Neutrophil extracellular traps mediate the crosstalk between glioma progression and the tumor microenvironment via the HMGB1/RAGE/IL-8 axis

Clinical datasets and tissue specimens

This study included 122 patients with glioma (WHO grade II, n = 37; grade III, n = 30; grade IV, n = 55) who received surgery between January 2012 and December 2019 at the Department of Neurosurgery at the Second Affiliated Hospital of Harbin Medical University. Clinicopathological data were collected as described in our previous study^23–25. Surgically removed samples were confirmed by 2 pathologists. Full pre-treatment blood counts were performed on all patients as well as healthy volunteers (as the control group, n = 30). Informed consent was obtained from patients involved in this study. The study protocol was approved by the Clinical Research Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (Approval No. KY2017-068), and all investigations were conducted in accordance with the Declaration of Helsinki.

Cell culture

The human glioma cell line LN229 was purchased from the Chinese Academy of Sciences Cell Bank. HG7 primary cells were obtained from an adult female patient who previously had GBM²⁴. All tumor cells were cultured in Dulbecco’s modified Eagle’s medium/F12 (Corning, Armonk, NY, USA) supplemented with 10% fetal bovine serum (BD Biosciences, San Jose, CA, USA) and 1% antibiotics (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere with 5% CO₂ and 95% air.

Neutrophils were isolated as described in our previous study¹⁹. Isolated neutrophils were cultured in RPMI 1640 medium (Corning, Armonk, NY, USA) with or without 10% fetal bovine serum. The neutrophil viability was > 95%, as assessed with trypan blue dye exclusion assays, and the neutrophil purity was > 98%, as assessed with Giemsa staining.

Immunohistochemistry (IHC) assay

Briefly, 3-µm paraffin-embedded tissue sections were deparaffinized, rehydrated, treated with 0.3% hydrogen peroxide, and processed for antigen retrieval with heat induction for approximately 10 min²⁶. Primary antibodies to the following proteins were used for IHC staining: CD66b (1:100, Proteintech Group, Chicago, IL, USA), neutrophil elastase (NE) (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom), and IL-8 (1:100, Affinity Biosciences, Cincinnati, OH, USA). Tissues were then incubated with peroxidase conjugated goat anti-mouse/rabbit IgG secondary antibody (ZSGB-bio, Beijing, China). The slides were finally stained with 3,3´-diaminoben-zidine (DAB substrate kit, ZSGB-bio, Beijing, China) and counterstained with hematoxylin. In negative controls, the primary antibody was replaced with phosphate buffered saline. The specimens were analyzed under a light microscope (Nikon, Tokyo, Japan) by pathologists.

Semiquantitative analysis was performed according to IHC scores^24,27. The proportion of stained cell counts per field was scored as 0, 1, 2, and 3 if there was no positive staining, < 10% positive staining, 10%–30% positive staining, and > 30% positive staining, respectively. IHC scoring of cytokine expression was classified into 4 categories (0, 1, 2, and 3) according to the staining intensity (none, weak, moderate, and strong).

Immunofluorescence assays

Tumor cells (LN229 or HG7, 1 × 10⁵) with or without treatments were seeded on coverslips and allowed to grow overnight. Isolated neutrophils (5 × 10⁵) with or without treatments were seeded on coverslips precoated with 0.001% poly-L-lysine for 3 h. Cells were then fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 5 min, blocked with 50% goat serum in phosphate buffered saline for 30 min at 37 °C, and incubated with primary antibody against NE (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom), myeloperoxidase (MPO; 1:100, Proteintech Group, Chicago, IL, USA), citH3 (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom), CXCR2 (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom), Ki67 (1:100, Proteintech Group, Chicago, IL, USA), p65 (1:100, Proteintech Group, Chicago, IL, USA), RAGE (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom), or IL-8 (1:100, Affinity Biosciences, Cincinnati, OH, USA) in blocking buffer overnight at 4 °C. Cells were washed and incubated with Alexa Fluor 594- or 488-conjugated secondary antibody (1:1000, Life Technologies, Waltham, MA, USA). TRITC-labeled Phalloidin (1:200, Yeasen, Shanghai, China) was used to stain the F-actin in the cells. For labeling of DNA, cells were counterstained with DAPI (5 µg/mL, Invitrogen, Carlsbad, CA, USA). The images were acquired with a fluorescence microscope (ECLIPSE Ti, Nikon, Tokyo, Japan).

