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:

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.