonist, triggering other non-canonical signaling pathways that activate NF-κB without full TLR4 dimerization. This could, in turn, stimulate the production of inflammatory cytokines (e.g., IL-1β or TNF-α), reinforcing NF-κB activation through a positive feedback loop.

The limitations of TLR4-targeted therapies underscore the potential advantages of an eVim/CitVim-directed strategy. Unlike TLR4, a broadly expressed receptor involved in both physiological and pathological processes, eVim and CitVim are DAMPs with localized expression under pathological conditions. Targeting eVim/CitVim allows for selective modulation of inflammatory pathways without fully blocking TLR4 signaling, which is crucial for maintaining innate immunity. This approach reduces the risk of immune suppression and related vulnerabilities to infections. Although TLR4 antagonists have demonstrated acceptable safety profiles in clinical trials, their efficacy has been limited, likely due to redundancy in immune pathways and compensatory mechanisms [65, 66]. By focusing on eVim/CitVim as upstream activators, therapeutic strategies can achieve greater precision and effectiveness in controlling pathological inflammation while preserving TLR4’s essential functions.

Conclusions

Overall, our study shows the efficiency of eVim to activate neutrophils and suggests that this activation can lead to extravasation and augmentation of inflammatory processes. The data also highlight the role of TLR4 in the differential response of neutrophils to eVim and CitVim. CitVim exhibits enhanced activation of some neutrophil functions, whereas it is less effective than eVim in others, and antibodies targeting Vim can mitigate the inflammatory response caused by CitVim. These findings offer insights into potential strategies for manipulating vimentin-related processes to control inflammation and reduce tissue damage in inflammatory disorders.

Materials and methods

Neutrophil isolation

Blood was collected from healthy donors with the approval of the Bioethics Committee at the Medical University of Bialystok (APK.002.8.2023). Neutrophils were isolated by density gradient centrifugation using PolymorphPrep (Progen, Heidelberg, Germany). Cells were counted on a hemocytometer and suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (#P30-8500, PAN Biotech) and 1% antibiotic antimycotic solution (#A5955, Sigma-Aldrich, St. Louis, MO, USA).

Cell culture and treatment

Human umbilical vein endothelial cells (HUVECs, #CRL-1730™, Sigma-Aldrich) were maintained in Endothelial Cell Growth Medium (#211–500, San Diego, CA, USA). Wild-type mouse embryonic fibroblasts (vim +/+ mEFs) and their variant lacking vimentin expression (vim-/- mEFs) were grown in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution. Cells were grown in a humidified atmosphere with 5% CO₂ at 37 °C.

For the experiments, cells were treated with recombinant human vimentin (#10028-H08B, SinoBiological, Wayne, PA, USA) and recombinant human citrullinated vimentin (#21942, Cayman Chemicals, Ellsworth, MI, USA), both at a concentration range of 0.1 to 20 µg/mL. Lipopolysaccharide from Escherichia coli O26:B6 (LPS, 1 µg/mL, #L8274, Sigma-Aldrich) and phorbol 12-myristate 13-acetate (PMA, 100 nM, #10008014, Cayman Chemicals) were used as positive controls.

Vimentin staining

To evaluate whether Vim and CitVim can bind the surface of cells, mEFs -/- and HUVECs were grown on a glass coverslip. When cells reached the desired confluence, Vim and CitVim (both at 1 µg/mL) were introduced or not (CT) to the cells for 1 h. Then, the unbound protein was washed 3 times with PBS, and cells were fixed in 3.7% paraformaldehyde (PFA) for 30 min at RT. Next, cells were permeabilized with 0.1% Triton X-100 for 15 min at RT, followed by 30 min incubation with 0.1% bovine serum albumin (BSA) to block nonspecific binding of the secondary antibody. Cells were stained with rabbit anti-vimentin (#ab45939, Abcam, Cambridge, UK) or mouse anti-citrullinated vimentin (#22054, Cayman Chemicals) at a dilution of 1:1,000 for, RT. The cells were further stained with a secondary anti-rabbit (#ab150081, Abcam) and anti-mouse (#ab150115, Abcam) antibody conjugated with Alexa Fluor 488 dye and Alexa Fluor 647, respectively, at a dilution of 1:1,000 for 1 h at RT in the dark. The cell nuclei were counterstained with Hoechst 33,342 (#R37605, Invitrogen). The coverslips were mounted with an antifade fluorescence mounting medium (Abcam) and examined on a confocal microscope.

Since endothelial cells express intracellular vimentin, vital staining was performed to visualize extracellular vimentin. To do so, anti-Vim antibodies (at 1:500) were introduced to the non-fixed cells for 1 h. Cells were incubated with antibodies at 4 °C to minimize possible endocytosis of the antibodies. Then, the antibodies were washed thrice with PBS, and the cells were fixed. After fixation, vital stained cells were permeabilized and stained with secondary antibody, Texas Red™-X phalloidin and Hoechst 33,342, to visualize eVim, actin, and nuclei, respectively.

Adhesion assay

To assess if the presence of extracellular eVim/CitVim on the surface of the cells determines the adhesion of the immune cells, vim-/- mEFs and mEF +/+ were grown in 96-well black cell culture plates with optical bottom until full confluency was reached. Then, vim-/- mEFs were preincubated with eVim and CitVim (both at the range of 0.1–20 µg/mL) for 1 h, and cells were washed three times with PBS. Then, 3 × 10⁵/well of neutrophils were introduced to the confluent monolayer of fibroblasts for 1 h. After 1 h incubation, unbounded neutrophils were washed off with PBS. Cells were fixed with 3.7% PFA, permeabilized using 0.1% Triton X-100, and blocked with 0.1% BSA. Next, the cells were stained with mouse anti-myeloperoxidase (MPO) antibody (1:500, #MA1-80878, Sigma-Aldrich) for 1 h at RT. The cells were stained with a secondary anti-mouse antibody conjugated with Alexa Fluor 647 dye (1:1,000, #ab150115, Abcam) for 1 h at RT in the dark. Actin was stained with AlexaFluor488-phalloidin (#R37110, Invitrogen), and nuclei were stained with Hoechst 33,342.

In a parallel experiment, neutrophils were live-stained with calcein AM (1 µM, #C1430, Invitrogen) for 30 min in the dark. Then, neutrophils were washed with PBS by gentle centrifugation. Next, 3 × 10⁵/well of neutrophils were introduced to the confluent monolayer of fibroblasts for 1 h. vim-/- mEFs cells were preincubated with eVim or CitVim as before for 1 h. Unbounded neutrophils were washed off with PBS. The fluorescence (494/517 nm) from calcein AM stained neutrophils was recorded on the Varioskan Lux microplate reader (Thermo Fisher Scientific). The background fluorescence was determined for each condition and subtracted from the total fluorescence values before data analysis. The results were compared to the untreated control, normalized to 1.0, and presented as a fold change in the adhesion of the neutrophils. LPS (1 µg/mL) was added to the neutrophils as a positive control.

