key: cord-0902253-7apmq5pm
authors: Kao, Ting‐I; Chen, Po‐Jen; Wang, Yi‐Hsuan; Tseng, Hsin‐Hui; Chang, Shih‐Hsin; Wu, Tian‐Shung; Yang, Sien‐Hung; Lee, Yen‐Tung; Hwang, Tsong‐Long
title: Bletinib ameliorates neutrophilic inflammation and lung injury by inhibiting Src family kinase phosphorylation and activity
date: 2021-07-14
journal: Br J Pharmacol
DOI: 10.1111/bph.15597
sha: f5276fcf9cf489ad177dcb55180517f4d300bf17
doc_id: 902253
cord_uid: 7apmq5pm

BACKGROUND AND PURPOSE: Neutrophil overactivation is crucial in the pathogenesis of acute lung injury (ALI). Bletinib (3,3′‐dihydroxy‐2′,6′‐bis(p‐hydroxybenzyl)‐5‐methoxybibenzyl), a natural bibenzyl, extracted from the Bletilla plant, exhibits anti‐inflammatory, antibacterial, and antimitotic effects. In this study, we evaluated the therapeutic effects of bletinib in human neutrophilic inflammation and LPS‐mediated ALI in mice. EXPERIMENTAL APPROACH: In human neutrophils activated with the formyl peptide (fMLP), we assessed integrin expression, superoxide anion production, degranulation, neutrophil extracellular trap (NET) formation, and adhesion through flow cytometry, spectrophotometry, and immunofluorescence microscopy. Immunoblotting was used to measure phosphorylation of Src family kinases (SFKs) and downstream proteins. Finally, a LPS‐induced ALI model in male BALB/c mice was used to investigate the potential therapeutic effects of bletinib treatment. KEY RESULTS: In activated human neutrophils, bletinib reduced degranulation, respiratory burst, NET formation, adhesion, migration, and integrin expression; suppressed the enzymic activity of SFKs, including Src, Lyn, Fgr, and Hck; and inhibited the phosphorylation of SFKs as well as Vav and Bruton's tyrosine kinase (Btk). In mice with ALI, the pulmonary sections demonstrated considerable amelioration of prominent inflammatory changes, such as haemorrhage, pulmonary oedema, and neutrophil infiltration, after bletinib treatment. CONCLUSION AND IMPLICATIONS: Bletinib regulates neutrophilic inflammation by inhibiting the SFK‐Btk‐Vav pathway. Bletinib ameliorates LPS‐induced ALI in mice. Further biochemical optimisation of bletinib may be a promising strategy for the development of novel therapeutic agents for inflammatory diseases.

the enzymic activity of SFKs, including Src, Lyn, Fgr, and Hck; and inhibited the phosphorylation of SFKs as well as Vav and Bruton's tyrosine kinase (Btk) . In mice with ALI, the pulmonary sections demonstrated considerable amelioration of prominent inflammatory changes, such as haemorrhage, pulmonary oedema, and neutrophil infiltration, after bletinib treatment.

Conclusion and Implications: Bletinib regulates neutrophilic inflammation by inhibiting the SFK-Btk-Vav pathway. Bletinib ameliorates LPS-induced ALI in mice.

Further biochemical optimisation of bletinib may be a promising strategy for the development of novel therapeutic agents for inflammatory diseases.

acute lung injury, acute respiratory distress syndrome, bletinib, inflammation, neutrophil, Src family kinase 1 | INTRODUCTION Neutrophils, the most abundant granulocytes in circulation, are responsible for eliminating pathogens through degranulation, enabling neutrophil elastase (NE) release, respiratory burst with superoxide production, and neutrophil extracellular trap (NET) formation (Brinkmann et al., 2004; Mantovani et al., 2011; Phan et al., 2018) .

Thus, neutrophils are key effectors of both adaptive and innate immune systems (Mantovani et al., 2011) . During inflammation, adhesion and migration are both crucial steps of neutrophil recruitment, which is regulated by the conformational change of macrophage-1 antigen (Mac-1; also known as αMβ2 and CD11b-CD18) on the cell surface of neutrophils (Carrigan et al., 2007; Kolaczkowska & Kubes, 2013; Li et al., 2018; Morisaki et al., 1991) . Dysregulated activation and recruitment of neutrophils can cause damage to host tissue through the release of an excessive amount of proteolytic enzymes, ROS, and NETs, resulting in various morbidities, including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis; Nemeth & Mocsai, 2012) , infectious diseases, such as sepsis (Sonego et al., 2016) , inflammatory diseases, such as chronic obstructive pulmonary disease (Noguera et al., 2001) , atherosclerosis (Soehnlein, 2012) and acute lung injury (ALI; Grommes & Soehnlein, 2011) and other major diseases, including cancers (Grecian et al., 2018; Jorch & Kubes, 2017) .