For immunofluorescence staining of glioma tissues, the tissue sections were deparaffinized, rehydrated, and processed with antigen retrieval as described above. Slides were then incubated with primary antibodies against MPO (1:100, Proteintech, Chicago, IL, USA) and citH3 (1:100, Abcam, Cambridge, Cambridgeshire, United Kingdom) followed by Alexa Fluor 594- or 488-conjugated secondary antibodies (1:1000, Life Technologies, Waltham, MA, USA). Tissue sections were counterstained with DAPI (5 µg/mL, Invitrogen, Carlsbad, CA, USA) and visualized under a fluorescence microscope.

Cell proliferation and colony formation assays

A Cell-Counting Kit 8 (CCK-8; Dojindo Corp., Kumamoto, Japan) was used to test relative cell growth for different time intervals (0, 24, 48, and 72 h) according to the manufacturer’s instructions. The absorbance (optical density at 450 nm) of each 96-well plate was measured with a Tecan infinite F50 microplate reader. For colony formation assays, glioma cells were seeded in 6-well plates (500 cells per well) with the indicated treatments for 2 weeks. Cell colonies were fixed with 4% formaldehyde and stained with 0.1% crystal violet for 10 min, and the number of colonies was counted. All assays were performed in triplicate.

Cell invasion and wound healing assays

Cell invasion assays were performed with the Boyden chamber invasion method²¹. A Boyden chamber (Corning, NY, USA) containing 8 µm pores was coated with Matrigel (Corning, NY, USA). Glioma cells (5 × 10⁴) were seeded into the upper chamber, and PMA-primed NETs-formation neutrophils (2.5 × 10⁵) on coverslips were seeded in the lower chamber with or without the indicated treatments. After incubation in 37 °C under 5% CO₂ and 95% air, the non-invading tumor cells were wiped off the upper surface of the membrane, and the cells on the bottom side of the membrane were fixed in methanol for 10 min, then stained with crystal violet. The invading cells were counted under a light microscope.

For wound healing assays, tumor cells were grown to 70%–80% confluence on a 6-well tissue culture plate. Cell monolayers were manually wounded by scraping cells with a P200 pipette tip. Debris was removed, and the edge of the scratch was smoothed by washing the cells once with growth medium. Cells were then cultured in culture medium containing a lower percentage of serum (1%) to minimize the effect of cell proliferation on migration, and were then treated as indicated. The method was consistent with that in previous study^28,29. The wound width was measured at different time intervals (12, 24, or 48 h) to evaluate the wound healing ability of glioma cells.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted with TRIzol^® reagent (Life Technologies, Waltham, MA, USA). The cDNAs were synthesized with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Conventional PCR was performed with a Bio-Rad system, and 1% agarose gel electrophoresis was used to detect the PCR products. The primer sequences used were as follows:

IL-8 forward: 5′-CTGCGCCAACACAGAAATTA-3′

IL-8 reverse: 5′-CATCTGGCAACCCTACAACA-3′

β-actin forward: 5′-CTCGCCTTTGCCGATCC-3′

β-actin reverse: 5′-GAATCCTTCTGACCCATGCC-3′.

β-actin was used as the reference control.

Western blot

Western blot assays were performed with standard procedures³⁰. Briefly, cells with or without treatments were harvested and lysed with RIPA buffer (Beyotime, Shanghai, China) with the proteinase inhibitor PMSF (1 mM) and a phosphatase inhibitor cocktail (1 mM, Roche, Basel, Switzerland). The protein concentration was detected with a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins (20 µg/sample) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After being blocked with 5% BSA in TBS-Tween, the membranes were incubated with primary antibodies overnight at 4 °C, followed by horseradish-peroxidase-conjugated secondary antibodies (1:4000, ZSGB-BIO, Beijing, China). Primary antibodies including anti-Cyclin D1 (Proteintech Group, Chicago, IL, USA), anti-Cyclin D2 (Proteintech Group, Chicago, IL, USA), anti-phospho-ERK1/2 (Cell Signaling Technology, CST, Massachusetts, USA), ERK1/2 (CST, Massachusetts, USA), anti-phospho-PI3K (CST, Massachusetts, USA), anti-PI3K (CST, Massachusetts, USA), anti-phospho-AKT (CST, Massachusetts, USA), anti-AKT (CST, Massachusetts, USA), and anti-β-actin (ZSGB-BIO, Beijing, China) were used at a dilution of 1:1000. Specific bands were detected with ECL Western blot substrate (Beyotime, Shanghai, China) with a ChemiDoc^TM MP Imaging System (Bio-Rad, Berkeley, CA, USA).

