Expression of neutrophil extracellular trap-related proteins and its correlation with IL-17 and TNF-α in patients with oral lichen planus

Patients and methods

Tissue and peripheral blood samples were obtained from individuals visiting the Department of Oral Mucosal Diseases at the Affiliated Stomatology Hospital of Nanjing Medical University, Jiangsu Province, between May 2021 and September 2022. According to the guidelines of the 5th edition of Oral Mucosa, edited by Qianming (2020), and the recommendations proposed by van der Meij & van der Waal (2003), a total of 20 patients with EOLP and 20 patients with NEOLP were recruited. Exclusion criteria: 1) having other established oral mucosal diseases; 2) having more severe systemic diseases, tumors; 3) having used antibiotics within 1 month and immunological agents within 3 months; 4) certain drugs or silver amalgam fillings that may cause moss-like reactions; 5) severe tobacco and alcohol addiction. Healthy subjects were included as having no systemic disease, no oral mucosal disease, and no antibiotics or immunosuppressive agents within 3 months. Within the erosion group, there were eight males and 12 females, with an average age of (46.8 ± 11.0) years. The NEOLP group consisted of nine male and 11 females, with an average age of (44.3 ± 13.6) years. The healthy control (HC) group (n = 20) were selected from patients undergoing treatment in the implant department during the same period, including 10 males and 10 females, with an average age of (39.7 ± 3.9) years. None of the subjects exhibited oral mucosal diseases or serious systemic illnesses, nor had they taken antibiotics or immunosuppressants within 3 months. There were no significant differences in age or gender distribution among the groups (p > 0.05). Informed consent was obtained in writing from all participants before sample collection. All procedures were approved by the Ethics Committee of the School of Stomatology, Nanjing Medical University, under approval number No. 281 (2019).

Immunofluorescence analysis of tissues

After the initial routine hematoxylin-eosin (HE) staining of the first section from each group, the remaining sections underwent tissue immunofluorescence (IF) staining to assess the levels of NET expression. Formalin-fixed and paraffin-embedded tissue sections (4 μm) were placed on adhesive slides (CITOGLAS, Jiangsu, China) and subjected to deparaffinization at 65 °C for 1 h. Deparaffinization was achieved using the xylene method, followed by rehydration with ethanol. Subsequently, the tissue sections were washed twice with phosphate-buffered saline (PBS; Gibco, Waltham, MA, USA), and antigen retrieval was performed using sodium citrate buffer (0.01 M, Phygene, Fujian, China) heated for 20 min. To minimize natural fluorescence, the sections were treated with 3% hydrogen peroxide for 10 min at room temperature. After two PBS washes, the sections were blocked with goat serum for 45 min at 37 °C and then incubated overnight at 4 °C with anti-MPO antibody (1:100, ab208670, Abcam, Cambridge, MA, USA). Following removal of the primary antibody and two PBS washes, the sections were incubated with CoraLite488-labeled goat anti-mouse IgG (H+L) secondary antibody (Proteintech, Wuhan, China) for 45 min at 37 °C in the dark. After two additional PBS washes, nuclei were stained with DAPI (Beyotime, Jiangsu, China) for 2 min. Following two more PBS washes, the slides were sealed with an anti-fluorescence quenching sealer (Beyotime, Jiangsu, China) and examined under a laser confocal microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) at 20× magnification. Excitation wavelengths for the blue and red channels were set to 405 and 561 nm, respectively. Images were captured randomly using ZEN software (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany), and the expression levels of NET-related proteins in tissues were analyzed semi-quantitatively by using ImageJ software version 1.53 m (National Institutes of Health, Maryland, USA).

Cell isolation, identification and IF analysis

Freshly drawn peripheral blood was collected and neutrophils were isolated within 2 h using the Human Neutrophil Isolation Solution Kit (TBDScience, Tianjin, China). A total of 6 mL of human neutrophil isolate was added to a 15 mL tube and peripheral blood was placed above the liquid level and centrifuged at 650 g for 30 min. The liquid in the tube was divided from top to bottom into plasma, monocytes, neutrophils and erythrocytes along with other cells. Approximately 1 mL of plasma was aspirated into lyophilization tubes (Thermo Fisher Scientific, Waltham, MA, USA) and stored in a refrigerator at −80 °C for subsequent experiments. The lower layer containing neutrophils was washed with HBSS washing solution, centrifuged at 350 g for 10 min, and the supernatant was discarded. The remaining cells were lysed using erythrocyte lysis solution, centrifuged at 250 g for 8 min, and the supernatant was discarded. Cell components were resuspended in 1 mL of RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% Australian fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and penicillin-streptomycin double antibody (HYclone, Logan, UT, USA). Cell viability (>95%) and purity (>95%) were confirmed by flow cytometry after cell counting.