Acute respiratory distress syndrome (ARDS), the clinical term for ALI, occurs most often in the course of sepsis and severe pneumonia, including coronavirus disease 2019 (COVID-19; Park et al., 2019; Sohrabi et al., 2020) . The mortality rate of ARDS remains as high as 30%-40%; moreover, other than lung-protective ventilation, few specific effective therapeutic procedures have been developed (Matthay et al., 2019) . ALI is pathologically characterised by diffuse alveolar damage along with neutrophil infiltration and inflammatory oedematous fluid accumulation in the bronchoalveolar space (Grommes & Soehnlein, 2011) . Neutrophil activation and recruitment contribute to ALI pathogenesi. Moreover, in patients with ALI induced by ischaemia/reperfusion or LPS, the inhibition of the phosphorylation and activity of Src family kinases (SFKs) and NET formation, respectively, improve clinical condition and increase survival (Grommes & Soehnlein, 2011; Oyaizu et al., 2012; Pedrazza et al., 2017) .

The SFKs are non-receptor intracellular protein tyrosine kinases, some of which, including Lyn, Fgr, and Hck, are mainly expressed in human neutrophils (Futosi & Mocsai, 2016) . SFKs modulate several functions of neutrophils, such as ROS production, degranulation, adhesion, NET formation, integrin activation, and migration towards inflamed sites (Fumagalli et al., 2013; Mocsai et al., 1999; Nani et al., 2015; Rohwedder et al., 2019; Sarantos et al., 2008) .

N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) is a chemotactic peptide recognised by neutrophils through their formyl peptide receptors (FPRs), which triggers SFK phosphorylation and thus neutrophil activation (Fumagalli et al., 2007) . SFKs regulate the signal transduction in fMLP-activated neutrophils and the phosphorylation of downstream signal proteins such as JNK, ERK, Vav, and Bruton's What is already known • Neutrophil overactivation plays a key role in acute lung injury (ALI) pathogenesis.

• Bletinib mitigates neutrophilic inflammation by inhibiting the SFK-Btk-Vav pathway.

• Bletinib can alleviate LPS-mediated ALI. tyrosine kinase (Btk; El-Hashim et al., 2017; Fumagalli et al., 2013; Tsai et al., 2019) .

In traditional Chinese medicine, Bletilla tubers have been used to treat pulmonary, gastrointestinal, and dermatological inflammatory and haemorrhagic diseases for thousands of years (He, Xiong, et al., 2016) . Bletinib [3,3 0 -dihydroxy-2 0 ,6 0 -bis(p-hydroxybenzyl)-5-methoxybibenzyl, molecular formula: C 29 H 28 O 5, MW: 456.538; Figure 1a ] is a natural bibenzyl compound that was first extracted from Bletilla striata bulbs by Takagi et al. in 1983 . Bletinib has antibacterial, antifungal, antiallergic, and antimitotic potential (Matsuda et al., 2004; Morita et al., 2005; Takagi et al., 1983; Yang et al., 2012) . In our previous study, although bletinib demonstrated anti-inflammatory effects on human neutrophils, the underlying mechanism remained unclear .

In this study, we investigated the effects of bletinib on neutrophil functions, such as ROS production, degranulation, adhesion, migration, NET formation, and integrin expression, to test the hypothesis that bletinib regulates the inflammatory condition of activated human neutrophils. Moreover, we elucidated the mechanism and signal transduction pathway underlying the bletinib-mediated modulation of neutrophilic inflammation, using a model of ALI induced by LPS in mice.

Bletinib was prepared as described by our group . The air-dried whole plants of B. formosana (10.0 kg) were extracted with ethanol at 60 C and refluxed for 8 h. The extracts were evaporated to provide 818.0 g (dry weight) of extract. The extract samples were fractionated with Diaion HP-20 column chromatography using a stepwise H 2 O-ethanol gradient elution to obtain five fractions (BF1-BF5). The BF4 (52.4 g) was refractionated on a silica gel column and eluted with a gradient of CHCl 3 and MeOH to obtain 11 fractions (BF4-1-BF4-11). Repeated column chromatography of BF4-7 over silica gel with n-hexane-diisopropyl ether (1:4) yielded 18 fractions (BF4-7-1 -BF4-7-18). Purification of BF4-7-11 by column chromatography over silica gel eluted with CHCl 3 -diisopropyl ether (6:1) to F I G U R E 1 Bletinib suppresses superoxide anion production, ROS generation, and NE release in fMLP-stimulated human neutrophils. (a) Chemical structure of bletinib. Neutrophils (6 Â 10 5 cellsÁml À1 ) were incubated with 0.1% DMSO or 0.3-3 μM bletinib for 5 min before activation with fMLP (0.1 μM) in the presence of CB. (b) Superoxide anion production was measured using ferricytochrome c reduction method. (c) The change in chemiluminescence was monitored in real time using an ELISA reader and is illustrated (left panel) and the percentage of peak chemiluminescence is displayed (right panel). (d) The intracellular ROS levels and mean fluorescence intensity (MFI) of DHR123 in the neutrophils are monitored through flow cytometry. (e) NE release was induced by fMLP along with the NE substrate and then evaluated spectrophotometrically. All data are shown as box (interquartile range) and whiskers (min, max) plots, with medians (n = 5 or 6). *P < 0.05, significantly different from the DMSO + fMLP group give bletinib (190 mg). The structure of bletinib was determined by NMR and mass spectroscopic methods (Figure 1a ). The purity of bletinib was > 97% ( Figure S1 ). Bletinib was dissolved in DMSO for further experimentation. The control concentration of DMSO used in the cell experiments was 0.1%, which did not affect the parameters measured. 