Flow cytometry

Flow cytometry assays were conducted to detect relative changes in ROS in neutrophils^19,31. Briefly, isolated neutrophils were resuspended in Hank’s solution (Solarbio, Beijing, China) at a concentration of 1 × 10⁶ cells/mL, and loaded with the fluorescent probe 2, 7-dichlorodihydrofluorescein diacetate (DCFH2-DA, 10 µM, Beyotime, Shanghai, China). After 3 washes to remove unloaded probe, cells were incubated with the indicated treatments. DCF relative fluorescence intensities in cells were quantified with flow cytometry (Becton Dickinson FACSCanto™ II, USA). Data were obtained in BD FACSDiva Software and analyzed in FlowJo X Software.

Chemotaxis assays

Chemotaxis of isolated neutrophils was assayed in triplicate with a modified Boyden chamber with 8.0 µm pores (Corning, NY, USA). Freshly isolated neutrophils (1 × 10⁶ cells/mL) were resuspended in chemotaxis buffer (RPMI 1640, Corning, Armonk, NY, USA, with 10 mM HEPES and 0.5% BSA). Then 100 µL of neutrophils was placed in the upper chamber, and the lower chamber was filled with 500 µL of chemokine buffer [NETs-primed tumor cell conditioned medium (CM) or different concentrations of IL-8]. Cells that migrated into the lower chamber were manually quantified with a Neubauer improved cell counting chamber. The chemotactic index was evaluated as the ratio of migrated cells in each experimental condition to those in control buffer.

Enzyme-linked immunosorbent assay (ELISA)

Cytokine levels in conditioned medium were determined with a quantitative sandwich enzyme immunoassay technique (IL-8 ELISA kit, Jianglai, Shanghai, China) according to the manufacturer’s instructions. Briefly, CM without phenol red was centrifuged for 20 min at 1,000 g. Then 50 µL of standard solution or 10× diluted samples were pipetted into the wells of 96-well plates precoated with anti-IL-8 monoclonal antibody, and a horseradish-peroxidase-labeled detection antibody was added to the wells. 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was used to develop the color, and reactions were stopped with stop solution. The optical density was acquired with a microplate reader at a wavelength of 450 nm. Results were calculated according to the standard curve.

Statistical analysis

Statistical results are presented as the mean ± SD. Student’s t-test was used to assess differences in the variable groups. Overall survival curves were used to describe the survival distributions, and the log-rank test was used for comparison. Chi-squared test was used to evaluate statistical differences in IHC staining of cytokines between groups. R packages, such as ggplot2 and KMsurv were used to produce figures. All statistical analyses were performed in SPSS 19.0 software (SPSS, Chicago, IL, USA) or GraphPad Prime 8.0 (La Jolla, CA, USA). P values less than 0.05 were considered statistically significant.

Neutrophil infiltration and NETs formation are induced in high-grade glioma

Activation and recruitment of neutrophils to tumor sites have been considered to correlate with greater tumor malignancy in several types of cancer^32,33. In the present study, we examined the relationship between circulating neutrophil counts and glioma malignancy. A total of 122 patients with glioma (WHO grade II, n = 37; grade III, n = 30; grade IV, n = 55) and 30 healthy volunteers were enrolled in our study. We found that the number of circulating pretreatment leukocytes was significantly higher in patients with glioma than in people in the healthy control group, and the highest level was found in grade IV (Figure 1A, Healthy vs. Grade IV: P = 1.28E-10, Grade II vs. Grade IV: P = 3.81E-09, Grade III vs. Grade IV: P = 4.97E-03). Given that the elevation in resting white blood cells was unchanged, the elevated number of circulating neutrophils primarily contributed to this phenomenon (Figure 1B, Healthy vs. Grade IV: P = 2.90E-10, Grade II vs. Grade IV: P = 1.34E-09, Grade III vs. Grade IV: P = 2.51E-03). To further evaluate the correlation between pretreatment neutrophil counts and the prognosis of glioma patients, we estimated the survival of glioma patients with different neutrophil to lymphocyte ratio (NLR). Patients with high NLR (≥ 4) exhibited a poorer survival rate than those with low NLR (< 4) (Figure 1C, P = 1.40E-02). We also found a positive correlation between the circulating neutrophil count and the infiltrated neutrophil scores (Figure 1D, IHC score 0 vs. IHC score 3: P = 4.28E-03, IHC score 1 vs. IHC score 3: P = 3.92E-02, IHC score 2 vs. IHC score 3: P = 1.68E-02).

