IL-17 mediated macrophage polarization increased inammatory damage in SWI-ALI models

Background Seawater inhalation induced acute lung injury (SWI-ALI) is the common accident in daily naval training. To investigate the mechanism of SWI-ALI will help to improve the treatment effect. Alveolar macrophages (AM) is the majority of alveolar, also paly the key role in SWI-ALI repair. IL-17 also paly the key role in the innate immunity process. Method In this study, we used seawater induced the ALI in mouse model. And the lungs and serum were exacted at D1, D3, D7 and D14. The AM polarization were tested by ow cytometry. The IL-17 concentration were tested by ELISA. Then the IL-17 function were conrmed by in vitro test. The mouse alveolar epithelial cell and mouse AM were co-cultured. The test compared the wound healing effect of MAE with and without IL-17.


Abstract Background
Seawater inhalation induced acute lung injury (SWI-ALI) is the common accident in daily naval training.
To investigate the mechanism of SWI-ALI will help to improve the treatment effect. Alveolar macrophages (AM) is the majority of alveolar, also paly the key role in SWI-ALI repair. IL-17 also paly the key role in the innate immunity process.

Method
In this study, we used seawater induced the ALI in mouse model. And the lungs and serum were exacted at D1, D3, D7 and D14. The AM polarization were tested by ow cytometry. The IL-17 concentration were tested by ELISA. Then the IL-17 function were con rmed by in vitro test. The mouse alveolar epithelial cell and mouse AM were co-cultured. The test compared the wound healing effect of MAE with and without IL-17.

Result
The AM switch into M1 and IL-17A increased were found after seawater dosing. And the IL-17a supplement attenuated wound healing of alveolar epithelial cells through improve the polarization of AM were con rmed in vitro model.

Conclusion
The high IL-I7 micro-environment will increased the in ammatory damage through induced macrophage polarization in acute lung injury. The IL-17 antagonists have the potential to increase clinical effect in SWI-ALI treatment.

Background.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by an acute in ammatory process in the airspaces and lung parenchyma. Seawater inhalation induced acute lung injury (SWI-ALI) is a common accident in daily naval training. The alveolar epithelial and pulmonary capillary endothelial impaired caused by seawater inhalation injury resulting in the signi cant hypoxemia [1][2]. Due to these patients are critically ill and frequently have coexisting conditions including sepsis and multiple organ failure, the patients are at higher risk of respiratory failure [3][4][5]. There is considerable experimental and clinical evidence that the changes of cytokines and immunity process play the key role in SWI-ALI development. But the conventional therapies such as mechanical ventilation are not adequate for all SWI-ALI treatments. So, to investigate the mechanism of SWI-ALI in ammation will help to nd novel therapy [6].
Macrophages play key roles during mammalian development and tissue injury repair [7][8]. The resident tissue macrophages cloud recruited macrophages or monocytes from bone marrow after organ injury induced by infection, autoimmune disorders, mechanical or toxic injuries, and various other causes [9]. Alveolar macrophages (AM) are the most important component of the alveoli, accounting for 80-90% of the alveolar cells. The AM polarized and cytokine released is the rst trigger of SWI-ALI induced In ammatory process [10]. The activated macrophage can recruit the monocytes and neutrophils to improve the lung injury repair [11]. So, we propose the macrophage polarization and the relevant cytokines change will affect the SWI-ALI repair.
In this study we established the SWI-ALI model. The signi cant change of IL-17 and macrophage polarization were found at D3 and D7 after seawater injected. The IL-17 and macrophage relationship were con rmed in vitro.

Material And Methods
Animal and Ethics Statement 60 Female BALB/c nude mice (6-8 weeks old) were maintained in a light and temperature-controlled room. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Bioduro (China) Co., Ltd under SPF protocol. The ethic number was HRJ-FFS-ON-20200127-01.

Mouse primary cells isolation and culture condition
Mouse primary alveolar epithelial cells (MAE) and AM were isolated from health BALB/C mice Lung were excised and stored at cold PBS before used. The lung single cell suspensions (SCS) were prepared by Lung Dissociation Kit, mouse (130-095-927, Miltenyi). The AM were selected by CD45 dynabeads(11153D, Thermo) and puri ed by maintained in RPMI-1640(22400-097, Thermo) with M-CSF (10ng/mL) for two weeks. And MAE were negative selected by CD45 dynabeads. MAE was maintained in DMEM (11965-175, Thermo) medium. 10% fetal bovine serum (16000-044, Gibco) and with 10,000 units penicillin and 10 mg streptomycin/mL (V900929, Sigma-Aldrich) were supplemented into all above medium. The cells were incubated in humidi ed atmosphere containing 5% CO 2 at 37℃. SWI-ALI model SWI-ALI models were established via intratracheal instillation 100 mL seawater to each mouse. The seawater was con gured by added sea salt (S9883, Sigma-Aldrich) into sterile ddH 2 O. Control group were received intratracheal instillation PBS with the same volume. Five mice were sacri ced at D1, D3, D7 and D14.
Serum cytokine detection Serum sample were exacted from jaws vein of mice. The serum sample were stored at -80 ℃ before used. IL-17a detection kit were purchased from Biolegend (432504). ELISA procedure were tested as standard protocol. Each sample were tested three replicates.