A total of 24 male BALB/c mice were randomly divided into four groups (6 mice per group): vehicle alone, bletinib alone, LPS control, and bletinib treatment (bletinib + LPS). The mice were starved overnight and then injected i.p. with 50 μl of bletinib (25 mgÁkg À1 ) or 50 μl of vehicle (10% DMSO). ALI was induced through intratracheal spraying of 50 μl of LPS (from Escherichia coli O111:B4; 2 mgÁkg À1 ) or 50 μl of 0.9% saline (in vehicle and bletinib alone group) under general anaesthesia with xylazine (6 mgÁkg À1 ) and Zoletil 50 (tiletamine and zolazepam, 1:1; 30 mgÁkg À1 ). Five hours later, mice were killed (CO 2 asphyxia), the lungs removed and frozen at À80 C or fixed with 10% formalin. The serum levels of glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), creatinine (CRE), and blood urea nitrogen (BUN) were determined by automated clinical chemistry analyser (Dri-Chem NX500i, Fujifilm ® , Japan). The level of blood neutrophils was measured using an automated haematology analyser (XT-1800i, Sysmex Corporation, Kobe, Japan).

For the LPS-induced mortality model, mice were injected i.p. with a single 50-μl dose of LPS (from Escherichia coli O111:B4; 5 mgÁkg À1 ) or 0.9% saline (vehicle alone group). The mice were monitored for 5 days to determine survival.

The collected lung tissues were washed with PBS and fixed with 10% formalin for 24 h. The samples were subsequently dehydrated, embedded with paraffin, sliced into 3-μm-thick sections with a microtome, and placed on glass slides. These sections were stained using haematoxylin and eosin (H&E) and corresponding antibodies. Then, images were acquired through light microscopy, as described previously (Yuan et al., 2006) . The severity of histological lung injury was For immunofluorescence staining, tissue sections were incubated with antibodies against H3 (citH3; citrulline R2 + R8 + R17) and

Ly6G at dilutions of 1:800 and 1:200, respectively. Anti-IgG secondary antibodies labelled with a fluorescent dye (Alexa Fluor 488 for citH3 or Alexa Fluor 568 for Ly6G) were used at dilutions of 1:1000 and 1:500, respectively. Immunofluorescence images were acquired through confocal microscopy (LSM 510 Meta, Zeiss).

The mouse lung tissues were suspended in a 0.5% hexadecyltrimethylammonium bromide buffer (pH 6.0) and then homogenised through sonication. To evaluate MPO activity, the MPO substrate buffer (containing PBS, 0.0005% hydrogen peroxide, and 0.2 mgÁml À1 o-dianisidine hydrochloride) was added to the homogenised tissue, and the light absorbance at 460 nm was detected through spectrophotometry, after which MPO activity was calculated with reference to the standard curve of human MPO activity (Yu et al., 2006) .

Total RNA was extracted from mouse lung tissues using homogenization (MagNA Lyser, Roche, Basel, Switzerland) and TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The mRNA level of IL-1β and GAPDH was determined using iScript cDNA Synthesis Kit, iQ™ SYBR ® Green Supermix and quantitative PCR with CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) which was conducted at 95 C for 3 min, followed by 40 cycles of denaturation at 95 C for 15 s, and annealing/extension at 56 C for 30 s. Primers for mouse IL-1β (5 0 -TGGACCTTCCAGGATGAGGACA-3 0 and 5 0 -GTTCATCTCGGAGCC TGTAGTG-3 0 ) and mouse GAPDH (5 0 -AAGGAGTAAGAAACCCT GGAC-3 0 and 5 0 -GATGGAAATTGTGAG GGAGATG-3 0 ) were used.

The study was conducted with the approval of the Institutional Review Board of Chang Gung Memorial Hospital (IRB No. 201601 111A3) in accordance with the Declaration of Helsinki. After written informed consent was obtained, whole blood samples were drawn from healthy individuals aged 20-30 years who had not taken any medication within the previous 2 weeks. Neutrophils were then isolated using the standard procedures for Ficoll-Hypaque gradient centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes. The isolated neutrophils-containing >98% living cells, confirmed through Trypan blue staining-were then suspended in Ca 2+ -free HBSS (pH 7.4) and stored at 4 C until use .