NET-related proteins were detected using cellular IF, and the expression levels of NETs were quantified. The cell concentration was adjusted to 5×10⁵/mL. Polylysine-treated cell slides (BOSTER, Wuhan, China) were placed in 24-well plates (NEST, Wuxi, China), and each well was seeded with 1 mL of cell suspension. The plates were then incubated overnight at 37 °C with 5% CO₂. After removing the supernatant, the cells were washed three times with PBS and fixed by adding pre-warmed 4% paraformaldehyde (Biosharp, Beijing, China) at 37 °C. Goat serum was used for blocking. The primary antibody was applied and incubated overnight at 4 °C, followed by incubation with the secondary antibody at 37 °C for 45 min in the dark. Nuclei were stained with DAPI, and the slides were sealed with an anti-fluorescence quenching sealer, following the same procedure as the previous tissue IF staining. Subsequently, the images were examined under a laser confocal microscope at 20× magnification, with excitation wavelengths set to 405 nm for the blue channel and 561 nm for the red channel. The number of neutrophils and NET-related proteins was recorded separately using ZEN software to calculate the formation rate of NETs.

Quantitative analysis of NETs in peripheral blood

The expression levels of NET-related proteins were quantified using a fluorophotometric assay. A 96-well plate (NEST) was utilized, with each well receiving 100 μL of cell suspension, and two replicate wells were set up. SYTOX^TM Green Nucleic Acid Dye (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well to achieve a final concentration of 0.4 μM. The plate was then incubated in the dark at 37 °C in a 5% CO₂ environment for 5 min. Subsequently, the optical density (OD) value was read at 560 nm using a microplate spectrophotometer (Molecular Devices, San Jose, CA, USA).

Co-culture of plasma and neutrophils and detection of NETs formation

Neutrophils from the HC group were co-cultured with plasma from each respective group to evaluate the impact of different plasma inflammatory environments on NETosis. The experiment comprised four distinct groups: a blank group (neutrophil culture medium only), the HC group (plasma from the HC group), the NEOLP group (plasma from the NEOLP group), and the EOLP group (plasma from the EOLP group). Neutrophil suspensions from the HC group were aliquoted into EP tubes (Biosharp, Harjumaa, Estonia) at 1 mL per tube for a total of four tubes. These tubes were centrifuged at 1,000 rpm for 5 min, after which the supernatant was discarded. Subsequently, the corresponding culture medium and plasma were added to the tubes, and the tubes were then incubated overnight at 37 °C in a 5% CO₂ environment.

Both the culture medium and plasma were clear and free of turbidity, and the cells exhibited good condition under an inverted microscope without any signs of contamination. Following centrifugation at 1,000 rpm for 5 min, the supernatant was aspirated into EP tubes and stored in the freezer at −20 °C for subsequent experiments. The cellular fraction was resuspended in 1 mL of culture medium and dispensed into a 96-well plate at 200 μL per well, with two replicate wells for each condition. Subsequently, 20 μL of SYTOX^TM Green fluorescent dye was added to each well, and the plate was incubated at 37 °C with 5% CO₂ for 5 min. The optical density (OD) value was then measured at 560 nm using a microplate spectrophotometer.

Enzyme-linked immunosorbent assay

The levels of IL-17 and TNF-α in the supernatant of each cell group after co-culture were quantified using enzyme-linked immunosorbent assay (ELISA). The supernatants were removed from −20 °C storage to room temperature and subsequently centrifuged at 2,000 rpm for 20 min. A standard curve was prepared as per the kit instructions, and the human IL-17 ELISA kit and human TNF-α ELISA kit (Mlbio, Shanghai, China) were used according to the manufacturer’s instructions. Two replicate wells were established for each sample, and the absorbance readings at 450 nm were recorded using a multifunctional enzyme marker.