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Figure 1

The infiltration of neutrophils and the formation of neutrophil extracellular traps (NETs) are associated with the malignancy of gliomas. (A) White blood cell counts of samples from healthy patients and patients with different WHO grades of gliomas. (B) Neutrophil cell counts of samples from healthy patients and patients with gliomas in different grades. (C) Kaplan–Meier survival analysis of patients with low neutrophil to lymphocyte ratio (NLR) and those with high NLR. (D) Neutrophil cell counts of samples with different immunohistochemistry (IHC) scores of NE. (E) IHC assays of CD66b, NE, and MPO in patients with different grades of gliomas (left panel), and corresponding IHC scores (right panel). (F) Immunofluorescence assays of MPO and citH3 in patients with different WHO grades of glioma. Significant results are presented as * P < 0.05, ** P < 0.01, or *** P < 0.001.

To clarify the involvement of neutrophils in promoting glioma progression, we analyzed the infiltration of neutrophils in glioma tissue by using IHC. CD66b, NE and MPO, defined as neutrophil markers, had elevated expression in grade IV glioma tissues compared with grade II or grade III glioma tissues (Figure 1E, CD66b: Grade II vs. Grade III: P = 3.00E-04, Grade II vs. Grade IV: P = 2.00E-04, Grade III vs. Grade IV: P = 7.30E-03; NE: Grade II vs. Grade III: P = 2.46E-02, Grade II vs. Grade IV: P = 2.90E-03, Grade III vs. Grade IV: P = 4.88E-02; MPO: Grade II vs. Grade III: P = 6.90E-03, Grade II vs. Grade IV: P = 6.00E-04, Grade III vs. Grade IV: P = 1.51E-02). Immunofluorescence analysis revealed higher levels of NETs in grade IV glioma tissues than in grade II or grade III glioma tissues, as determined by staining for MPO and citrullinated histone H3 (citH3) (Figure 1F). These data indicated that neutrophil infiltration and NETs formation were significantly associated with increased glioma malignancy.

NETs secreted by TINs are involved in glioblastoma cell proliferation and invasion

NETs are mesh-like structures extruded by activated neutrophils in certain conditions. Fresh neutrophils isolated from healthy volunteers were primed with phorbol myristate acetate (PMA, 30 nM) to produce NETs (Figure 2A), and the formation of NETs was confirmed by immunofluorescence staining (Figure 2B). To explore the potential implications of NETs in glioma progression, we then co-cultured neutrophils, NETs, or NETs and DNase I (100 U/mL, Sigma, Saint Louis, MO, USA) with glioma cells (LN229). In CCK-8 assays, NETs significantly promoted the glioma cell proliferation rate, and this effect was abrogated in the NETs and DNase I group, because the NETs were digested by DNase I (Figure 2C, Control vs. NETs: P = 4.85E-05, Neutrophil vs. NETs: P = 1.94E-04, NETs combined with DNase I vs. NETs: P = 3.49E-04). In agreement with this effect, NETs incubation elevated the expression of the proliferation markers Ki67, Cyclin D1, and Cyclin D2 in glioma cells (Figure 2D and 2E). Colony formation assays also indicated an oncogenic effect of NETs on glioma cells (LN229 and HG7) (Figure 2F, LN229: Control vs. NETs: P = 3.87E-03, NETs combined with DNase I vs. NETs: P = 3.16E-03; HG7: Control vs. NETs P = 4.30E-02, NETs combined with DNase I vs. NETs: P = 3.79E-02). Wound healing assays demonstrated that NETs enhanced the migration ability of glioma cells (Figure 2G). To investigate the effect of NETs on glioma cell invasiveness, we performed Transwell assays by seeding PMA-primed neutrophils in the lower chamber, to form NETs, and glioma cells in the upper chamber. As shown in Figure 2H, NETs significantly promoted tumor cell invasiveness, and this phenomenon was suppressed by NETs digestion with DNase I (LN229: 12 h, Control vs. NETs: P = 8.54E-01, NETs combined with DNase I vs. NETs: P = 7.68E-01, 24 h, Control vs. NETs: P = 8.16E-01, NETs combined with DNase I vs. NETs: P = 8.59E-01, 48 h, Control vs. NETs: P = 6.58E-04, NETs combined with DNase I vs. NETs: P = 3.21E-03; HG7: 12 h, Control vs. NETs: P = 6.53E-01, NETs combined with DNase I vs. NETs: P = 8.15E-01, 24 h, Control vs. NETs: P = 8.33E-01, NETs combined with DNase I vs. NETs: P = 9.07E-01, 48 h, Control vs. NETs: P = 5.59E-04, NETs combined with DNase I vs. NETs: P = 1.44E-03).

