Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation
Neutrophilic inflammation is central to chronic inflammatory diseases such as rheumatoid arthritis and chronic obstructive pulmonary disease ( COPD ) , which impose an increasing social and economic burden on our aging population . Treatment of COPD by next-generation combination therapy with inhaled corticosteroids and newer bronchodilators are viewed as maintenance pharmacotherapies but they do not specifically target cellular inflammation . The anti-inflammatory phosphodiesterase-4 inhibitor , roflumilast , targets systemic inflammation associated with COPD and reduces moderate to severe exacerbations in severe disease , possibly via effects on eosinophils ( Martinez et al . , 2018; Rabe et al . , 2018 ) . In these diseases , clearance of neutrophils by apoptosis is dysregulated particularly during exacerbations ( Pletz et al . , 2004; Sapey et al . , 2011 ) , but to date it has not been possible to therapeutically modify this , indeed corticosteroids can supress neutrophil apoptosis and hence perpetuate inflammation ( Liles et al . , 1995 ) . Recognising the urgent need for new therapies , we interrogated neutrophil inflammation and survival pathways using an unbiased approach focusing on potentially druggable kinases . Neutrophil persistence in tissues , caused by a delay in apoptosis , can result in a destructive cellular phenotype , whereby neutrophils have greater potential to expel histotoxic factors such as proteases and oxidative molecules onto surrounding tissue . This can occur either actively ( by degranulation ) or passively ( by secondary necrosis ) . In COPD , among other diseases , delayed apoptosis is considered to be a key part of the pathogenesis , occurring either as a result of pro-survival factors that are present in the lung microenvironment or an innate apoptosis defect ( Brown et al . , 2009; Haslett , 1999; Pletz et al . , 2004; Zhang et al . , 2012 ) . Despite this mechanistic understanding , there are no effective treatment strategies in clinical use to specifically reverse this cellular mechanism . Accelerating neutrophil apoptosis has been shown to promote the resolution of inflammation in multiple experimental models ( Burgon et al . , 2014; Chello et al . , 2007; Ren et al . , 2008; Rossi et al . , 2006 ) . A number of studies highlight the importance of protein kinases in regulating neutrophil apoptosis ( Burgon et al . , 2014; Rossi et al . , 2006; Webb et al . , 2000 ) and therefore reveal potential therapeutically targetable pathways for inflammatory disease . A growing class of clinically-exploited small molecule kinase inhibitors are being intensively developed ( Wu et al . , 2015 ) , making this a timely investigation . Using parallel unbiased screening approaches in vitro and in vivo , we here identify inhibitors of the ErbB family of receptor tyrosine kinases ( RTKs ) as potential therapeutic drivers of inflammation resolution . The ErbB family consist of four RTKs with structural homology to the human epidermal growth factor receptor ( EGFR/ErbB1/Her-1 ) . In an in vivo zebrafish model of inflammation , we show that inhibition of ErbBs , pharmacologically and genetically , reduced the number of neutrophils at the site of injury . Furthermore , ErbB inhibitors reduced inflammation in a murine peritonitis model and promoted neutrophil apoptosis and clearance by macrophages in the mouse lung . This study reveals an opportunity for the use of ErbB inhibitors as a treatment for chronic neutrophilic inflammatory disease .
Using a well-characterised transgenic zebrafish inflammation model ( Henry et al . , 2013; Renshaw et al . , 2006 ) , we adopted a chemical genetics approach , which has great potential for accelerated drug discovery ( Jones and Bunnage , 2017 ) . We initiated inflammation by controlled tissue injury of the zebrafish tail fin and screened a library of kinase inhibitors in order to establish which kinases could be exploited to enhance inflammation resolution in vivo ( Figure 1—figure supplement 1A ) . We quantified the ability of a library of 367 publicly available kinase inhibitors ( PKIS ) ( Elkins et al . , 2016 ) to reduce neutrophil number at the site of injury during the resolution phase of inflammation . The screen identified 16 hit compounds which reduced neutrophil number at the site of injury in the zebrafish model ( Figure 1A ) . For each compound the degree of kinase inhibition had been