Extracellular superoxide anion production in activated neutrophils was assessed through the reduction of ferricytochrome c. After incubating the isolated human neutrophils (6 Â 10 5 cellsÁml À1 ) with Ca 2+ (1 mM) and ferricytochrome c (0.5 mgÁml À1 ) at 37 C, the cells were then incubated with 0.1% DMSO or 0.3-10 μM bletinib for 5 min. The cells were then pretreated with cytochalasin B (1 or 2 μgÁml À1 ) for 3 min and then stimulated with fMLP, MMK-1, or sodium fluoride (NaF), or directly activated with phorbol-12-myristate-13-acetate (PMA). The change in absorbance at 550 nm was detected continuously using a spectrophotometer (U-3010, Hitachi, Tokyo, Japan), and superoxide anion levels were calculated using a method described previously (Hwang et al., 2003) .

Human neutrophils (2.5 Â 10 6 cellsÁml À1 ) were labelled using 2 μM dihydrorhodamine 123 (DHR123) at 37 C for 10 min and then incubated with DMSO or bletinib for 5 min and then stimulated with fMLP (0.1 μM) and cytochalasin B (0.5 μgÁml À1 ) for 15 min. The fluorescence intensity was detected through flow cytometry to evaluate intracellular superoxide anion production of human neutrophils.

Total ROS produced by neutrophils was assessed using a luminolamplified chemiluminescence method described previously (Bedouhene et al., 2017) . In brief, human neutrophils (2 Â 10 6 cellsÁml À1 ) were preincubated with 6 UÁml À1 HRP and 37.5 μM luminol in a 96-well plate at 37 C for 5 min. Cells were incubated with DMSO or bletinib for 5 min, followed by stimulation with 0.1 μM fMLP. Chemiluminescence was then detected and analysed in real time on a 96-well chemiluminometer (Tecan Infinite F200 Pro;

Männedorf, Switzerland).

Azurophilic degranulation was determined on the basis of NE release from human neutrophils, as reported previously (Sklar et al., 1982) . In brief, human neutrophils (6 Â 10 5 cellsÁml À1 ) were incubated with DMSO or bletinib after treatment with 1 mM CaCl 2 and 100 μM NE substrate (methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide) at 37 C for 5 min. Cells were stimulated with fMLP with cytochalasin B (0.5 μgÁml À1 ), LTB 4 , with cytochalasin B ((2 μgÁml À1 ), NaF with cytochalasin B (2 μgÁml À1 ), or MMK-1, with cytochalasin B (0.5 μgÁml À1 ) for 10 min before determination of NE release through measuring the change of absorbance at 405 nm on a spectrophotometer.

2.11 | Analysis of NET formation 2.11.1 | Quantification of extracellular DNA Human neutrophils (10 6 cellsÁml À1 ) resuspended in HBSS with 2.5 μM Sytox green were incubated with DMSO or bletinib for 10 min and stimulated using 10 nM PMA or 10 μgÁml À1 LPS for 3 h. The fluorescence intensity was quantified on a Tecan Infinite 200 reader at 485-535 nm.

Neutrophils (3 Â 10 5 cellsÁml À1 ) were incubated with DMSO or bletinib for 10 min before being activated with 10 nM PMA for 2 h.

Neutrophils were fixed with 4% paraformaldehyde and treated with 5% goat serum blocking buffer for 1 h and then treated with 5 μgÁml À1 anti-MPO (Abcam) and 5 μgÁml À1 anti-NE (Merck Millipore) antibodies for 1 h. These cells were then treated with the Alexa 488 or 568-labelled secondary goat anti-rabbit antibody for another 1 h.

Thereafter, the cells were washed with PBS and treated with 1 ngÁml À1 Hoechst 33342 and ProLong Gold antifade reagent (Invitrogen, CA, USA). Immunofluorescence microscopy and scanning electron microscopy were both used to observe the NET formation of activated neutrophils, as described previously Remijsen et al., 2011) .

Human neutrophils (10 6 cellsÁml À1 ) were labelled with Hoechst 33342 and then incubated with DMSO or bletinib for 5 min. After centrifugation, the cells were resuspended and activated with 0.1 μM fMLP with cytochalasin B (1 μgÁml À1 ) for 10 min before incubation with bEnd.3 cells (ATCC Cat# CRL-2299, RRID:CVCL_0170) at 37 C for 30 min.

After they were washed with HBSS, the cells were fixed with 4% paraformaldehyde, and the neutrophils that adhered to the bEnd.3 cells were detected and quantified on a motorised inverted microscope (Olympus, Japan), as described previously (Chen, Wang, et al., 2016) .