Polyacrylamide hydrogels

Polyacrylamide hydrogels with a stiffness of 30 kPa were prepared using previously described methods [67]. 40% acrylamide (1170 µL, #1610140, Bio-rad) and 2% bis-acrylamide solutions (594 µL, #1610142, Bio-rad) were formulated in distilled water (2736 µL) to a total volume of 4.5 mL, divided into 500 µL aliquots. To initiate polymerization, 1.5 µL of TEMED (#1610800, Bio-rad) and 3 µL of 10% (w/v) ammonium persulfate (#1610700, Bio-rad) were added to 500 µL aliquots. Immediately after the addition of polymerization initiators, 190 µl of the solution was pipetted to a 20-mm round glass coverslip (adhesive) pretreated with 5% 3-aminopropyltrimethoxysilane and 0.5% (v/v), glutaraldehyde. The solution was covered with a 24 × 60 mm glass coverslip (non-adhesive) siliconized with 10% SurfaSil solution (#TS42800, Thermo Fisher). After 30 min, the top coverslip was removed, and hydrogels were covalently linked to ligands by incubating the gels for 1 h with 50 µL of collagen I, vimentin, or citrullinated vimentin (all proteins at 0.1 mg/mL in 50 mM HEPES pH 8.5) after activating the gel surface with the UV-sensitive Sulfo-SANPAH cross-linker. Before experiments, hydrogels were sterilized in UV light and preincubated with a complete cell culture medium containing antibiotics. The stiffness of the gel substrates was verified using a rheometer (data not shown).

To assess the adhesion and activation of human neutrophils on the surface of 30 kPa polyacrylamide hydrogels coated with collagen, vimentin, or citrullinated vimentin, 2 × 10⁵ of neutrophils in 200 µL of complete DMEM were added on top of the hydrogels for 1 h. Then, unbounded neutrophils were washed off with PBS. Bright-field images of neutrophils remaining on hydrogels were captured using an inverted microscope. Cell morphology, number of adherent neutrophils, and aggregation index were further processed from the images using ImageJ Fiji Software. The aggregation index is the number of neutrophils within the cluster (at least 3 cells stick together) per 100 cells. After bright-field image acquisition, cells on top of hydrogels were fixed, permeabilized, and blocked. The cells on hydrogels were stained with rabbit anti-NOX2/gp91phox (1:200, #BS-3889R, Invitrogen) for 1 h, at RT. The cells were stained with a secondary anti-mouse antibody conjugated with Alexa Fluor 647 (1:1,000, #ab150115, Abcam) and a secondary anti-rabbit conjugated with Alexa Fluor 488 (1:1,000, #ab150081, Abcam) for 1 h, at RT in the dark. Nuclei were stained as before with Hoechst 33,342. The fluorescence intensity of NOX2/gp91phox was determined in ImageJ Fiji software and processed according to the equation:

$$\eqalign{& \>Fluorescence\>intensity \cr & = Integrated\>density - (Cell\>area \times \>Background\>fluorescence) \cr} $$

The results were compared to the values in collagen control and presented as a fold change in NOX2 intensity.

2D migration assay on Transwell inserts

The migration of neutrophils was assessed on Transwell inserts with a 5 μm pore size semi-permeable membrane (#3421, Corning, NY, USA). The membrane was incubated overnight with 0.1 mg/mL of collagen, vimentin, or citrullinated vimentin at 37 °C. The membrane was washed with PBS. 3 × 10⁵ of neutrophils in 200 µL of the medium was added for 2 h to the upper chamber of the Transwell insert, and the bottom chamber was filled with 500 µL of media. After 2 h, media from the bottom chamber was collected, and the number of neutrophils that migrated through the membrane was counted on a flow cytometer using Muse Count & Viability Kit (#LUMIMCH100102, Merck, NJ, USA) according to the producer’s instructions. The upper chamber was washed with PBS thrice, fixed in 3.7% PFA, stained with Hoechst 33,342, and imaged on a fluorescence microscope.

Microfabrication of the 3D microfluidic device

We followed a previously described method with some modifications to fabricate the microfluidic device [68]. Initially, a 3-inch silicon wafer was prepared, and the SU-8 2025 epoxy-based photoresist (MicroChem) was carefully poured onto it at 95 °C. The wafer with the photoresist was then left overnight for curing. Next, a photomask was precisely aligned and applied over the wafer, which was then exposed to a 200-W UV lamp for 2 h. This exposure ensured the polymerization of the photoresist, forming the desired microstructures. After exposure, any unpolymerized photoresist was dissolved using propylene glycol methyl ether acetate (PGMEA) (MicroChem). Polydimethylsiloxane (PDMS) (#2065622, Ellsworth) was used to create negative molds for the microfluidic channels. PDMS was poured over the silicon master, replicating the microstructures present on the wafer. Subsequently, positive stamps made of PDMS were cast using the same procedure, generating the microfluidic channels on glass coverslips. Before proceeding with gel fabrication, the hydrogel reservoir of the device was prepared. It was filled with 5 M sulfuric acid (#258105, Sigma-Aldrich) and left for 90 min. Afterward, thorough washing with distilled water was performed to remove residual acid. Finally, the reservoir was filled with a solution containing 50 µg/mL collagen type I, and sterilization was achieved using UV light exposure.

3D transendothelial migration assay

As previously described [69], each microfluidic device was filled with 60 µL of a hydrogel formulation containing the following components: 6 µL of 10x phosphate-buffered saline (PBS) (#5493, Sigma), 6 µL of 0.1 M sodium hydroxide (NaOH) (#BA0981118, POCh SA), 18 µL of distilled water, and 30 µL of a 10 mg/mL collagen type I solution. This formulation resulted in a gel with a final 5 mg/mL collagen concentration. All components were kept on ice until ready for mixing and then injected into each device’s hydrogel reservoir. To create cylindrical voids resembling human vessels, two freshly coated with sterile 1% BSA 180-mm-diameter acupuncture needles were inserted into the needle guides of the device. The devices were then transferred to a 37 °C environment for 10 min to facilitate gel polymerization. To prevent drying of the hydrogel, phosphate-buffered saline (PBS) was pipetted onto the reservoir ports. After 2 h of incubation, the acupuncture needles were carefully removed from the hydrogel, forming cylindrical voids. Human umbilical vein endothelial cells (HUVECs) were injected into one of the channels at 10 million cells per mL density and left to incubate for 10 min. Subsequently, the devices were inverted for an additional 10 min to ensure complete coverage of the cylindrical voids by the HUVECs. The cell-seeded devices were then placed in wells of 6-well culture plates containing 7 mL of complete growth medium and incubated in a cell culture incubator for 24 h. Once the HUVEC monolayer had fully covered the cylindrical voids after 24 h, 20 µL of human neutrophils at a concentration of 1 × 10⁶ cells/mL in medium, with or without vimentin, citrullinated vimentin or lipopolysaccharide (LPS) (all at 1 µg/mL), were introduced into the freshly washed lumen of endothelial cells and incubated for 4 h. Following the 4-hour incubation, the channels were gently washed with media and then fixed with 3.7% paraformaldehyde (PFA) for immunofluorescence staining. After fixation, the hydrogels were carefully detached from the device hydrogel reservoir and placed in 1.5 mL centrifuge tubes. To permeabilize the cells within the hydrogel, 0.2% Triton X-100 was added and incubated for 30 min at room temperature. Subsequently, the hydrogels were blocked with 3% BSA for 30 min at 37 °C. The hydrogels were then incubated overnight at 4 °C with a rabbit anti-neutrophil elastase antibody (#ab21595, Abcam). After thorough washing, the hydrogels were incubated with the appropriate secondary antibody at a dilution of 1:1000, along with Texas Red™-X Phalloidin and Hoechst 33,342, as previously mentioned. Finally, the hydrogels were imaged using a scanning confocal system.

