Neutrophil-Derived IL-17 Promotes Ventilator-Induced Lung Injury via p38 MAPK/MCP-1 Pathway Activation

Ventilator-induced lung injury (VILI) is one of the most common complications of mechanical ventilation and can severely affect health. VILI appears to involve excessive inflammatory responses, but its pathogenesis has not yet been clarified. Since interleukin-17 (IL-17) plays a critical role in the immune system and the development of infectious and inflammatory diseases, we investigated here whether it plays a role in VILI. In a mouse model of VILI, mechanical ventilation with high tidal volume promoted the accumulation of lung neutrophils, leading to increased IL-17 levels in the lung, which in turn upregulated macrophage chemoattractant protein-1 via p38 mitogen-activated protein kinase. Depletion of neutrophils decreases the production IL-17 in mice and inhibition of IL-17 significantly reduced HTV-induced lung injury and inflammatory response. These results were confirmed in vitro using RAW264.7 macrophage cultures. Our results suggest that IL-17 plays a pro-inflammatory role in VILI and could serve as a new target for its treatment.


INTRODUCTION
Mechanical ventilation is widely used to support patients with acute respiratory failure and other severe diseases. However, it is associated with ventilator-induced lung injury (VILI), a syndrome that can cause or exacerbate lung injury through volu-, baro-and biotrauma (1)(2)(3). VILI can lead to overwhelming inflammatory responses, such as activation of the innate immune system and release of inflammatory molecules, which increase alveolar edema and pulmonary vascular permeability (4). We previously found that alveolar macrophages (AM) played an important role in promoting inflammation in the occurrence and development of VILI-mediated lung inflammation (5). VILI-induced inflammation and the release of inflammatory factors appear to involve phosphorylation of p38 mitogen-activated protein kinase (MAPK) (6), but many details of the processes behind VILI remain unclear.
Interleukin (IL)-17, a pro-inflammatory cytokine that was first detected in human peripheral blood (7,8), contributes to inflammatory, tumor, and autoimmune diseases (9), as it can induce the production of chemokines and inflammatory molecules such as tumor necrosis factor-a, thereby activating innate immune responses and recruiting immunecells (10). Increased IL-17 levels in the serum, sputum, and broncho alveolar lavage fluid (BALF) of asthmatic patients positively correlate with disease severity (11)(12)(13). Moreover, IL-17 was found to aggravate lung inflammation and reduce lung function during H1N1 influenza virus infection and Klebsiella pneumoniae-induced pneumonia (14, 15).
The p38 MAPK, a member of the MAPK superfamily, is activated by various pro-inflammatory and stressful stimuli, mediating the release of inflammatory cytokines involved in the regulation of various diseases (16)(17)(18)(19). Macrophage chemoattractant protein-1 (MCP-1), also known as C-C chemokine ligand 2 (CCL2), is an important downstream molecule of p38 MAPK activation that modulates the recruitment of inflammatory cells into damaged organs and tissues (20). MCP-1 may also exacerbate lung injury in patients with polycystic kidney disease and acute respiratory distress syndrome (21,22).
In this study, we aimed to determine whether IL-17 plays a role in VILI. In particular, we asked whether IL-17 influences VILI by activating p38 MAPK/MCP-1 signaling.

Animals
Male C57BL/6 mice aged 6-8 weeks with a body weight of 22 ± 5 g were purchased from the Experimental Animal Center of Guangxi Medical University. All mice were raised at 20-25°C and 30-70% relative humidity and provided with proteincontaining feed and purified water. The study was approved by the Experimental Animal Committee of Guangxi Medical University, and all animal experiments were performed in accordance with local and international ethical guidelines. Ageand weight-matched controls were used in all experiments.