A microchemotaxis chamber with 3-μm filters (Millipore) was used to evaluate chemotactic migration of neutrophils. Neutrophils (5 Â 10 6 cellsÁml À1 ) treated with bletinib or DMSO for 5 min were placed in the top chamber, and 0.1 μM fMLP or 0.1 μgÁml À1 IL-8 was added into the bottom chamber. The number of neutrophils that migrated from the top to the bottom chamber after incubation at 5% CO 2 for 1 h was counted on a MoxiZ automatic cell counter (ORFLO).

2.14 | Evaluation of surface CD18 and CD11b expression Neutrophils (5 Â 10 5 cellsÁml À1 ) were incubated with bletinib or DMSO for 5 min and then activated through incubation with 0.1 μM fMLP, with cytochalasin B (1 μgÁml À1 ) or 0.1 μgÁml À1 IL-8 with cytochalasin B (1 μgÁml À1 ) for 5 min. After centrifugation at 200Â g for 8 min at 4 C, the cells were resuspended in 5% BSA with FITCconjugated antibodies against CD18 or CD11b on ice in the dark for 15 min. The fluorescent intensity was then analysed through FACS (Tsai et al., 2017) .

The immunoblotting assay was conducted as described previously 

The kinase activity of SFKs was assessed using an ADP-Glo kinase assay kit (Promega, Fitchburg, USA) according to the manufacturer's instructions, as described previously . In brief, the kinase reaction was initiated by adding SFKs (Src, Lyn, Fgr, and Hck), their substrate-125 μM ATP, and 1-10 μM bletinib or 0.1-3 μM PP2 into the reaction buffer for 1 h. The ADP-Glo reagent was used to end the kinase reaction and remove the remaining ATP; next, the kinase, which converted ADP to ATP, detection reagent was added and incubated for 30 min (Kovacs et al., 2014; Lowell, 2004 

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018) . All data are presented as box-and-whiskers plots (median, min-max). One-way ANOVA and Dunnett's multiple comparison tests were employed for all experiments. The survival rate of the mice was analysed using the log-rank (Mantel-Cox) test. All statistical calculations were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Differences with P values <0.05 were considered to show statistically significant effects. Furthermore, bletinib did not exhibit ROS scavenging activity in the cell-free xanthine oxidase system ( Figure S2D ) or cytotoxicity ( Figure S3 ).

Human neutrophils were pretreated with 0.1% DMSO or 1-10 μM bletinib for 10 min and then incubated with or without 10 nM PMA. (a) NET formation was quantified using Sytox green, a nucleic acid stain. (b) Neutrophils were stained with antibodies against NE (red) or MPO (green) and then analysed with confocal microscopy. DNA was detected using Hoechst 33342 (blue). (c) Scanning electron microscopy images of neutrophils. Representative images are shown. All data are shown as box (interquartile range) and whiskers (min, max) plots, with medians; n = 5. *P < 0.05, significantly different from the DMSO + fMLP group Chemiluminescence and flow cytometry assay were performed to determine if bletinib affects ROS production in stimulated neutrophils.

The results of luminol-amplified chemiluminescence assay in stimulated neutrophils demonstrated that total ROS production (intracellular and extracellular) was significantly attenuated by bletinib in a concentration-dependent manner (Figure 1c) . Moreover, the results of flow cytometry, using DHR123, revealed that bletinib significantly suppressed intracellular ROS production in fMLP-activated neutrophils (Figures 1d and S2E ).

Degranulation, an important function of neutrophils during inflammation, was evaluated by measuring the release of NE. Bletinib inhibited the release of NE by fMLP-stimulated human neutrophils (IC 50 = 0.53 ± 0.07 μM) without affecting resting neutrophils (Figures 1e and S4A ).

In addition, bletinib down-regulated NE release from neutrophils stimulated with MMK-1, NaF or LTB 4 in a concentration-dependent manner ( Figures S4 and S5 , respectively).

NET, mainly composed of granular proteins, proteases, and chromatin filaments coated with histones, is crucial in sterile inflammation (Jorch & Kubes, 2017) . To elucidate and quantify the effects of bletinib on NET formation, neutrophils were stained with Sytox green after activation with 10 nM PMA and 10 μgÁml À1 LPS. Fluorescent spectrometry assay results demonstrated that NET formation, induced by PMA and LPS, was significantly reduced by bletinib (Figures 2a and S6 ). In addition, scanning electron microscopy and immunofluorescent staining demonstrated the presence of NETs and neutrophils co-stained with Hoechst 33342 and antibodies against MPO and NE in the NETs (Figure 2b,c) .

SFKs and the MAPK/ERK pathway play critical roles in the degranulation, respiratory burst, NET formation, and migration of neutrophils (Hakkim et al., 2011; Minuz et al., 2018; Romero et al., 2010) . 3 and 4a-f ).

SFKs are non-receptor tyrosine kinases present in neutrophils, with predominant expression of Src, Fgr, Hck, and Lyn (Ear et al., 2017; Kovacs et al., 2014) . SFKs are responsible for the generation of the inflammatory environment in vivo. The cell-free ADP-Glo kinase assay results demonstrated that both bletinib and PP2, a selective inhibitor of SFKs (Hanke et al., 1996) , inhibited the kinase activity of Src, Fgr, Hck, and Lyn, in a concentration-dependent manner (Figure 4g-j) .