Fungal and bacterial preparation

Laboratory strains of Candida albicans (C. albicans 1408) were obtained from the Polish Collection of Microorganisms, Polish Academy of Science in Wroclaw, Poland. The C. albicans strain was plated on Sabouraud dextrose with chloramphenicol agar (#PS192, Biomaxima, Lublin, Poland). The clinical strain of Escherichia coli (E. coli RS218) was plated on MacConkey agar plates (#PS10, Biomaxima). Both strains were grown routinely at 37 °C. Hemocytometer counts were used to estimate the quantity of yeast and bacteria. For coculture with human neutrophils, exponentially growing cells were suspended and diluted to the desired cell number in cell culture media without antibiotics and antimycotics.

Phagocytosis assays

C. albicans 1408 was stained with calcofluor white (#F3543, Sigma-Aldrich) at 0.25 µg/mL for 30 min, RT. E. coli RS218 was stained with Fluorescein isothiocyanate (FITC, #F7250, Sigma-Aldrich). Briefly, bacterial cells were resuspended in 0.1 M sodium bicarbonate buffer, pH 9.0. Freshly made FITC (2 µg/mL final concentration) was added to the bacterial suspension for 30 min, RT, in the dark. Then, cells were washed by centrifugation thrice in PBS. Both yeast and bacteria were finally resuspended in cell culture media at a desired concentration.

Prestained, C. albicans cells at MOI (multiplicity of infection, number of germ cells per single neutrophil) of 10 and E. coli at MOI of 100 were added for 2 h to 1 × 10⁵ neutrophils seeded onto 0.01% poly-l-lysine (Sigma-Aldrich) treated 96-well black cell culture plates with the optical bottom. Neutrophils were treated simultaneously with yeast or bacteria and vimentin (eVim– 1 µg/mL) or citrullinated vimentin (CitVim– 1 µg/mL). Alternatively, neutrophils were preincubated with Vimentin (pre-eVim) and Citrullinated vimentin (pre-CitVim) at 1 µg/mL for 1 h in cell culture media. Proteins were washed off, and C. albicans and E. coli were added to the neutrophils for 2 h. After 2 h of incubation, neutrophils were washed with PBS, fixed with 3.7% PFA, and blocked with 0.1% BSA. Neutrophils were stained with Wheat Germ Agglutinin (WGA at 5 µg/mL) conjugated with Alexa Fluor 647 (#W32466, Invitrogen) for 10 min at RT. After staining, cells were examined by fluorescence microscopy. The results are presented as the percentage of cells taking up or adherent to fungal/bacterial cells (engaged neutrophils) and the total number of fungal/bacterial cells taken up per 100 cells (phagocytic index). Data were obtained from 3 separate experiments by analyzing 5 to 10 individual randomly taken images per replicate (at least 500 neutrophils/replicate).

Intracellular survival assay

Neutrophils (1 × 10⁶ cells/well in a 24-well plate) were treated as in the phagocytosis assay. After 2 h of incubation with yeast or bacteria, the cultures were collected using cell scrapers and transferred to Eppendorf tubes. The neutrophils were lysed by sonication for 10 min. Serial dilutions were plated on Sabouraud or MacConkey agar for yeast/bacterial outgrowth assessment and left for 24 h of incubation at 37 °C.

NETosis assay

To assess the formation of neutrophil extracellular traps (NETs), 1 × 10⁵ cells were seeded onto 0.01% poly-l-lysine functionalized sterilized glass coverslips. Neutrophils were untreated or treated with eVim (0.1–20 µg/mL), CitVim (0.1–20 µg/mL), or PMA (100 nM) for 4 h at 37 °C and 5% CO₂. After incubation, the cells were washed with PBS, fixed in 3.7% PFA, permeabilized with 0.1% Triton X-100, and blocked in 0.1% bovine serum albumin, as mentioned above. Next, the cells were stained with rabbit anti-neutrophil elastase (NE) antibody (#ab21595; Abcam) at a dilution of 1:500 for 1 h, at RT. The cells were stained with an appropriate secondary anti-rabbit antibody at a dilution of 1:1,000 for 1 h, at RT in the dark. The cell nuclei were counterstained with Hoechst 33,342. The coverslips were mounted with an antifade fluorescence mounting medium (#ab104135, Abcam) and examined by confocal microscopy. The number of cells producing NETs was manually counted. At least five images per coverslip were taken randomly and analyzed (n = 3).

RNA isolation and gene expression

Total RNA was extracted using the universal RNA purification kit (#E3598-02; EURx, Gdansk, Poland) from 5 × 10⁶ neutrophils per well seeded in 6-well cell culture plates. Cells were untreated or treated with eVim (0.1–20 µg/mL), CitVim (0.1–20 µg/mL), or PMA (100 nM) for 2 h (for phagocytosis-related genes) or 4 h (for NET- and Inflammation-related genes) at 37 °C and 5% CO₂. The cells were scratched, transferred to Eppendorf tubes, and centrifuged, and the supernatant was discarded. The concentration and purity of the isolated RNA were evaluated using a Qubit 4 fluorometer (Thermo Fisher Scientific). cDNA was synthesized using the iScript cDNA synthesis kit (#1708891; Bio-Rad). Reverse transcription-quantitative PCR (qRT-PCR) was performed with 100 ng of cDNA in a 20-µL reaction mixture containing SsoAdvanced universal SYBR green supermix (#1725274; Bio-Rad) using phagocytosis PrimePCR plates (#10047255; Bio-Rad) and VEGF signaling and activation PrimePCR plates (#10025756, Bio-Rad) on the CFX Opus real-time PCR detection system (Bio-Rad) with the following amplification program: 2 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. The gene expression levels were reported as the relative quantity, expressed using the comparative cycle threshold (CT) method (2^−ΔΔCt), and presented as log₂FC.

For quantifying NETosis-related mRNAs, the following primer pairs were used:

H3.3 C

Forward 5’-TCCAGAGGTTGGTGAGGGAGAT-3’.

Reverse 5’-TAGCGTGGATGGCACACAGGTT-3’.

NOX2

Forward 5’-CAAGATGCGTGGAAACTACCTAAGAT-3’.

Reverse 5’-TCCCTGCTCCCACTAACATCA-3’.

TLR4

Forward 5’-CCCTGAGGCATTTAGGCAGCTA-3’.

Reverse 5’-AGGTAGAGAGGTGGCTTAGGCT-3’.

NE

Forward 5’-TCCACGGAATTGCCTCCTTC-3’.

Reverse 5’-CCTCGGAGCGTTGGATGATA-3’.

GAPDH

Forward 5’-GTCTCCTCTGACTTCAACAGCG-3’.

Reverse 5’-ACCACCCTGTTGCTGTAGCCAA-3’.