Mouse Model of VILI
Before tracheal incubation, all mice were fasted for 8h and left without water for 4 h. Mice were then anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg); they were administered in 1/3 of the first dose to maintain anesthesia every 45 min. A sterile 20G intravenous trocar was used for tracheal intubation at a depth of 1.2 ± 0.5 cm. The catheter was fixed in the trachea to prevent one-lung ventilation and was connected to a small-animal ventilator (catalog no. SAR-1000, CWE) for mechanical ventilation with air. Nonventilated mice were used as controls, while mechanically ventilated mice were ventilated with either normal tidal volume (NTV, 7 mL/kg) or high tidal volume (HTV, 20 mL/kg).
To explore the source of IL-17 in the mice treated with HTV, the mice were injected intraperitoneally with anti-Ly6G antibody (400 µg; BP0075, BioXCell) one day before ventilation to deplete neutrophils in vivo. To assess the role of IL-17 in the p38 MAPK/ MCP-1 pathway after HTV ventilation, mice were randomly divided into four groups (H1-4) depending on whether ventilation lasted 1, 2, 3, or 4 h. One day before ventilation, the mice were injected intraperitoneally with anti-IL-17 antibody (400 µg; BE0173, BioXCell), and at 30 min before anesthesia 200 µg anti-IL-17 antibody was administered through intratracheal instillation. Recombinant mouse IL-17 (rmIL-17; 0.5 mg, catalog no. 576006, Biolegend) was administered through intratracheal instillation 30 min before anesthesia. HTV-ventilated mice administered with saline served as controls.

Cell Strains
RAW264.7 mononuclear macrophage leukemia cells were purchased from the American Type Culture Collection. Cells were grown in Dulbecco's Modified Eagle's medium (DMEM; catalog no. C11995500BT, Gibco), and the optimal infection conditions and multiplicity of infection were determined. In the optimized procedure, a 500 mL suspension of RAW246.7 cells and 20 mL of HitransG A infection-enhancing solution were added to each well of a 24-cell well plate. After cells reached 50% confluence, they were co-cultured with 20 mL of HitransG P infection-enhancing solution and lentivirus infection solution. After 16 h, the medium was replaced with fresh DMEM medium. At 72 h after transfection, the transfection efficiency was determined by fluorescence microscopy. Transfected cells were selected using 2 mg/mL puromycin, which was reduced to 1 mg/ mL for further screening.
To clarify the mechanism of IL-17 in vitro, wild-type RAW264.7 cells were stimulated with rmIL-17(100 ng/mL) and cultured for 1, 2, 3, 4, or 5 h. Non-stimulated wild-type RAW264.7 cells served as controls. RAW264.7 cells transfected with negative-control lentivirus and stimulated with rmIL-17 for 4 h are referred to below as the CN group. RAW264.7 cells transfected with lentivirus encoding short hairpin RNA (shRNA) against p38 MAPK (Shanghai Jikai Gene Technology, Shanghai, China) and cultured with rmIL-17 for 4 h are referred to below as the shRNA group.

Inflammatory Response
After the animal model was established, all mice were sacrificed by carotid artery bleeding, and their lungs were excised and weighed immediately (wet weight) and again after drying in an oven at 60°C for 72 h (dry weight). All operations were conducted carefully to minimize the risk of inflammatory activation.
The total protein concentration in BALF was estimated using the bicinchoninic acid (BCA) assay (P0012S, Beyotime) to assess pulmonary permeability. The levels of IL-17 and MCP-1were assayed in BALF and culture medium were detected using the ELISA kits (CSB-E04608m, Cusabio Biotech and EK0568, Boster) according to the manufacturers' instructions.

Histopathological Analysis
The pathological damage in mouse lungs after HTV ventilation was assessed after hematoxylin-eosin (H&E) staining. Briefly, the collected mouse lungs were fixed with 3.7% paraformaldehyde, embedded in paraffin, and cut into 5-mm sections. After dewaxing, the sections were stained and the tissue morphology was observed with a microscope. The degree of lung injury was evaluated by applying a four-point scale (0, no injury; 1, minor injury; 2, moderate injury; 3, severe injury) to each of the following parameters (6,23,24): alveolar congestion, hemorrhage, inflammatory cell infiltration, and alveolar wall thickening. These individual scores were summed to give the total score of lung damage.