F I G U R E 3 Bletinib inhibits ERK and SFKs phosphorylation in fMLP-stimulated neutrophils. Neutrophils were preincubated with 0.1% DMSO or 10 μM bletinib and then stimulated with 0.1 μM fMLP. All the immunoblotting experiments were performed under the same conditions. Summary data from immunoblots for phosphorylated and total (a) SFKs, (b) Akt (S473), (c) ERK, (d) JNK, and (e) p38 are presented as box (interquartile range) and whiskers (min, max) plots, with medians (n = 6). *P < 0.05, significantly different from the DMSO + fMLP group 3.6 | Bletinib reduces adhesion and transmigration of activated human neutrophils Adhesion and transmigration are both crucial steps in the neutrophil recruitment cascade during inflammation (Kolaczkowska & Kubes, 2013) . Here, after incubation with bletinib (1-10 μM) or DMSO for 5 min and stimulation with fMLP, we incubated the Hoechst 33342-labelled neutrophils (10 6 cellsÁml À1 ) with bEnd.3 cells in 37 C for 30 min. Neutrophils adherent to bEnd.3 cells were detected and counted by fluorescence microscopy and our results demonstrated that bletinib inhibited the adhesion function of fMLPactivated neutrophils (Figure 5a,b) . We also used a chemotaxis chamber and a cell counter to quantitate the number of migrating neutrophils and found that bletinib significantly reduced fMLP-induced transwell migration of neutrophils (Figure 5c ). Furthermore, IL-8 serves as a chemotactic factor for the attraction of neutrophils. IL-8-induced neutrophil migration was also inhibited by bletinib ( Figure S7A ).

Mac-1 is a complement receptor composed of CD11b (integrin α M ) and CD18 (integrin β 2 ) and facilitates leukocyte recruitment during inflammation (Li et al., 2018; Wolf et al., 2018) . Here, we determined the surface expression of CD11b and CD18 through flow cytometry and found that bletinib significantly attenuated the expression of both F I G U R E 4 Bletinib inhibits phosphorylation and enzymic activity of SKFs. (a-f) Phosphorylation of SFKs, namely, (a) Src, (b) Lyn, (c) Fgr, and (d) Hck, and downstream proteins, (e) Btk and (f) Vav, was determined through immunoblotting. (g-j) The ADP-Glo kinase assay kit was used to evaluate the enzymic activity. (g) Src, (h) Lyn, (i) Fgr, or (j) Hck (1.5 ngÁml À1 ) was incubated with DMSO, 1-10 μM bletinib, or 0.1-3 μM PP2, and then 125 μM ATP (substrate) was added to the reaction mixture for 60 min, which was followed by enzymic activity detection. Data are shown as box (interquartile range) and whiskers (min, max) plots, with medians (n = 6). *P < 0.05, significantly different from the DMSO + fMLP group CD11b and CD18 in fMLP-stimulated human neutrophils ( Figure 6 ). Furthermore, the CD11b expression induced by IL-8 was decreased by bletinib in human neutrophils ( Figure S7B ).

Endotoxins such as LPS are the most common causes of ALI in bacterial infection and inflammatory diseases Dreyfuss & Ricard, 2005; Tsai et al., 2015) . To investigate the anti-inflammatory effects of bletinib in vivo, we used BALB/c mice treated with bletinib (25 mgÁkg À1 ) or DMSO administered i.p., followed by intratracheal spraying of LPS for 5 h. The exterior photos and HE-stained histopathological features of lungs revealed that LPS induced haemorrhage and erythema, interalveolar septal thickening, and pulmonary interstitial oedema formation. Infiltration of inflammatory MPO-and Ly6Gpositive cells infiltration (specific markers of neutrophils), protease release (NE), cytokine production (IL-1β), oxidative stress-induced lipid peroxidation (4-HNE), vascular permeability (occludin), MPO activity, and total protein levels were observed after LPS administration. Distortion of the pulmonary architecture was significantly suppressed in the bletinib treatment group (Figures 7, S8, and S9A ).

Furthermore, LPS-activated phosphorylation of Vav (p-Vav) was also attenuated by bletinib (Figure 7) . Furthermore, LPS-induced NET formation (Ly6G + citH3 + cell accumulation) was considerably reduced after bletinib treatment (Figure 8a ). Bletinib did not alter the serum level of GPT, GOT, CRE, and BUN in mice ( Figure S9B ), suggesting that bletinib treatment did not cause toxicity to liver or kidney. The therapeutic ability of bletinib to increase survival was further observed in LPS-primed mice. BALB/c mice were injected with LPS (5 mgÁkg À1 ), and their survival rate was monitored for 5 days. All mice in the LPS group died within 2 days, whereas the bletinib-treated mice (25 mgÁkg À1 ) exhibited significantly prolonged survival after LPS injection (log-rank test; Figure 8b ).