Protein preparation and western blotting

5 × 10⁶ of human neutrophils were seeded in 6-well plates and were untreated or treated with eVim (1 µg/mL), CitVim (1 µg/mL), or PMA (100 nM) for 4 h. After 4 h, the supernatant was aspirated, cells detached using scrappers, transferred to Eppendorf tubes containing supernatant, and centrifuged. The supernatant was discarded, and the whole-cell lysate was prepared using RIPA lysis buffer (#89901, ThermoFisher) with Pierce Protease Inhibitor (#A32963, ThermoFisher) added freshly before use. Cells were lysed for 20 min on ice and centrifuged at 14,000 rpm for 20 min at 4 °C. Next, supernatants were transferred to fresh tubes, and the Bradford (#5000006, Bio-Rad) assay was performed to determine protein concentration. Lysates were subjected to electrophoresis using 10% sodium dodecyl sulfate–polyacrylamide (SDS-PAGE) at an amount of 15 µg protein per lane. After SDS-PAGE separation, proteins were blotted onto methanol-activated polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked for 1 h in 5% nonfat dry milk in TBS-T (150 mM NaCl, 50 mM Tris-base, 0.05% Tween 20, pH = 7.4). Blocked protein blots were incubated with anti-TLR4 (1:1,000; #ab13556, Abcam), anti-NOX2/gp91phox (1:200, #BS-3889R, Invitrogen), anti-neutrophil elastase (1:1,000; #ab21595, Abcam), anti-Cit H3 (1:1,000; #ab10799, Abcam), and anti-β-actin (1:5,000; #A5441, Sigma-Aldrich), in TBS-T at 4 °C overnight, followed by incubation with goat anti-rabbit IRDye 800CW IgG and goat anti-mouse IRDye 800CW IgG secondary antibody in TBS-T (1:10,000) at room temperature for 1 h, in the dark. Protein blots were visualized with the Odyssey LiCor Imaging System (LiCor Biosciences). Band intensities were quantified using Image Studio Acquisition Software. Data are presented as relative intensity of protein of interest bands in eVim, CitVim, or PMA-treated samples compared to the untreated samples and normalized β-actin.

Cytokine and chemokine secretion

The Bio-Plex Pro human cytokine assay assessed the secretion of IL-4, IL-6, IL-8, IL-10, IFN-γ, TNF-α, and GM-CSF (Bio-Rad Laboratories, Hercules, USA). IL-1β was evaluated using an IL-1 beta Human ELISA Kit (Invitrogen). Neutrophils (3 × 10⁵/well) were cultured on 96-well culture plates for 4 h with vimentin and citrullinated vimentin. LPS and heat-inactivated Pseudomonas aeruginosa (3 × 10⁶/mL) were positive controls. For the assay with anti-vimentin antibodies, neutrophils were simultaneously stimulated with CitVim at 1 µg/mL and various antibodies, namely; rabbit anti-vi (#ab45939, Abcam), Pritumumab (#MA5-41862, Invitrogen), mouse anti-cell surface vimentin (#H00007431-M08, Invitrogen) and anti-CitVim (#22054, Cayman Chemical) at a dilution range of 1:1,000–1:100 for 4 h. Mouse (Ms IgG) and rabbit (Rb IgG) isotype IgG control was used at 0.1 mg/mL. Cytokine and chemokine levels were assessed in the supernatant collected and centrifuged to remove remaining neutrophils.

ROS formation and TLR4 inhibition

For the generation of reactive oxygen species (ROS), neutrophils were probed with DCFH-DA (2′-7′-dichlorofluorescein diacetate; 20 µM, Sigma-Aldrich) in culture medium untreated, or treated with vimentin, citrullinated vimentin and PMA for 4 h in the dark. The anti-TLR4 antibody (#ab13556, Abcam) at 1:100 dilution and TLR4 inhibitor (TAK-242, # 614316, Sigma-Aldrich) at 1 µM was added to the treated and untreated cells. The background fluorescence was determined for each condition and subtracted from the total fluorescence values before data analysis. The results were compared to the values in untreated condition (0), set as 1.

Colocalization studies

We conducted confocal microscopy analysis to determine the surface localization of eVim, CitVim, and TLR4 in human neutrophils. Initially, 2 × 10⁵ neutrophils were cultured on poly-l-lysine-treated glass coverslips at 37 °C. Subsequently, eVim and CitVim (at a concentration of 1 µg/mL) were introduced or omitted (CT) to the cells for a 1 h incubation period. After incubation, the cells were carefully washed with PBS, and the surface presence of eVim and CitVim was vitally stained using a mouse anti-Vim antibody (for eVim, #ab8069, Abcam) and mouse anti-CitVim antibody (for CitVim), both diluted at 1:500, for 1 h at 4 °C. Following vital staining, the cells were fixed with 3.7% paraformaldehyde (30 min, RT), blocked with 0.1% BSA (30 min, RT), and permeabilized with 0.1% Triton X100 (15 min, RT). Next, neutrophils were incubated with an anti-TLR4 antibody (1:500 dilution) for 1 h at RT. After washing off the primary antibody, secondary antibodies (1:500 dilution) were introduced to the cells for 1 h at room temperature in the dark. Specifically, an anti-mouse antibody conjugated with AlexaFluor 488 was used for eVim and CitVim, while an anti-rabbit antibody conjugated with AlexaFluor 647 was employed for TLR4. Following a final wash, stained cells were mounted with an anti-fade solution and sealed with a glass coverslip. Subsequently, samples were scanned using a Stellaris 5 confocal microscope (Leica) with a 60× oil immersion objective. The degree of colocalization was quantitatively assessed using Mander’s coefficients through the JACoP plugin in FIJI ImageJ.

Functionalization of AFM tips and substrates

Silicon nitride AFM tips (Bruker, MSCT-C) with a nominal spring constant of 0.01 N/m were employed to investigate the interaction between eVim/CitVim functionalized on cantilevers and TLR4 (#ab233665, Abcam) immobilized on a mica surface. All silicon nitride tips were functionalized following a modification of established protocols [29, 70]. In brief, tips were silanized with 2% v/v 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) in acetone for 10 min at room temperature (~ 22 °C). Subsequently, tips were rinsed with deionized water (DI water) and immersed in 0.5% v/v glutaraldehyde in DI water for 30 min at 25 °C. After another round of DI water rinsing, tips were incubated in 50 µL of eVim or CitVim solution at 100 µg/mL for 30 min at room temperature. Unbound proteins were washed away with DI water. To block any remaining aldehyde groups on the probe surface, the tip was treated with 100 µg/mL BSA in PBS for 1 min at room temperature. The tips were rinsed and then submerged in PBS buffer (to be used within 6 h).

For protein immobilization, mica surfaces were freshly cleaved and incubated with 0.01% poly-L-lysine overnight. The mica was washed with DI water and then incubated with 10 µL of recombinant TLR4 at 100 µg/mL for 30 min at room temperature. Unbound protein was washed off. A series of control experiments were conducted to assess the binding’s specificity. Specifically, tip AFM and mica surfaces were functionalized with BSA, or the protein addition step was omitted.

AFM force measurements

AFM was employed to assess single-molecule binding interactions between eVim/CitVim and TLR4 on a mica surface, utilizing a NanoWizard 4 BioScience JPK Instruments Bruker atomic force microscope operating in force spectroscopy mode. The investigation of eVim/CitVim–TLR4 interactions involved recording AFM force–distance (FD) curves. TLR4 immobilized on mica surfaces was probed in PBS at room temperature. Up to 40 force maps, each comprising 16 × 16 points and corresponding to a scan area of 5 μm × 5 μm, were acquired for each sample, covering multiple random spots on mica or confluent cell surfaces. A set point of 0.15 nN was consistently applied for all measurements, and a 5 μm/s AFM cantilever approach/retraction speed was employed to probe TLR4 immobilized on mica.

For the analysis of specific adhesion events, a worm-like chain (WLC) model, offering reaction forces and loading rate (LR) data (processed using JPK data processing and the JPK built-in analysis software), was employed. The retraction part of the obtained curves was utilized to classify and determine eVim/CitVim and TLR4 interactions. Curves exhibiting specific adhesion displayed a distinctive “shape,” amenable to fitting using the WLC model. JPK software facilitated the WLC model fitting in the segment of the force curve, indicating polymer stretching, and corresponding loading rate (LR) values were simultaneously determined. Forces represent averages derived from all collected force–distance curves where adhesion forces were observed.