Flow Cytometry
Flow cytometry was used to detect the accumulation of neutrophils in the lung tissues of mice and the expression of IL-17 in neutrophils, Th17 cells, and T cells. Lung sections were treated with 200 U/mL Type I collagenase (950 mL, 17018029, Gibco), DNaseII(20 U,4942078001, Merck), and FBS (50 mL,10099141, Gibco)to prepare single-cell suspensions. Red blood cell lysis solution (1mL) was then added to each cell suspension to remove red blood cells and adjust the density to 5 × 10 6 mL −1 . Cells were labelled using antibodies against Ly6G (catalog no.REA256, Miltenyi Biotec), CD4 (catalog no. GK1.5, Miltenyi Biotec), and CD90 (catalog no. 105201, Biolegend). After washing with PBST buffer, cells were ruptured and intracellular proteins were labelled for 20 min using anti-IL-17 antibody (catalog no. 560522, BD Horizon) and analyzed at 4°C using a flow cytometer.

Statistical Analysis
Statistical analysis was performed using SPSS 22.0 software (IBM, USA). All data were reported as mean ± standard deviation (SD). Differences in parametric data that showed a normal distribution were assessed for significance using one-way analysis of variance and the Bonferroni test. Differences associated with a two-tailed P < 0.05 were considered statistically significant.

HTV Ventilation Upregulates IL-17 in Lung Tissues
To assess the effect of HTV ventilation on IL-17 expression, we determined the levels of IL-17 in non-ventilated, NTV, and HTV ventilated mice. The IL-17 levels in lungs were significantly higher in the HTV group than in the control and NTV groups ( Figures 1A-C). Compared with CON group, the concentration of IL-17 in BALF also increased in HTV group ( Figure 1D). These results suggest that IL-17 is closely associated with the development of VILI.

IL-17 Is Mainly Produced by Lung Neutrophils After HTV Ventilation
IL-17 is expressed mainly by Th17 cells and neutrophils (25,26). Flow cytometry showed no significant difference in the numbers of CD4 + IL-17 + cells among control, NTV and HTV groups ( Figure 2A). IL-17 in the HTV group did not co-localize with the Th17 cell surface marker CD4 or the T cell surface marker CD90 ( Figure 2B), suggesting that IL-17 is not produced by Th17 cells and T cells during VILI. In contrast, the number of Ly6G + neutrophils was significantly higher in the HTV group than in the other two groups ( Figure 3A). Immunofluorescence shows that IL-17 co-localizes with the neutrophils surface marker Ly6G ( Figure 3B). Furthermore, when neutrophils were depleted in mice ( Figures 3C, F), compared with the  HTV group, the levels of IL-17 in lung tissue ( Figure 3D), BALF ( Figure 3E) and plasma ( Figure 3G) were significantly reduced in the HTV+anti-Ly6G group. These results indicate that IL-17 is produced mainly by Ly6G + neutrophils that are recruited to the lungs at the onset of VILI.

IL-17 Inhibition Attenuates VILI-Induced Inflammation
The wet weight to dry weight ratio (W/D) of mouse lungs reflects the degree of lung edema, as the fluid that accumulates in the lung interstitium, alveolar cavity, and small bronchi increases lung tissue wet weight. Moreover, lung inflammation leads to the secretion of several inflammatory molecules in the alveolar cavity. We took advantage of these phenomena to assess whether inhibiting the function of IL-17 using an anti-IL-17 antibody ( Figure 4A) would reduce pulmonary edema and total protein levels in BALF. Indeed, treating the HTV group with anti-IL-17 antibody led to significantly lower pulmonary edema and total BALF protein ( Figures 4B, C), as well as milder alveolar congestion, alveolar interstitial thickening, and neutrophil infiltration ( Figure 4D). These results suggest that blocking IL-17 can mitigate inflammation caused by VILI.