The plant B. formosana belongs to the Orchidaceae family and is widely distributed in Taiwan. Its tuber is extensively used in traditional Chinese medicine to treat pulmonary, gastrointestinal, and dermatological inflammatory diseases . Appropriate fermentation processes can be applied to enhance antioxidant activities and F I G U R E 5 Bletinib inhibits adhesion and transmigration of fMLP-activated human neutrophils. Hoechst 33342-labelled neutrophils (10 6 cellsÁml À1 ) were treated with 0.1% DMSO or 1-10 μM bletinib for 5 min, followed by no stimulation or stimulation with 0.1 μM fMLP, with 1 μgÁml À1 cytochalasin B for another 5 min. Neutrophils were then incubated with bEnd.3 cells at 37 C for 30 min. The neutrophils adherent to bEnd.3 cells were detected and enumerated through fluorescent microscopy. (a) Representative histograms of fluorescent microscopy. (b) Adherent neutrophils: Enumeration and quantification. (c) Human neutrophils were treated with DMSO or 1-10 μM bletinib for 5 min in the upper chemotaxis chamber and then were either not activated or activated with 0.1 μM fMLP for another 60 min. Migrated neutrophils were measured using a cell counter. Data are shown as box (interquartile range) and whiskers (min, max) plots, with medians (n = 5). *P < 0.05, significantly different from the DMSO + fMLP group total phenolic content of B. formosana (Dong et al., 2014) . In the present study, bletinib, a natural compound extracted from B. formosana, was found to significantly ameliorate neutrophilic inflammation by inhibiting SFK phosphorylation and activity and the related downstream signal transduction pathway.

Neutrophils have pivotal roles in innate immunity, and in the progression of infectious and inflammatory conditions. For instance, during bacterial infection, neutrophils are recruited, by chemotaxis, to inflamed tissues by endotoxin components, such as fMLP and LPS. Upon activation and recruitment, neutrophils produce superoxide anion and release proteolytic enzymes, such as elastase, to attenuate the progression of bacterial infection (Phan et al., 2018) .

However, excessive superoxide anion production is cytotoxic and damages host tissue (Fridovich, 1986) . For instance, although ROS is an essential component of the inflammatory process (Tintinger et al., 2009) , its overproduction contributes to various acute and chronic diseases, such as ALI (Dreyfuss & Ricard, 2005; Grommes & Soehnlein, 2011; Kellner et al., 2017) , coronary artery disease (Belaidi et al., 2016) , chronic Helicobacter pylori infection (Beceiro et al., 2016) , pulmonary hypertension (Chen, Li, et al., 2016) , and diabetes mellitus and related complications (He, Fang, et al., 2016; Miranda-Diaz et al., 2016; Wu et al., 2016) . ARDS/ALI is a major complication of pulmonary oedema and severe pneumonia, such as COVID-19, which can have a high mortality without appropriate treatment (Guan et al., 2020; Sohrabi et al., 2020) . Numerous neutrophils are present in the bronchoalveolar lavage fluid of patients with ARDS/ALI, and a number of experimental ALI models in mice have confirmed the therapeutic effects of neutrophil depletion (Nemeth et al., 2020) . Kellner et al. (2017) revealed that ROS enhances adhesion molecule and proinflammatory cytokine expression, resulting in pulmonary oedema and tissue damage. Researchers worldwide have been searching for compounds with antioxidant and anti-inflammatory effects to serve as potential therapeutic agents to treat the life-threatening diseases listed above (Boeing et al., 2020; Chen et al., 2015; Hwang et al., 2015; Liao et al., 2015) .

In the present study, bletinib significantly decreased superoxide anion production (Figures 1b and S2) , ROS production (Figure 1c,d) , and degranulation (Figures 1e and S5 ) in stimulated human neutrophils. However, bletinib did not exhibit either superoxide scavenging potential ( Figure S2D ) in the xanthine/xanthine oxidase cell-free system or cytotoxicity ( Figure S3 ). These findings indicated that bletinib reduced oxidative stress and protease release through regulating cellular signalling rather than through cytotoxicity or free radical scavenging activity.