Dot blot binding assay

Dot blot binding assays were conducted to validate the binding capabilities of recombinant Vim and CitVim proteins to TLR4, employing a modified approach based on previous methodologies [71, 72]. Various quantities (1.0, 2.0, and 4.0 µg) of eVim, CitVim, and BSA were transferred in 10 µL volumes onto NC membranes with a pore size of 0.2 μm (Bio-Rad). Following the transfer, the membranes were dried and then blocked with 5% skim milk in Tris-buffered saline–Tween 20 (0.1%) (TBS-T) for 2 h at room temperature. Subsequently, the membranes were washed with TBS-T (3 × 15 min) and incubated with whole cell lysate of human neutrophils, equivalent to 5 × 10⁶ cells per blot, in TBS-T with gentle shaking overnight at 4 °C. After extensive washing with TBS-T, the blots were probed with the respective primary antibodies (anti-Vim, anti-CitVim, or anti-TLR4) at a 1:500 dilution in TBS-T overnight at 4 °C. Following another round of washing, the blots were incubated for 2 h at room temperature with an IRDye 800CW-conjugated goat anti-rabbit or goat anti-mouse secondary antibody at a dilution of 1:5,000. Images were visualized using the Odyssey LiCor Imaging System (LiCor Biosciences), and spot intensities were quantified using Image Studio Acquisition Software.

TLR4/MD2-Mediated recognition of vimentin

HEK293-NEE cells, obtained as a gift from the National Cancer Institute, are an NF-kB reporter cell line that expresses GFP when NF-kB translocates to the nucleus to trigger gene expression. These cells were expanded and cultured in DMEM + 10% FBS. Cells (8 × 10⁵ /well) were plated in 0.5 mL DMEM + 10% FBS in a 24-well plate. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. 16 h after transfection, media was replaced with fresh DMEM + 10% FBS + 0, 1, 5, 10, or 20 µg/mL vimentin, followed by a 24 h incubation. Cells were harvested 40 h post-transfection with PBS + EDTA and resuspended in PBS + BSA for flow cytometry analysis.

A single cell population was established by evaluating FSC height vs. area, where a transfected cell population was determined by gating for cells expressing miRFP720. Within this transfected cell population, GFP mean fluorescence intensity was measured.

hMD2 Gene block- inserted into pcDNA backbone using EcoRI and HindIII cGCCACCATGgGGAATTCgggcggaTTACCATTTCTGTTTTTTTCCACCCTGTTTTCTTCCATATTTACTGAAGCTCAGAAGCAGTATTGGGTCTGCAACTCATCCGATGCAAGTATTTCATACACCTACTGTGATAAAATGCAATACCCAATTTCAATTAATGTTAACCCCTGTATAGAATTGAAAGGATCtAAAGGATTATTGCACATTTTCTACATTCCAAGGAGAGATTTAAAGCAATTATATTTCAATCTCTATATAACTGTCAACACCATGAATCTTCCAAAGCGCAAAGAAGTTATTTGCCGAGGATCTGATGACGATTACTCTTTTTGCAGAGCTCTGAAGGGAGAGACTGTGAATACAACAATATCATTCTCCTTCAAGGGAATAAAATTTTCTAAGGGAAAATACAAATGTGTTGTTGAAGCTATTTCTGGGAGCCCAGAAGAAATGCTCTTTTGCTTGGAGTTTGTCATCCTACACCAACCTAATTCAAATTAGTAGaagcttaagtttaaaccg.

Quantification and statistical analysis

Statistical analysis and data visualization were performed using Origin 2021 software. Gene-gene coexpression analysis using the STRING database and analysis of enriched pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) were analyzed using STRING v11.5 web software. Statistical comparisons were made using unpaired Student’s t-test (between two samples) and one-way ANOVA followed by Tukey’s post hoc test (among multiple samples). Differences were considered statistically significant at p < 0.05. We confirmed the validity of the biological replicates and enhanced the reliability of the data by repeatedly performing independent experiments using at least three independent samples. Data are generally presented as mean ± SD of n = x experiments, with x indicating in figure legends the number of independent experiments performed unless otherwise stated.

Data availability

No datasets were generated or analyzed during the current study.

Abbreviations

AFM:: Atomic Force Microscope
APTES:: 3-Aminopropyltriethoxysilane
BSA:: Bovine Serum Albumin
cDNA:: Complementary DNA
CitH3:: citrullinated Histone H3
CitVim:: Citrullinated Vimentin
Col:: Collagen
COVID-19:: Coronavirus Disease 2019
CSV:: Cell-surface vimentin
CTGF:: Connective Tissue Growth Factor
CTGF:: Control, Untreated Condition
DAMP:: Damage-associated Molecular Pattern
DCFH-DA:: 2′-7′-Dichlorofluorescein Diacetate
DMEM:: Dulbecco’s Modified Eagle’s Medium
DNA:: Deoxyribonucleic Acid
ECM:: Extracellular Matrix
eVim:: Extracellular Vimentin
FBS:: Fetal Bovine Serum
FD:: Force Distance
FITC:: Fluorescein Isothiocyanate
FLS:: Fibroblast-like Synoviocytes
GAPDH:: Glyceraldehyde 3-phosphate Dehydrogenase
GM-CSF:: Granulocyte-Macrophage Colony-Stimulating Factor
HUVECs:: Human Umbilical Vein Endothelial Cells
ICAM-1:: Intercellular Adhesion Molecule1
ICU:: Intensive Care Unit
IL:: Interleukin
IPF:: Idiopathic Pulmonary Fibrosis
KEGG:: Kyoto Encyclopedia of Genes and Genomes
LPS:: Lipopolysaccharide
LR:: Loading Rate
MAPK3:: Mitogen-Activated Protein kinase 3
MD2:: Myeloid Differentiation Factor 2
mEF:: mouse Embryonic Fibroblasts
MOI:: Multiplicity Of Infection
MPO:: Myeloperoxidase
NC:: Nitrocellulose
NE:: Neutrophil Elastase
NET:: Neutrophil Extracellular Traps
NF-κB:: Nuclear Factor Kappa B
NOX2/gp91phox:: NADPH Oxidase Subunit 2
oxLDL:: oxidized Low-Density Protein
PAD:: Peptidyl Arginine Deiminase
PBS:: Phosphate-Buffered Saline
PDMS:: Polydimethylsiloxane
PFA:: Paraformaldehyde
PGMEA:: Propylene Glycol Methyl Ether Acetate
PMA:: Phorbol 12-myristate 13-acetate
PRRs:: Pathogen Recognition Receptors
PTM:: Post-Translational Modification
qRT-PCR:: Reverse Transcription-quantitative Polymerase Chain Reaction
RA:: Rheumatoid Arthritis
RIPA:: Radioimmunoprecipitation Assay Buffer
RNA:: Ribonucleic acid
ROS:: Reactive Oxygen Species
RT:: Room Temperature
SD:: Standard Deviation
SDS-PAGE:: Sodium Dodecyl Sulfate-Polyacrylamide
TAK-242:: Selective TLR-4 Inhibitor
TGF-β:: Transforming Growth Factor-β
TLR4:: Toll-Like Receptor 4
TNF-α:: Tumor Necrosis Factor-α
VEGF:: Vascular Endothelial Growth Factor
Vim:: Vimentin
WGA:: Wheat Germ Agglutinin
WLC model:: Worm-Like Chain Model