SWI-ALI in vitro model
SWI-ALI in vitro model was established by MAE and AM co-culture. 20000 MAE were seeded into each well and allowed to growth over night. The wound was created in the center of each well using a scratcher. AM were added into macrophage group and macrophage+IL-17a group after wound made at 1:10 ratio of effect: target (E:T). IL-17a 10ng/mL were added into IL-17a group and macrophage+IL-17a group and control group. The wounds were photographed and measured every three hours for two days. After two days the supernatants were collected and detected by uorescence-activated cell sorting (FACS).

Statistical analysis
Statistical signi cant analysis of the data was using the analysis of variance and the Student's t-test. And all data were presented as the mean ± standard deviation (SD). ELISA was calculated by 4 parameters logistic regression. The tests were conducted by using the GraphPad Prism 7.0 software (GraphPad software, USA).

Result
Serum concentration of IL17 increased signi cantly after SWI-ALI.
The serum concentration of SWI-ALI group and control group were showed in gure 1. IL-17 attenuated the wound healing at SWI-ALI in vitro model.
The wound width changing of each group were show in gure 3. The wounds of control group, macrophage group and IL-17a group were completed healed before 39h, 33h and 36h. The wounds of macrophage and IL-17a group were not completed healed after 2d. The nal wound width of macrophage+ IL-17a group was 751.79±180.42mm2.

IL-17 can induce AM polarization
The AM typing from the co-culture supernatants of each group were showed in gure 4. The M1 percent of macrophage group and macrophage+IL-17a group were 0.85±0.47 and 16.77±2.60. The M1 percent of macrophage group was higher than the macrophage+IL-17a group (P<0.01).

Discussion
IL-17 is a typical In ammatory cytokines family which played the key role in various in ammation induced by direct injury or pathogen invasion [12][13][14]. IL-17 family`s cytokines were secreted by T helper 17(Th17) cells during in ammation [15]. The secreted IL-17 further activates a complex network of cytokine interactions to help repair the injury. But the IL-17 is a double-edged sword. The high IL-17 concentration also distributed to autoimmunity disease like Systemic Lupus Erythematosus [14]. IL-17a is a member of IL-17 family which can bind IL-17Ra to perform the biological effect. IL-17Ra were higher expression at broblast and lung [16]. As the reports, high IL-17 were found at patient`s bronchoalveolar lavage uid (BALF) after SWI-ALI [17][18].
In this study, we used seawater to established SWI-ALI model. The IL-17a were increasing rapidly after SWI-ALI and existed more than a week. The high serum IL-17a level shows the innate immunity triggered immediately. Activated Th17 cells recruit neutrophil though the secreted IL-17 to heal the impaired epithelium and airway [19]. But the persist secreting IL-17 also remain the increasing in amed damage. So, we want to con rm whether the IL-17 improve or attenuated SWI-ALI repair.
AM is the majority part of alveolar and the macrophage is classical organ damage restorer. The activated macrophage is the main source of IL-17 [20]. Plasticity is the characteristics of macrophages. Macrophages show different phenotype at different micro-environments. Macrophage can polar into the in ammatory M1 or anti-in ammatory M2 [21][22]. The FACS show the AM polarization were increased during SWI-ALI process. The M1 percent were highest at the D3 after SWI-ALI. And the polarization of macrophages rapidly disappeared after D7. Zhou and his colleagues report that the M1 macrophages can impair the keratinocyte migration via TNF-α [23]. So, the increased M1 percent antagonize epithelial repair.
The relationship of IL-17 and macrophage polarization were con rmed by SWI-ALI in vitro model. The supplement of IL-17a increased M1 percent and attenuated the wound healing of PAE. But the macrophage alone show improves the wound healing effect. And the IL-17a alone not in uent the wound healing. It`s seem that the IL-17 via induce the macrophage polarization to increase the SWI-ALI damage during in ammation. This show a potential that the IL-17 antagonist could help the clinical treatment in SWI-ALI.

Conclusion
This study found that the IL-17 and M1 percent of AM increased during the SWI-ALI in ammation. And con rm that the IL-17 can affect the macrophage switch to M1 to increase the in ammatory damage. This may help to improve the clinical effect of SWI-ALI treatment. The serum concentration of SWI-ALI group and control group. The signi cant different (P<0.001) between control group and seawater group were showed as ***.