established ( Elkins et al . , 2016 ) ( Figure 1A ) . A number of kinases were inhibited by the 16 compounds , with Abelson murine leukaemia viral homolog 1 ( ABL1 ) , Platelet-derived growth factor receptor ( PDGFR ) α , PDGFRβ , p38α and ErbB4 being the top five most frequently targeted kinases overall . In addition to frequency of target , we also interrogated selectivity of compound . The most selective compounds , that is those that strongly inhibited individual kinases or kinase families , targeted the kinases YES , ABL1 , p38 and the ErbB family . Apoptosis is an important mechanism contributing to inflammation resolution; we therefore sought to identify kinases common to both inflammation resolution and neutrophil apoptosis pathways . Circulating neutrophils have a short half-life in vivo ( Summers et al . , 2010 ) and undergo spontaneous apoptosis in the absence of growth factors in vitro . We re-screened PKIS library compounds in a human neutrophil apoptosis assay for their ability to accelerate apoptosis ( Figure 1—figure supplement 1B ) . PKIS compounds were screened at 62 µM in order to maximise the chance of identifying ‘hits’ and resulted in 62 compounds that accelerated neutrophil apoptosis ≥2 fold compared to DMSO control ( Figure 1B and Supplementary file 1 ) . Secondary screening of top 38 compounds ( chosen from the 62 hits based on greatest selectivity for kinase targets ) was carried out at 10 µM in order to reduce false positives . This yielded 11 compounds that accelerated neutrophil apoptosis ≥2 fold over control ( as indicated by dashed green line , Figure 1C ) . Representative flow cytometry dot plots illustrating Annexin-V and ToPro-3 profiles for these hit compounds are shown in Figure 1—figure supplement 2 . Kinases targeted by these compounds included DYRK1B , KIT , EGFR , ErbB2 and ErbB4 , PDGFR , CDK6 and p38 ( Figure 1C , inset ) . The identification of known regulators of neutrophil survival ( p38 , PI3K ) was encouraging support for the screen design and execution . We found that members of the ErbB family of RTKs were the next most frequently inhibited kinase family , being targeted by three highly selective compounds out of the 11 hits ( Figure 1C , inset ) . Since inhibitors of the ErbB family were common hits in both zebrafish and human screens , we hypothesised that targeting ErbBs may be a potential strategy to reduce inflammation . To address a role for ErbB antagonists in regulating neutrophil apoptosis we tested a range of clinical and non-clinical ErbB-targeting compounds . We show that among inhibitors of ErbBs that are in clinical use , the EGFR inhibitor , gefitinib , is the most effective in promoting neutrophil apoptosis , reaching significance at 50 µM ( Figure 2A ) . The ErbB2-selective inhibitor , CP-724714 ( Jani et al . , 2007 ) also promoted neutrophil apoptosis in a dose-dependent manner ( Figure 2B ) as did Erbstatin and tyrphostin AG825 , selective for EGFR and ErbB2 respectively ( Osherov et al . , 1993; Umezawa and Imoto , 1991 ) ( Figure 2C–D ) . Since caspase-dependent apoptosis is an anti-inflammatory and pro-resolution form of cell death , engagement of the apoptosis programme was verified biochemically by measuring phosphatidylserine ( PS ) exposure by Annexin-V staining ( Figure 2—figure supplement 1A–C ) . Furthermore , the pan-caspase inhibitor Q-VD-OPh ( Wardle et al . , 2011 ) completely abrogated Erbstatin and tyrphostin AG825-driven neutrophil apoptosis , confirming the caspase dependence of inhibitor mediated cell death ( Figure 2—figure supplement 1D–E ) . COPD is a chronic inflammatory disease associated with functionally defective circulating neutrophils , including a resistance to undergoing apoptosis during exacerbations ( Pletz et al . , 2004; Sapey et al . , 2011 ) . To show ErbB inhibition is effective in driving apoptosis in subjects with systemic inflammation , we isolated neutrophils from the blood of patients with COPD and age-matched healthy control subjects . Erbstatin and tyrphostin AG825 significantly increased apoptosis of neutrophils from both COPD patients and healthy control subjects in a dose dependent manner at both 6 hr ( Figure 2E–F ) and 20 hr ( data not shown ) . ErbB inhibition overcomes neutrophil survival stimuli . Neutrophils are exposed to multiple pro-survival stimuli at sites of inflammation , which could undermine the therapeutic potential of anti-inflammatory drugs . Factors that raise intracellular cAMP