References

Rosales C. Neutrophil: a cell with many roles in inflammation or several cell types? Front Physiol. 2018;9:113.
Article PubMed PubMed Central Google Scholar
Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, Gizinski A, Yalavarthi S, Knight JS, et al. NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med. 2013;5(178):ra17840–40.
Article Google Scholar
Pittman K, Kubes P. Damage-associated molecular patterns control neutrophil recruitment. J Innate Immun. 2013;5(4):315–23.
Article CAS PubMed PubMed Central Google Scholar
Gaudet AD, Popovich PG. Extracellular matrix regulation of inflammation in the healthy and injured spinal cord. Exp Neurol. 2014;258:24–34.
Article CAS PubMed Google Scholar
Li D, Wu M. Pattern recognition receptors in health and diseases. Signal Transduct Target Therapy. 2021;6(1):291.
Article CAS Google Scholar
Zhou X, Jin S, Pan J, Lin Q, Yang S, Ambe PC, et al. Damage associated molecular patterns and neutrophil extracellular traps in acute pancreatitis. Front Cell Infect Microbiol. 2022;12:927193.
Article CAS PubMed PubMed Central Google Scholar
Huang H, Tohme S, Al-Khafaji AB, Tai S, Loughran P, Chen L, et al. Damage‐associated molecular pattern–activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury. Hepatology. 2015;62(2):600–14.
Article CAS PubMed Google Scholar
Hernandez C, Huebener P, Schwabe RF. Damage-associated molecular patterns in cancer: a double-edged sword. Oncogene. 2016;35(46):5931–41.
Article CAS PubMed PubMed Central Google Scholar
Pogoda K, Byfield F, Deptuła P, Cieśluk M, Suprewicz Ł, Skłodowski K et al. Unique role of Vimentin Networks in Compression Stiffening of cells and protection of nuclei from compressive stress. Nano Lett. 2022.
Ridge KM, Eriksson JE, Pekny M, Goldman RD. Roles of vimentin in health and disease. Genes Dev. 2022;36(7–8):391–407.
Article CAS PubMed PubMed Central Google Scholar
Sadeghi N, Fazli G, Bayat AA, Fatemi R, Ebrahimnejhad N, Salimi A, et al. Cell surface vimentin detection in Cancer cells by peptide-based monoclonal antibody. Avicenna J Med Biotechnol. 2023;15(2):68.
PubMed PubMed Central Google Scholar
Amraei R, Xia C, Olejnik J, White MR, Napoleon MA, Lotfollahzadeh S et al. Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells. Proceedings of the National Academy of Sciences. 2022;119(6).
Walker J, Bleaken B, Romisher A, Alnwibit A, Menko A. In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype. Mol Biol Cell. 2018;29(13):1555–70.
Article CAS PubMed PubMed Central Google Scholar
Li FJ, Surolia R, Li H, Wang Z, Liu G, Kulkarni T, et al. Citrullinated vimentin mediates development and progression of lung fibrosis. Sci Transl Med. 2021;13(585):eaba2927.
Article CAS PubMed PubMed Central Google Scholar
Miao H, Cui Y, Lu Y, Sun T, Dou J, Ren Y, et al. Serum vimentin predicts mortality in pediatric severe sepsis: a prospective observational study. Int J Infect Dis. 2022;121:141–7.
Article CAS PubMed Google Scholar
Van Steendam K, Tilleman K, Deforce D. The relevance of citrullinated vimentin in the production of antibodies against citrullinated proteins and the pathogenesis of rheumatoid arthritis. Rheumatology. 2011;50(5):830–7.
Article PubMed PubMed Central Google Scholar
Witalison E, Thompson P, Hofseth L. Protein arginine deiminases and Associated 566 citrullination: physiological functions and diseases Associated with Dysregulation. Curr 567 Drug Targets. 2015;16(700–710):568.
Google Scholar
Bang H, Egerer K, Gauliard A, Lüthke K, Rudolph PE, Fredenhagen G, et al. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthr Rhuem. 2007;56(8):2503–11.
Article CAS Google Scholar
Thiam HR, Wong SL, Qiu R, Kittisopikul M, Vahabikashi A, Goldman AE et al. NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture. Proceedings of the National Academy of Sciences. 2020;117(13):7326-37.
Darrah E, Andrade F. Rheumatoid arthritis and citrullination. Curr Opin Rheumatol. 2018;30(1):72.
Article CAS PubMed PubMed Central Google Scholar
Suprewicz Ł, Swoger M, Gupta S, Piktel E, Byfield FJ, Iwamoto DV, et al. Extracellular Vimentin as a target against SARS-CoV‐2 host cell Invasion. Small. 2022;18(6):2105640.
Article CAS Google Scholar
Steinmetz NF, Maurer J, Sheng H, Bensussan A, Maricic I, Kumar V, et al. Two domains of vimentin are expressed on the surface of lymph node, bone and brain metastatic prostate cancer lines along with the putative stem cell marker proteins CD44 and CD133. Cancers. 2011;3(3):2870–85.
Article CAS PubMed PubMed Central Google Scholar
Bucki R, Iwamoto D, Shi X, Kerr KE, Byfield FJ, Suprewicz L et al. Extracellular vimentin is sufficient to promote cell attachment, spreading, and motility by a mechanism involving N-acetyl glucosamine-containing structures. bioRxiv. 2022:2022.11. 28.518249.
Raad H, Derkawi RA, Tlili A, Belambri SA, Dang PM-C, El-Benna J. Phosphorylation of gp91 phox/NOX2 in Human Neutrophils. NADPH Oxidases: Methods and Protocols. 2019:341– 52.
Choi EY, Santoso S, Chavakis T. Mechanisms of neutrophil transendothelial migration. Front Bioscience: J Virtual Libr. 2009;14:1596.
Article CAS Google Scholar
Gomes NE, Brunialti MKC, Mendes ME, Freudenberg M, Galanos C, Salomão R. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood. Braz J Med Biol Res. 2010;43:853–8.
Article CAS PubMed Google Scholar
Karlsson A, Nixon JB, McPhail LC. Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3‐kinase. J Leukoc Biol. 2000;67(3):396–404.
Article CAS PubMed Google Scholar
Fan J, Frey RS, Malik AB. TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase. J Clin Investig. 2003;112(8):1234–43.
Article CAS PubMed PubMed Central Google Scholar
Deptuła P, Fiedoruk K, Wasilewska M, Suprewicz Ł, Cieśluk M, Żeliszewska P, et al. Physicochemical Nature of SARS-CoV-2 spike protein binding to human Vimentin. ACS Applied Materials & Interfaces; 2023.
Yang J, Petitjean SJL, Koehler M, Zhang Q, Dumitru AC, Chen W, et al. Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor. Nat Commun. 2020;11(1):4541.
Article CAS PubMed PubMed Central Google Scholar
Horton M, Charras G, Lehenkari P. Analysis of ligand–receptor interactions in cells by atomic force microscopy. J Recept Signal Transduction. 2002;22(1–4):169–90.
Article CAS Google Scholar
Koehler M, Aravamudhan P, Guzman-Cardozo C, Dumitru AC, Yang J, Gargiulo S, et al. Glycan-mediated enhancement of reovirus receptor binding. Nat Commun. 2019;10(1):4460.
Article PubMed PubMed Central Google Scholar
Lostao A, Medina M. Atomic force microscopy: single-molecule imaging and force spectroscopy in the study of flavoproteins ligand binding and reaction mechanisms. Flavins Flavoproteins: Methods Protocols. 2021;157:78.
Google Scholar
Rubel C, Gómez S, Fernández GC, Isturiz MA, Caamaño J, Palermo MS. Fibrinogen-CD11b/CD18 interaction activates the NF‐κB pathway and delays apoptosis in human neutrophils. Eur J Immunol. 2003;33(5):1429–38.
Article CAS PubMed Google Scholar
Mitra A, Satelli A, Xia X, Cutrera J, Mishra L, Li S. Cell-surface V imentin: a mislocalized protein for isolating csV imentin + CD 133– novel stem‐like hepatocellular carcinoma cells expressing EMT markers. Int J Cancer. 2015;137(2):491–6.
Article CAS PubMed Google Scholar
Päll T, Pink A, Kasak L, Turkina M, Anderson W, Valkna A, et al. Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface. PLoS ONE. 2011;6(12):e29305.
Article PubMed PubMed Central Google Scholar
Menko AS, Romisher A, Walker JL. The pro-fibrotic response of mesenchymal leader cells to lens wounding involves hyaluronic acid, its receptor RHAMM, and vimentin. Front Cell Dev Biology. 2022;10.
Thiagarajan PS, Yakubenko VP, Elsori DH, Yadav SP, Willard B, Tan CD, et al. Vimentin is an endogenous ligand for the pattern recognition receptor Dectin-1. Cardiovascular Res. 2013;99(3):494–504.