NETs are web-like structures of nuclear chromatin coated with granular proteins of neutrophils, such as NE and MPO, which can trap F I G U R E 6 Bletinib decreases CD11b (integrin α M ) and CD18 (integrin β 2 ) expression in fMLPactivated neutrophils. Neutrophils were incubated with 0.1% DMSO or 1-10 μM bletinib for 5 min and then either not stimulated or stimulated with 0.1 μM fMLP and with 1 μgÁml À1 cytochalasin B for another 5 min. The mean fluorescence intensity (MFI) of FITC-labelled antibodies against (a) CD11b and (b) CD18 was detected through flow cytometry. Data are shown as box (interquartile range) and whiskers (min, max) plots, with medians (n = 6). *P < 0.05, significantly different from the DMSO + fMLP group and eliminate invading pathogens (Brinkmann et al., 2004) . However, NETs are also involved in non-infectious conditions, such as systemic lupus erythematosus, rheumatoid arthritis, atherosclerosis, chronic obstructive pulmonary disease and diabetes (Jorch & Kubes, 2017; Uddin et al., 2019) . Moreover, NET-microparticle complexes are potent inducers of neutrophil recruitment (Wang et al., 2019) . Pedrazza et al. (2017) also suggested that excessive NET release causes damage to lung tissues, as inhibition of NET formation increased survival in their LPS-induced ALI model. NETs may be cytotoxic to pulmonary endothelium and epithelium directly and may have a crucial role in both tumour dissemination and defence (Twaddell et al., 2019) . In addition, NET formation and ROS production are both triggered and regulated by SFKs (Nani et al., 2015) .

Bletinib significantly reduced NET formation and release (Figures 2   and S6 ) and had therapeutic effects in our LPS-induced ALI model in mice. Haemorrhage, protease release, pulmonary interstitial oedema formation, cytokine production, oxidative stress accumulation, tight junction destruction, SFKs signalling activation, NET formation, and neutrophil infiltration were all significantly decreased in vivo (Figures 7, 8, and S8) . Therefore, we focused on investigating whether bletinib attenuates oxidative stress, NET formation, and ALI by inhibiting SFK phosphorylation and activity.

SFKs are non-receptor tyrosine kinases initially known for their role in malignant tumour progression and their potential in regulation of the immune system has emerged in the past decade (Kovacs et al., 2014; Parsons & Parsons, 2004) . The phosphorylation of cellular proteins by SFKs, including Src, Fgr, Hck, and Lyn, occurs immediately after neutrophil activation. SFKs also play a critical role in modulating several effector functions of neutrophils, such as ROS production, degranulation and NET formation (Ear et al., 2017) . In particular, during cerebral ischaemia, Src is responsible for inducing the up-regulation of the activity of ERK and its target transcription factors (Hu et al., 2009 ). In the current study, bletinib inhibited the phosphorylation of SFKs, including Src, Lyn, Hck, and Fgr, and directly suppressed their enzymic activities in fMLP-activated human neutrophils (Figures 3 and 4) .

Btk is a Tec family non-receptor TK and, by contrast, Vav is a Rho family guanine nucleotide exchange factor that regulates activation of F I G U R E 7 Bletinib reduces LPS-induced ALI in mice. BALB/c mice (n = 6 in each group) were treated with the vehicle (10% DMSO) or 25 mgÁkg À1 bletinib, given i.p., followed by intratracheal spraying of LPS, for 5 h. (Figures 1, S2, and S5) . Therefore, our data suggest that SFKs may make a greater contribution to the neutrophil activation mediated by agonists of FPR1. However, we cannot rule out the possiblity that bletinib interacts with other FPR1-mediated signal pathways.

SFKs, which may regulate neutrophil responses to fMLP stimulation, are essential for β 2 integrin-mediated neutrophil adhesion and transmigration (Baruzzi et al., 2008; Evangelista et al., 2007; Sarantos et al., 2008) . In the present study, bletinib abolished human neutrophil adhesion to bEnd.3 cells and fMLP-induced transmigration of neutrophils ( Figure 5 ). Mac-1 integrin is crucial in mediating neutrophil recruitment, including adhesion and transmigration Li et al., 2018; Sule et al., 2019; Wolf et al., 2018) . Therefore, we investigated whether bletinib affects integrin expression in neutrophils. Our results demonstrated that bletinib significantly reduced surface expression of CD11b and CD18 in neutrophils ( Figure 6 ) and neutrophil infiltration in mice (Figure 7) , indicating that bletinib regulates neutrophil adhesion and transmigration by inhibiting SFK phosphorylation and activity and Mac-1 surface expression.

Additionally, the number of circulating neutrophils is increased by LPS in mice, and this effect was suppressed by bletinib treatment ( Figure S9C ), suggesting that bletinib may prevent the release of neutrophils from bone marrow.

In conclusion, to our knowledge, this is the first study to demonstrate bletinib, a natural compound extracted from B. formosana, is a SFK inhibitor: Bletinib strongly inhibits superoxide anion production, ROS production, degranulation, NET formation, adhesion, transmigration, and CD11b/CD18 integrin expression in activated human neutrophils, all mediated by the inhibition of the phosphorylation and enzymic activity of SFKs. In addition, in our murine LPS-induced ALI model, bletinib exhibited therapeutic effects, suggesting the potential of bletinib as a novel therapeutic agent of choice for ARDS. 

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis, Immunoblotting and Immunochemistry, Natural Products Research and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

All authors declare no conflict of interests.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

https://orcid.org/0000-0002-5780-3977

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