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Figure 2

Neutrophil extracellular traps (NETs) promote proliferation, migration, and invasion of glioblastoma cells. (A) Isolation of neutrophils from blood samples of healthy volunteers. (B) Immunofluorescence assay of the confirmation of NETs formation. (C) CCK-8 assays of glioma cells treated with neutrophils, NETs, or NETs combined with DNase I (NETs & DNase I). (D) Immunofluorescence assays of Ki67 in glioma cells treated with or without NETs. (E) Western blot validation of cyclin D1 and cyclin D2 in LN229 and HG7 cells treated with or without NETs. (F) Colony formation assays of LN229 and HG7 cells treated with NETs or NETs combined with DNase I (NETs & DNase I). The statistics are shown in the histogram. (G) Wound-healing assays of LN229 cells treated with or without NETs. (H) Transwell invasion assays of LN229 and HG7 cells treated with NETs or NETs combined with DNase I (NETs & DNase I). The statistics are shown in the histogram. Significant results are presented as * P < 0.05, ** P < 0.01, or *** P < 0.001. Non-significant results are presented as ns.

HMGB1 derived from NETs binds to RAGE and activates the NF-κB signaling pathway in glioblastoma

NETs formation can result in the extracellular exposure of granular and nuclear proteins, of which HMGB1 is considered a key mediator in chronic inflammation process^34,35. HMGB1 has also been reported to be involved in stem cell proliferation and migration^36,37. Immunofluorescence staining results demonstrated that the mesh-like structures of NETs were decorated with HMGB1 (Figure 3A). Because RAGE is the major receptor for HMGB1 in mediating chronic sterile inflammation³⁸, we proposed that NETs might exert oncogenic effects in an HMGB1/RAGE dependent manner in glioma. As shown in Figure 3B, RAGE expression was detected in glioma tissues by IHC, and intense RAGE staining was found in high-grade glioma. We then co-cultured NETs and LN229 cells with or without RAGE inhibitor (FPS-ZM1, 10 µM, Merck, Darmstadt, Germany) to block RAGE’s binding ability, and assessed the cell proliferation rate with CCK-8 assays. We found that FPS-ZM1 clearly suppressed NETs-induced tumor cell proliferation (Figure 3C, Control vs. NETs: P = 1.84E-05, FPS-ZM1 combined with NETs vs. NETs: P = 6.58E-04).

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Figure 3

Neutrophil extracellular traps (NETs) mediate the malignancy of glioma cells via the HMGB1/RAGE/NF-κB axis. (A) Identification of HMGB1 by immunofluorescence assays on NETs. (B) The different RAGE expression in low-grade gliomas and high-grade gliomas. (C) CCK-8 assay of gliomas treated with NETs or NETs combined with FPS-ZM1 (FPS-ZM1 & NETs). (D) Immunofluorescence assays of HMGB1 and RAGE in glioma cells treated with NETs or recombinant HMGB1. (E) Expression of p-ERK1/2, t-ERK1/2 and p-IκB in glioma cells treated with recombinant HMGB1 for different time periods. (F) Expression of p-ERK1/2, t-ERK1/2, and p-IκB in glioma cells treated with NETs, recombinant HMGB1 or FPS-ZM1. (G) Nuclear translocation of NF-κB in LN229 and HG7 cells treated with NETs or NETs combined with FPS-ZM1 (FPS-ZM1 & NETs). Significant results are presented as *** P < 0.001.