Article CAS Google Scholar
Takashi S, Okubo Y, Horie S. Contribution of CD54 to human eosinophil and neutrophil superoxide production. J Appl Physiol. 2001;91(2):613–22.
Article CAS PubMed Google Scholar
Wang JH, Sexton DM, Redmond HP, Watson R, Croke DT, Bouchier-Hayes D. Intercellular adhesion molecule-1 (ICAM-1) is expressed on human neutrophils and is essential for neutrophil adherence and aggregation. Shock (Augusta Ga). 1997;8(5):357–61.
Article CAS PubMed Google Scholar
Woodfin A, Beyrau M, Voisin M-B, Ma B, Whiteford JR, Hordijk PL, et al. ICAM-1–expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia. Blood J Am Soc Hematol. 2016;127(7):898–907.
CAS Google Scholar
Kim H-M, Wang M, Kim S, Park J, Hong S-T. Removal of Extracellular Vimentin in Blood Confers Resistance to Viral Infection. 2022.
Su L, Pan P, Yan P, Long Y, Zhou X, Wang X, et al. Role of vimentin in modulating immune cell apoptosis and inflammatory responses in sepsis. Sci Rep. 2019;9(1):5747.
Article PubMed PubMed Central Google Scholar
Martinez-Vargas M, Cebula A, Brubaker LS, Seshadri N, Lam FW, Loor M, et al. A novel interaction between extracellular vimentin and fibrinogen in fibrin formation. Thromb Res. 2023;221:97–104.
Article CAS PubMed Google Scholar
Ellis S, Way R, Nel M, Burleigh A, Doykov I, Kembou-Ringert J et al. Salivary IgA and vimentin differentiate in vitro SARS-CoV-2 infection: a study of 290 convalescent COVID-19 patients. Mucosal Immunol. 2023.
Kim S, Cho W, Kim I, Lee S-H, Oh GT, Park YM. Oxidized LDL induces vimentin secretion by macrophages and contributes to atherosclerotic inflammation. J Mol Med. 2020;98(7):973–83.
Article CAS PubMed Google Scholar
Yu MB, Guerra J, Firek A, Langridge WH. Extracellular vimentin modulates human dendritic cell activation. Mol Immunol. 2018;104:37–46.
Article CAS PubMed PubMed Central Google Scholar
Nieminen M, Henttinen T, Merinen M, Marttila–Ichihara F, Eriksson JE, Jalkanen S. Vimentin function in lymphocyte adhesion and transcellular migration. Nat Cell Biol. 2006;8(2):156–62.
Article CAS PubMed Google Scholar
Thalla DG, Rajwar AC, Laurent AM, Becher JE, Kainka L, Lautenschläger F. Extracellular vimentin is expressed at the rear of activated macrophage-like cells: potential role in enhancement of migration and phagocytosis. Front Cell Dev Biology. 2022;10.
Engbersen DJ, van Beijnum JR, Roos A, van Beelen M, de Haan JD, Grinwis GC, et al. Vaccination against Extracellular Vimentin for treatment of Urothelial Cancer of the bladder in client-owned dogs. Cancers. 2023;15(15):3958.
Article CAS PubMed PubMed Central Google Scholar
Kim SY, Jeong S-J, Park J-H, Cho W, Ahn Y-H, Choi Y-H, et al. Plasma membrane localization of CD36 requires vimentin phosphorylation; a mechanism by which macrophage vimentin promotes atherosclerosis. Front Cardiovasc Med. 2022;9:792717.
Article CAS PubMed PubMed Central Google Scholar
Arrindell J, Desnues B. Vimentin: from a cytoskeletal protein to a critical modulator of immune response and a target for infection. Front Immunol. 2023;14.
Kim JS, Choi M, Choi JY, Kim JY, Kim JY, Song J-S, et al. Implication of the association of fibrinogen citrullination and osteoclastogenesis in bone destruction in rheumatoid arthritis. Cells. 2020;9(12):2720.
Article CAS PubMed PubMed Central Google Scholar
Shi L, Aymonnier K, Wagner DD. Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4. PLoS ONE. 2021;16(5):e0251726.
Article CAS PubMed PubMed Central Google Scholar
Myers LK, Ouyang Y-X, Patel JR, Odens HH, Woo-Rasberry V, Park J, et al. Role of Citrullinated Collagen in Autoimmune Arthritis. Int J Mol Sci. 2022;23(17):9833.
Article CAS PubMed PubMed Central Google Scholar
Snir O, Rieck M, Gebe JA, Yue BB, Rawlings CA, Nepom G, et al. Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA–DRB1* 0401–positive humanized mice and rheumatoid arthritis patients. Arthr Rhuem. 2011;63(10):2873–83.
Article CAS Google Scholar
Fan L, He D, Wang Q, Zong M, Zhang H, Yang L, et al. Citrullinated vimentin stimulates proliferation, pro-inflammatory cytokine secretion, and PADI4 and RANKL expression of fibroblast-like synoviocytes in rheumatoid arthritis. Scand J Rheumatol. 2012;41(5):354–8.
Article CAS PubMed Google Scholar
Shindo S, Pierrelus R, Ikeda A, Nakamura S, Heidari A, Pastore MR, et al. Extracellular release of Citrullinated Vimentin directly acts on osteoclasts to promote bone resorption in a mouse model of Periodontitis. Cells. 2023;12(8):1109.
Article CAS PubMed PubMed Central Google Scholar
Georganas C, Liu H, Perlman H, Hoffmann A, Thimmapaya B, Pope RM. Regulation of IL-6 and IL-8 expression in rheumatoid arthritis synovial fibroblasts: the dominant role for NF-κB but not C/EBPβ or c-Jun. J Immunol. 2000;165(12):7199–206.
Article CAS PubMed Google Scholar
Leifer CA, Medvedev AE. Molecular mechanisms of regulation of toll-like receptor signaling. J Leucocyte Biology. 2016;100(5):927–41.
Article CAS Google Scholar
Kircheis R. In Silico Analyses Indicate a lower potency for dimerization of TLR4/MD-2 as the reason for the Lower pathogenicity of Omicron compared to wild-type virus and earlier SARS-CoV-2 variants. Int J Mol Sci. 2024;25(10):5451.
Article CAS PubMed PubMed Central Google Scholar
Husebye H, Halaas Ø, Stenmark H, Tunheim G, Sandanger Ø, Bogen B, et al. Endocytic pathways regulate toll-like receptor 4 signaling and link innate and adaptive immunity. EMBO J. 2006;25(4):683–92.
Article CAS PubMed PubMed Central Google Scholar
Gangloff M. Different dimerisation mode for TLR4 upon endosomal acidification? Trends in biochemical sciences. 2012;37(3):92–8.
Tan Y, Zanoni I, Cullen TW, Goodman AL, Kagan JC. Mechanisms of toll-like receptor 4 endocytosis reveal a common immune-evasion strategy used by pathogenic and commensal bacteria. Immunity. 2015;43(5):909–22.
Article CAS PubMed PubMed Central Google Scholar
Rice TW, Wheeler AP, Bernard GR, Vincent J-L, Angus DC, Aikawa N, et al. A randomized, double-blind, placebo-controlled trial of TAK-242 for the treatment of severe sepsis. Crit Care Med. 2010;38(8):1685–94.
Article CAS PubMed Google Scholar
Opal SM, Laterre P-F, Francois B, LaRosa SP, Angus DC, Mira J-P, et al. Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial. JAMA. 2013;309(11):1154–62.
Article CAS PubMed Google Scholar
Bucki R, Iwamoto DV, Shi X, Kerr KE, Byfield FJ, Suprewicz Ł et al. Extracellular vimentin is sufficient to promote cell attachment, spreading, and motility by a mechanism involving N-acetyl glucosamine-containing structures. J Biol Chem. 2023;299(8).
Partyka PP, Godsey GA, Galie JR, Kosciuk MC, Acharya NK, Nagele RG, et al. Mechanical stress regulates transport in a compliant 3D model of the blood-brain barrier. Biomaterials. 2017;115:30–9.
Article CAS PubMed Google Scholar
Suprewicz Ł, Tran KA, Piktel E, Fiedoruk K, Janmey PA, Galie PA, et al. Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus. J Neuroinflamm. 2022;19(1):1–16.
Article Google Scholar
Chen Y, Zeng G, Chen SS, Feng Q, Chen ZW. AFM force measurements of the gp120–sCD4 and gp120 or CD4 antigen–antibody interactions. Biochem Biophys Res Commun. 2011;407(2):301–6.
Article CAS PubMed PubMed Central Google Scholar
Miranda-Ozuna JF, Hernández-García MS, Brieba LG, Benítez-Cardoza CG, Ortega-López J, González-Robles A, et al. The glycolytic enzyme triosephosphate isomerase of Trichomonas Vaginalis is a surface-associated protein induced by glucose that functions as a laminin-and fibronectin-binding protein. Infect Immun. 2016;84(10):2878–94.
Article CAS PubMed PubMed Central Google Scholar
Bastos CR, Mathias LA, Jusi MMG, Santos RFd, Silva GCPd, André MR, et al. Evaluation of dot-blot test for serological diagnosis of bovine brucellosis. Brazilian J Microbiol. 2018;49:564–8.
Article CAS Google Scholar