concentration ( [cAMP]i ) are present during inflammation , and elevated [cAMP]i is known to prolong neutrophil survival via activation of cAMP-dependent protein kinases ( Krakstad et al . , 2004; Vaughan et al . , 2007 ) . We show that neutrophil apoptosis was reduced by the cAMP analogue and site selective activator of PKA , N6-monobutyryl-cAMP ( N6-MB-cAMP ) , and that this was reversed by Erbstatin analog ( Figure 3A ) and Tyrphostin AG825 ( Figure 3B ) . Similar effects were observed in neutrophils from patients with COPD ( Figure 3C ) . GMCSF is a key neutrophil chemoattractant and pro-survival factor , and is closely associated with the severity of inflammation in disease ( Klein et al . , 2000; Wicks and Roberts , 2016 ) . We show that erbstatin and tyrphostin AG825 prevent GMCSF-mediated survival in COPD and age-matched healthy control neutrophils ( Figure 3D–E ) . GMCSF is known to promote neutrophil survival via the phosphatidylinositol 3-kinase ( PI3K ) /AKT pathway , ultimately leading to the stabilisation of the anti-apoptotic Bcl-2 family member , Mcl-1 ( Derouet et al . , 2004; Klein et al . , 2000 ) . To investigate potential mechanisms underpinning the ability of tyrphostin AG825 to prevent GMCSF-mediated survival , we assessed AKT-phosphorylation as a measure of PI3K activation and found that tyrphostin AG825 reduced GMCSF-induced AKT phosphorylation after 15 and 30 min of treatment becoming statistically significant at 15 mins ( Figure 3F ) . Tyrphostin AG825 accelerated the spontaneous downregulation of Mcl-1 and also prevented GMCSF-induced stabilisation of Mcl-1 ( Figure 3G ) . These data show ErbB inhibition engages neutrophil apoptosis even in the presence of inflammatory stimuli and therefore has the potential to drive apoptosis at inflammatory sites . To explore whether ErbB family members are phosphorylated in response to survival stimuli we studied the activated kinome in human neutrophils stimulated with N6-MB-cAMP ( Vaughan et al . , 2007 ) . A Kinex antibody microarray was performed to detect the phosphorylation of over 400 kinases and kinase-associated proteins and this data set was interrogated to seek evidence of activation of ErbB by N6-MB-cAMP . Of the phospho-specific antibodies , 17 yielded an increase over baseline control of ≥1 . 5 at 30 min and 8 at 60 min ( Table 1 ) . Among these targets , ErbB2 phosphorylation was detected at 30 min ( 1 . 94 > control ) and 60 min ( 1 . 53 > control , Table 1 ) . This suggests that ErbB is part of the neutrophil signalling response to survival stimuli . In support of this , we detected the presence of ErbB2 mRNA in human neutrophils by RT-PCR ( Figure 4A ) and a 60kD protein ( lower molecular weight ErbB family products are well-documented ( Jackson et al . , 2013; Guillaudeau et al . , 2012; Siegel , 1999; Ward et al . , 2013 ) which was upregulated by GMCSF and dbcAMP ( Figure 4B ) . ErbB3 was also detected in human neutrophils by ELISA ( Figure 4C ) , at levels similar to those observed in other tissues in literature ( Buta et al . , 2016 ) . We found ErbB3 expression was not regulated by growth factors , which may in part be due to regulation being primarily at the post-translational level . To determine the ability of ErbB inhibition to exert an effect on neutrophil number and apoptosis in vivo , we used three complementary animal models of acute inflammation . To specifically address whether tyrphostin AG825 was able to accelerate apoptosis of neutrophils in the mammalian lung , we used a murine model of LPS-induced airway inflammation ( Thompson et al . , 2014 ) . C57BL/6 mice nebulised with LPS developed an acute pulmonary neutrophilia after 48 hr , to a degree seen previously ( Figure 5A–B ) ( Thompson et al . , 2014 ) . Tyrphostin AG825 had no effect on percentage of , or absolute number of neutrophils or macrophages compared to DMSO control ( Figure 5A–B ) . Tyrphostin AG825 significantly increased the percentage of neutrophil apoptosis , both visualised as ‘free’ apoptotic cells ( closed circles ) and as a summation of both free apoptotic cells and apoptotic inclusions within macrophages in order to capture those that had been efferocytosed ( closed triangles , Figure 5C ) . Macrophage efferocytosis was also significantly elevated by tyrphostin AG825 , compared to vehicle control ( Figure 5D ) , determined by counting the number of macrophages containing apoptotic inclusions as a proportion of total macrophages ( Figure 5E ) . We next tested the anti-inflammatory potential of tyrphostin AG825 when administered once inflammation was established , which is more representative of the clinical scenario . Mice were i . p injected with zymosan to induce peritonitis and after 4 hr were treated ( i . p . ) with tyrphostin AG825 or vehicle control . Total cell counts in peritoneal lavage were 2 . 