Statistical analyses

The IF staining and the cell experiments were conducted in 20 cases. Negative controls without primary antibody were included in each staining batch to assess the specificity and background of the fluorescent signal. Student’s t-test was applied for comparisons between two groups, and ANOVA followed by Tukey’s post-hoc test for multiple comparisons. ImageJ 1.53 m software was used for semi-quantitative analysis of the relative fluorescence intensity of NET-related proteins in each group, and GraphPad Prism version 9 (Boston, CA, USA) was used for statistical analysis and mapping. A p-value of <0.05 was considered statistically significant.

HE staining and IF analysis of NET-related protein in OLP lesions

MPO is located on the azurophilic granules of neutrophils, playing a pivotal role in histone cleavage and chromatin decondensation, thereby contributing to NET formation. This section adopted tissue IF technology to qualitatively assess the expression of NETs in tissues by examining the DNA-MPO complex expression under laser confocal microscope observation (Byrd et al., 2019).

The results from both HE staining and IF staining revealed an obvious increase in MPO-positive staining (depicted in red) within NEOLP and EOLP tissues, co-localizing with DNA structures (depicted in blue) to form MPO-DNA complexes, indicating the presence of NET-related proteins. In contrast, elevated levels of NET-related proteins were not observed in the normal control group. The white arrows show the distribution of NETs. Combined with HE staining, the increased expression of NET-related proteins mainly appeared in the subepithelial and lamina propria regions of the mucosa (Fig. 1A).

Through semi-quantitative analysis of the relative fluorescence intensity of red fluorescence in each group, it was found that the expression of NET-related proteins in the NEOLP and EOLP groups was significantly higher than that in the HC group (p < 0.0001). Moreover, within the EOLP group, the expression was significantly higher than that in the NEOLP group (p < 0.0001) (Fig. 1B).

Qualitative and quantitative analysis of NET-related protein in peripheral blood

The results of cellular IF staining indicated a noticeable presence of MPO-DNA complexes in NEOLP and EOLP tissues, while their expression was not obvious in normal peripheral blood (Fig. 2A). The expression of NET-related proteins in the peripheral blood of NEOLP and EOLP patients was significantly higher than that in normal peripheral blood (p < 0.05). Interestingly, the expressions in the EOLP and NEOLP groups were similar, with no significant difference observed (p > 0.05) (Fig. 2B).

Furthermore, the results obtained from fluorescence spectrophotometry showed that the expression of NET-related cfDNA in the peripheral blood of NEOLP and EOLP patients was significantly higher than that in normal peripheral blood (p < 0.05). The expression of NET-related cfDNA in the peripheral blood of EOLP and NEOLP was similar, with no siginificant difference (p > 0.05) (Fig. 2C).

Fluorescence spectrophotometry of NET-related cfDNA in neutrophils after plasma-cell co-culture

The results of fluorescence spectrophotometry showed that the expression of NET-related cfDNA in the EOLP group was significantly higher than that in the HC group following plasma-cell co-culture (p < 0.05). However, there was no significant difference observed between the NEOLP group, HC group, and EOLP group (p > 0.05). When compared to the blank control group, the expression of NET-related cfDNA in the NEOLP and EOLP groups was significantly higher (p < 0.05), while no significant difference was observed between the HC group and the blank group (p > 0.05) (Fig. 3).

Expression levels of IL-17 and TNF-α in neutrophil supernatant (plasma) of each group

The ELISA results showed that the EOLP group had significantly higher levels of IL-17 and TNF-α compared to the HC and NEOLP groups, with no significant difference observed between the NEOLP and HC groups (Figs. 4A, 4B).

Monotonicity detection conducted using SPSS software showed that NETs had a positive monotonic relationship with IL-17 and TNF-α. Specifically, higher levels of IL-17 and TNF-α corresponded to increased formation of NETs (Figs. 4C, 4D). Spearman correlation analysis further confirmed these findings, with correlation coefficients of 0.568 and 0.655, respectively (p < 0.001), indicating a positive correlation between the formation of NETs and the levels of IL-17 and TNF-α.