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Funding

This work was supported by a Polish National Science Centre under a Preludium 21 grant (UMO-2022/45/N//NZ6/01454) awarded to Ł.S. and by the Medical University of Białystok to R.B. (B.SUB.23.326), and by NIH (R01EY035771) to P.A.J. Ł.S. was supported by the Foundation for Polish Science (FNP START 075/2024).

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Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Bialystok, 15-089, Poland
Łukasz Suprewicz, Krzysztof Fiedoruk, Karol Skłodowski, Magdalena Zakrzewska & Robert Bucki
Department of Biomedical Engineering, Rowan University, Glassboro, NJ, 08028, USA
Evan Hutt & Peter A. Galie
Centre of Regenerative Medicine, Medical University of Bialystok, Bialystok, 15-269, Poland
Alicja Walewska
Independent Laboratory of Nanomedicine, Medical University of Bialystok, Bialystok, 15-089, Poland
Piotr Deptuła
Institute of Medical Sciences, Collegium Medicum, Jan Kochanowski University of Kielce, Kielce, 25-369, Poland
Agata Lesiak & Sławomir Okła
Physics Department and BioInspired Institute, Syracuse University, Syracuse, NY, 13244, USA
Alison E. Patteson
Department of Physiology and Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, 19104, USA
Paul A. Janmey

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Łukasz Suprewicz
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Krzysztof Fiedoruk
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Karol Skłodowski
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Evan Hutt
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Magdalena Zakrzewska
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Alicja Walewska
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Piotr Deptuła
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Agata Lesiak
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Sławomir Okła
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Peter A. Galie
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Alison E. Patteson
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Paul A. Janmey
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Robert Bucki
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Contributions

Ł.S., K. F., E.H., P.A.G., A.E.P., P.A.J., and R.B. designed research; Ł.S., K.S., E.H., A.W., P.D., A.L., and S.O. performed research. Ł.S. and M.Z. contributed new reagents/analytic tools; Ł.S., P.D. analyzed the data; Ł.S. prepared figures; Ł.S. and R.B. wrote the paper.

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Correspondence to Robert Bucki.

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Procedures with human blood samples from healthy donors were performed in accordance with the approval of the Medical University of Bialystok ethics committee No. APK.002.8.2023. All donors signed an informed consent form, in accordance with the relevant ethics committee approval.

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The authors declare no competing interests.

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Suprewicz, Ł., Fiedoruk, K., Skłodowski, K. et al. Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils. Cell Commun Signal 23, 64 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02062-w

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Received: 02 May 2024
Accepted: 22 January 2025
Published: 05 February 2025
DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-025-02062-w

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Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils

Abstract

Background

Methods

Results

Conclusions

Introduction

Results

Extracellular vimentin on endothelial cell surfaces and enhanced adhesion and activation of human neutrophils

Extracellular vimentin promotes transendothelial migration of human neutrophils

Extracellular vimentin enhances phagocytosis and NETosis

Citrullinated vimentin boosts the inflammatory response of human neutrophils, and antibodies targeting vimentin diminish this response

In human neutrophils, eVim and CitVim serve as a ligand for toll-like receptor 4

Discussion

Conclusions

Materials and methods

Neutrophil isolation

Cell culture and treatment

Vimentin staining

Adhesion assay

Polyacrylamide hydrogels

2D migration assay on Transwell inserts

Microfabrication of the 3D microfluidic device

3D transendothelial migration assay

Fungal and bacterial preparation

Phagocytosis assays

Intracellular survival assay

NETosis assay

RNA isolation and gene expression

H3.3 C

NOX2

TLR4

NE

GAPDH

Protein preparation and western blotting

Cytokine and chemokine secretion

ROS formation and TLR4 inhibition

Colocalization studies

Functionalization of AFM tips and substrates

AFM force measurements

Dot blot binding assay

TLR4/MD2-Mediated recognition of vimentin

Quantification and statistical analysis

Data availability

Abbreviations

References

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval and consent to participate

Competing interests

Additional information

Publisher’s note

Electronic supplementary material

Supplementary Material 1

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About this article

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Keywords

Cell Communication and Signaling

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