To further demonstrate the binding of HMGB1 to RAGE on glioma cells, we incubated LN229 cells with NETs or recombinant HMGB1 (1 µg/mL, Sigma, St. Louis, MO, USA). The binding ability was confirmed by immunofluorescence staining (Figure 3D). We found that recombinant HMGB1 treatment increased the phosphorylation levels of extracellular regulated protein kinases (ERK1/2) and inhibitor of kappa-B (IκB) from 0.5 h after treatments in LN229 cells (Figure 3E). Moreover, phosphorylation of ERK1/2 and IκB mediated by NETs or HMGB1 was abrogated by blocking RAGE on glioma cells (Figure 3F). These effects were further examined by detecting NF-κB nuclear translocation through immunofluorescence staining (Figure 3G).

NETs stimulate the NF-κB signaling pathway to promote IL-8 secretion in glioblastoma

Aberrant activation of chronic inflammatory responses in the TME is a hallmark of tumor progression³⁹. NF-κB acts as a key transcription factor for cytokines that mediate tumor glioma progression, including IL-8, which is a proinflammatory CXC chemokine mainly responsible for the recruitment of neutrophils^40,41. Analysis of the IL-8 expression levels in The Cancer Genome Atlas (TCGA) glioma RNA-seq dataset (http://cancergenome.nih.gov/) showed that GBM and IDH wild-type patients had higher IL-8 expression levels than those in other groups (Figure 4A). Accordingly, the Kaplan–Meier survival curve indicated that patients with high-grade glioma have shorter survival times (Figure 4B, Low expression vs. High expression: P = 8.18E-13). The IL-8 expression in glioma tissues was also detected. As shown in Figure 4C and 4D, patients with high-grade glioma exhibited significantly elevated levels of IL-8 staining (Low vs. High: P = 1.00E-06). To confirm that the elevated IL-8 was induced by NETs-primed glioma cells, we co-cultured LN229 cells with neutrophils or NETs, and detected IL-8 expression in CM by ELISA. The data clearly showed that LN229 cells co-cultured with NETs elevated IL-8 concentration, as compared with those co-cultured with neutrophils or control group (normal LN229 cell culture medium) (Figure 4E, Control vs. NETs: P = 8.36E-05, Neutrophil vs. NETs: P = 8.86E-05, Control vs. Neutrophil: P = 1.79E-01). To further investigate the involvement of the RAGE/ERK/NF-κB axis in IL-8 production, we used FPS-ZM1, U0126 (10 µM, ERK1/2 inhibitor, Sigma, St. Louis, MO, USA) and JSH-23 (25 µM, NF-κB inhibitor, Hycultec GmbH, Beutelsbach, Germany) to inhibit RAGE, ERK, and NF-κB activation, respectively. We found that the inhibition of the RAGE/ERK/NF-κB axis significantly decreased the NETs or HMGB1 induced IL-8 production in glioma cells at both mRNA and protein levels (Figure 4F and G, Control vs. HMGB1: P = 2.26E-06, HMGB1 combined with FPS-ZM1 vs. HMGB1: P = 1.15E-05, HMGB1 combined with U0126 vs. HMGB1: P = 9.89E-05, HMGB1 combined with JSH-23 vs. HMGB1: P = 1.04E-04). These results indicated that HMGB1 in NETs induced IL-8 secretion through the RAGE/ERK/NF-κB axis in glioma cells.

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Figure 4

The IL-8 secretion of glioma cells is promoted via the HMGB1/RAGE/NF-κB axis. (A) IL-8 expression in different types of gliomas in the TCGA RNA-seq dataset. (B) Kaplan–Meier survival curve of TCGA glioma patients with low or high expression of IL-8. (C) Immunohistochemistry (IHC) assays of IL-8 in patients with low-grade or high-grade gliomas. (D) IHC score of IL-8 in low-grade or high-grade gliomas. (E) IL-8 secretion detected with ELISA in glioma cells treated with neutrophils or NETs. (F) The RNA level of IL-8 in LN229 cells treated with NETs, recombinant HMGB1, FPS-ZM1, U0126, or JSH-23. (G) IL-8 secretion detected with ELISA in glioma cells treated with or without FPS-ZM1, U0126, or JSH-23, followed by recombinant HMGB1 treatment (&FPS-ZM1, &U0126, or &JSH-23). Significant results are presented as *** P < 0.001, or ### P < 0.001. Non-significant results are presented as ns.