IL-17 Upregulates MCP-1 in Mouse Lung
Tissues via the p38 MAPK Pathway To explore whether the observed effects of IL-17 on the p38 MAPK/MCP-1 pathway in vitro also occur in vivo, HTV ventilated mice were injected with anti-IL-17 antibody orrmIL-17 before ventilation. Anti-IL-17 antibody significantly reduced MCP-1 and p-p38 MAPK expression ( Figures 6A-C), whereas rmIL-17 exerted the opposite effects ( Figures 6D, E). At the same time, neither treatment affected the total level of p38 MAPK in lung or BALF (Figures 6C, E). These results confirm that IL-17 can upregulate MCP-1 by promoting the phosphorylation of p38 MAPK.

DISCUSSION
VILI is a pathophysiological process that leads to overwhelming inflammatory responses when alveolar macrophages, neutrophils, alveolar epithelial cells, and endothelial cells migrate to the lungs (27)(28)(29) and when pro-inflammatory cytokines are expressed. Current treatment methods for VILI are mainly based on regulating the tidal volume, oxygen concentration, and positive end-expiratory pressure of the  ventilator (1,30,31). However, these therapies generally show low efficacy, highlighting the need to clarify the underlying mechanism of VILI in order to identify more effective targets.
In this study, we explored the role of IL-17 and the p38 MAPK/MCP-1 pathway in the pathogenesis of VILI. In our experiments in vitro and in vivo, HTV ventilation significantly increased the number of lung neutrophils, which secreted large amounts of IL-17, and this cytokine in turn upregulatedMCP-1 via the p38 MAPK pathway. Inhibition of IL-17 significantly reduced lung injury caused by HTV mechanical ventilation, suggesting that IL-17 may be a promising new target for the treatment of VILI-induced inflammation.  IL-17 receptors contain a conserved SEFIR domain, which binds to Act1 to activate downstream signal cascades (32,33). Downstream targets of IL-17 include the NF-kB signaling pathway (34,35), three MAPK pathways (JNK, ERK, and p38), and the phosphoinositide kinase pathway (36)(37)(38). Our findings about the role of IL-17 in VILI extend the list of conditions in which the cytokine can contribute to excessive inflammation, which include bacterial and fungal infections (39,40). In fact, in certain cases of bacterial infection, higher levels of IL-17 may be beneficial to eliminate pathogens (41)(42)(43). Thus, whether elevated IL-17 levels lead to injury or benefit may depend on the cells secreting the cytokine and on other factors.
Inflammatory cells infiltrate into affected tissues, where they trigger local immune responses. Neutrophils, a type of polymorphonuclear leukocytes, are the first inflammatory cells recruited to inflammatory sites, where they help eliminate pathogens through multiple mechanisms (44)(45)(46). Although neutrophils are short-lived, they can be activated several times during inflammation (47,48) and their chemotaxis is elevated in the lungs of patients with bacterial pneumonia or chronic bronchitis (49,50). Here, we found a significantly high number of lung neutrophils in HTV ventilated mice, and flow cytometry revealed that lung neutrophils rather than Th17 cells are the main source of IL-17 during VILI. Therefore, we speculate that lung neutrophils may promote pneumonia response after HTV ventilation by promoting inflammation on their own, as well as by producing IL-17 to trigger inflammatory responses in other cell types. Future studies should clarify the role of neutrophils in VILI.
Although IL-17 participates in VILI through the p38 MAPK/ MCP-1 pathway, there are still some limitations in this research that deserve improvement. First of all, HTV ventilation may cause respiratory alkalosis, we should continue to monitor and control the PCO2 and pH. However we had some difficulties in the monitoring methods mentioned above. Another limitation was there are some other factors that affect the severity of lung injury besides tidal volume. To reduce the influence of these factors, we should continuously monitor hemodynamics, PEEP or lung recruitment.

CONCLUSION
Our study suggests that IL-17 produced by lung neutrophils can act via the p38 MAPK/MCP-1 pathway to drive pulmonary inflammatory responses in VILI. Inhibition of IL-17 can effectively relieve VILI, suggesting that IL-17 may serve as a new target for attenuating lung inflammation induced by HTV ventilation.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by The Animal Care & Welfare Committee of Guangxi Medical University.