2 × 106 in PBS vs 1 . 7 × 107 in zymosan treated animals at 4 hr demonstrating established inflammation at this time point ( Navarro-Xavier et al . , 2010 ) . Importantly , tyrphostin AG825 does not induce leukopenia ( Figure 5F ) , however significantly fewer inflammatory cells were found in peritoneal lavage following tyrphostin AG825 treatment ( Figure 5G ) . The neutrophil chemoattractant and proinflammatory cytokine , KC , was reduced in tyrphostin AG825 treated mice , and concomitant with this , a trend for less IL-6 was also observed ( Figure 5H ) . IgM , which correlates with the number and activation of peritoneal B lymphocytes ( Almeida et al . , 2001 ) , is significantly reduced in tyrphostin AG825-treated mice ( Figure 5I ) . To further extend this observation , we tested the ability of ErbB inhibitors to modulate neutrophilic inflammation resolution as a whole , in a model which encompasses multiple mechanisms of neutrophil removal including both apoptosis and reverse migration . In the mpx:GFP zebrafish tail fin injury model ( Renshaw et al . , 2006 ) ( Figure 6A ) we were able to show that tyrphostin AG825 ( Figure 6B ) and CP-724714 ( Figure 6C ) significantly reduced the number of neutrophils at the site of injury at 4 and 8 hpi . Simultaneous gene knockdown of egfra and erbb2 via CRISPR/Cas9 ( referred to as ‘crispants’ ) also recapitulated this phenotype ( Figure 6D ) . Tyrphostin AG825 did not affect total neutrophil number ( Figure 6E ) , but egfra and erbb2 crispants had significantly fewer neutrophils ( Figure 6F ) . As demonstrated by TSA and TUNEL double staining ( Figure 6G ) , tyrphostin AG825 upregulated neutrophil apoptosis at both the site of injury ( Figure 6H ) and in the caudal hematopoietic tissue ( CHT ) of zebrafish ( Figure 6I ) . CHT neutrophil counts were unchanged between conditions ( data not shown ) . egfra and erbb2 crispants had increased numbers of apoptotic neutrophils at the site of injury , but this was not significant ( Figure 6J ) , perhaps suggesting the presence of compensatory mechanisms . These findings show that inhibiting ErbB RTKs accelerate neutrophil apoptosis in vitro and in vivo and enhance inflammation resolution , making ErbB inhibitors an attractive therapeutic strategy for inflammatory disease .
Neutrophils are powerful immune cells because of their destructive anti-microbial contents . A deleterious by-product of this is their remarkable histotoxic potential to host tissue , ordinarily held in check by the onset of apoptosis . The inappropriate suppression of neutrophil apoptosis underpins a number of chronic inflammatory diseases , and we are yet to have available an effective treatment strategy that can reverse this cellular defect in clinical practice . Here we show in human , mouse and zebrafish models of inflammation and neutrophil cell death that targeting the ErbB family of RTKs regulates neutrophil survival and resolves inflammation . Promoting neutrophil apoptosis is a desirable approach for the resolution of inflammation , since apoptosis functionally downregulates the cell , promotes rapid cell clearance by efferocytosis and engages an anti-inflammatory phenotype in phagocytosing cells ( Savill et al . , 1989; Whyte et al . , 1999 ) . As proof of principle , driving apoptosis experimentally promotes the resolution of inflammation across multiple disease models ( Chello et al . , 2007; Ren et al . , 2008; Rossi et al . , 2006 ) . Several compounds targeting the ErbB family have been approved as medicines for the treatment of cancer ( Singh et al . , 2016 ) . Our findings open up the possibility of repurposing well-tolerated ErbB inhibitors for patients with inflammatory disease , potentially addressing a currently unmet clinical need . The ErbB family are critical regulators of cell proliferation and are associated with the development of many human malignancies ( Roskoski , 2014 ) . In addition to the development of cancer , ErbB members have known roles in inflammatory diseases of the airway , skin and gut ( Davies et al . , 1999; Finigan et al . , 2011; Frey and Brent