IL-8 recruits neutrophils and mediates NETs formation via the PI3K/AKT/ROS axis in TINs

Chemotaxis assays were performed to determine whether the infiltration of neutrophils in glioma tissue was due to the attraction of IL-8. NETs-primed LN229 cell CM exhibited a higher ability to attract neutrophils compared to the control group (cell culture medium only) (Figure 5A, control vs. CM: P = 7.17E-07, CM vs. CM combined with anti-IL-8: P = 3.86E-06, CM vs. CM combined with IgG: P = 5.92E-01). This effect was significantly repressed by the addition of anti-IL-8 neutralizing antibody into the CM, whereas the addition of isotype control antibody had no suppressive effect (Figure 5A). The neutrophil attraction effect of IL-8 was further confirmed that with higher concentrations of recombinant IL-8, the chemotaxis index was upregulated [Figure 5B, Control vs. IL-8 (200 pg/mL): P = 7.73E-03, Control vs. IL-8 (400 pg/mL): P = 1.09E-03, Control vs. IL-8 (800 pg/mL): P = 4.03E-06, Control vs. IL-8 (1600 pg mL): P = 1.06E-06]. CXCR2, a cell surface receptor of IL-8⁴², was primarily expressed on myeloid lineage cells, including neutrophils in the Protein Atlas dataset (https://www.proteinatlas.org) (Figure 5C). With freshly isolated neutrophils incubated with recombinant IL-8, we found that IL-8 colocalized with CXCR2 on neutrophils (Figure 5D).

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Figure 5

IL-8 recruits neutrophils and mediates neutrophil extracellular traps (NETs) formation. (A) Recruitment of neutrophils detected with chemotaxis assays with NETs-primed conditioned medium (CM), CM combined with anti-IL-8 antibody (CM & anti-IL-8), or CM combined with isotype IgG (CM & IgG). (B) Recruitment of neutrophils, detected with chemotaxis assays with different concentrations of IL-8. (C) Expression levels of CXCR2 in different cell lines. (D) Binding affinity of IL-8 and CXCR2 on neutrophils, detected with immunofluorescence assays. (E) Expression of p-PI3K and t-PI3K in neutrophils treated with IL-8 or IL-8 combined with SB225002 (200 nM, CXCR2 inhibitor, Calbiochem). (F) Expression of p-AKT and t-AKT in neutrophils treated with IL-8, SB225002, or LY294002 (5 µM, PI3K inhibitor, EMD, Billerica, MA). (G) ROS activity in neutrophils treated with CM, IL-8, IL-8 combined with SB225002 (SB225002 & IL-8), IL-8 combined with LY294002 (LY294002 & IL-8), IL-8 combined with AKT inhibitor 1/2 (Akt inh. & IL-8; Akt inh., 10 μM, Calbiochem, San Diego, CA), IL-8 combined with DPI (DPI & IL-8; 10 μM, NADPH oxidase inhibitor, Sigma, Saint Louis, MO, USA), or PMA (30 nM). (H) Detection of NETs formation by immunofluorescence in neutrophils treated with CM, IL-8, IL-8 combined with SB225002 (SB225002 & IL-8), IL-8 combined with LY294002 (LY294002 & IL-8), IL-8 combined with AKT inhibitor 1/2 (Akt inh. & IL-8), IL-8 combined with DPI (DPI & IL-8), or PMA. Significant results are presented as *** P < 0.01. Non-significant results are presented as ns.

By incubating recombinant IL-8 with freshly isolated neutrophils, we observed elevated PI3K phosphorylation in neutrophils, and this effect was clearly abrogated by blocking CXCR2 (Figure 5E). In agreement with these results, we identified that IL-8 incubation led to the phosphorylation of AKT, a key mediator downstream of the PI3K signaling pathway (Figure 5F). We further explored the effect of NETs-primed glioma cell CM and recombinant IL-8 on ROS production in freshly isolated neutrophils. As shown in Figure 5G, the CM or IL-8 incubation resulted in robust production of ROS in neutrophils, and inhibition of the CXCR2/PI3K/AKT axis or NADPH oxidase reversed this effect, thus indicating their involvement in IL-8-induced ROS production. We also found that incubating NETs-primed glioma cell CM or recombinant IL-8 with freshly isolated neutrophils resulted in NETs formation, and this effect was suppressed by inhibition of CXCR2 or the PI3K/AKT/ROS axis (Figure 5H). These results advanced understanding of NETs in the crosstalk between glioma progression and the TME by regulating the HMGB1/RAGE/IL-8 axis (Figure 6).

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Figure 6

NETs play a role in the crosstalk between glioma progression and tumor microenvironment by regulating the HMGB1/RAGE/IL-8 axis is shown.