Polk , 2014; Hamilton et al . , 2003; Pastore et al . , 2008 ) . In the context of lung inflammation , ErbB2 is upregulated in whole lung lysates in murine bleomycin models of lung injury and EGFR ligands are increased in BAL from acute lung injury patients receiving mechanical ventilation ( Finigan et al . , 2011 ) , suggesting ErbB signalling axes may play a role in the process of airway inflammation in vivo . We show , in murine models where Tyrphostin AG825 was administered either at the time of inflammatory stimulus or once inflammation was established , an impact on cell number , proinflammatory cytokine production and neutrophil apoptosis , further validating the use of ErbB inhibitors to reduce inflammation . The benefit of EGFR inhibitors in reducing inflammation in ventilator-induced and OVA/LPS-induced lung injury rodent models is shown by others , further supporting the targetting of this pathway in inflammatory disease settings ( Bierman et al . , 2008; Shimizu et al . , 2018; Takezawa et al . , 2016 ) . Others have reported that neutrophils express members of the ErbB family ( Lewkowicz et al . , 2005 ) , particularly ErbB2 at low levels ( Petryszak et al . , 2016 ) and we show that they are phosphorylated and regulated following exposure to inflammatory stimuli . ErbBs have known roles in suppressing apoptosis of epithelial cells and keratinocytes , but this study is the first to show a role for ErbBs in survival signalling of myeloid cells . Little is known about the roles of ErbBs in neutrophil function . Erbstatin has been shown to inhibit neutrophil ROS production ( Dreiem et al . , 2003; Mócsai et al . , 1997; Reistad et al . , 2005 ) and chemotactic responses ( Yasui et al . , 1994 ) . Other kinase families have been found to play a role in neutrophil survival and neutrophilic inflammation , most notably the cyclin-dependent kinases ( CDKs ) ( Rossi et al . , 2006 ) . In accordance with this , compounds targeting CDKs were identified as drivers of neutrophil apoptosis in both our primary and secondary screens . Moreover , p38 MAPK inhibitor compounds were also identified in both zebrafish and human screens , and since this kinase is known to mediate survival signals , these findings give confidence to the robustness of the screen design and execution . The engagement of apoptosis by the ErbB inhibitors erbstatin and tyrphostin AG825 was confirmed both biochemically by phosphatidylserine exposure , and mechanistically by the caspase inhibitor Q-VD-OPh and loss of Mcl-1 . This suggests that inhibiting ErbBs as a therapeutic strategy may achieve an overall anti-inflammatory effect in in vivo systems , facilitating clearance by macrophages . In support of this , we provide evidence of increased efferocytosis in vivo following tyrphostin AG825 treatment , with no evidence of secondary neutrophil necrosis due to overwhelming macrophage clearance capacity , evidenced both morphologically and by TO-PRO-3 staining . The ability of ErbB inhibitors to promote neutrophil apoptosis even in the presence of multiple pro-survival stimuli emphasises the potential of ErbB inhibitors in the lung , at sites where inflammatory mediators are in abundance and where neutrophils are exposed to microorganisms . This is supported by the ability of tyrphostin AG825 to prevent early pro-survival signalling in response to GMCSF , including the phosphorylation of AKT . This precedes the onset of apoptosis , occurring at a time point ( 15 min ) where apoptosis is typically less than 1% . Others have shown the ability of erbstatin to prevent GMCSF-mediated activation of PI3K in human neutrophils , although the impact on cell survival was not studied ( al-Shami et al . , 1997 ) . Therefore , ErbBs may function as an early and upstream component of the survival pathway in neutrophils . Subsequent impact on Mcl-1 destabilisation by tyrphostin AG825 at 8 hr suggests a cellular mechanism by which these pro-apoptotic effects are mediated . The effects of ErbB inhibitors in driving spontaneous apoptosis suggest that , under certain circumstances , ErbB activity might be required for constitutive neutrophil survival . It is not clear what , if anything , engages ErbB signalling in culture . The rapid phosphorylation of ErbB2 following N6-MB-cAMP treatment ( 30 min ) suggests that perhaps a ligand is not required , or that the neutrophils can rapidly release ErbB agonists in an autocrine manner . Unlike all other ErbBs , ErbB2 monomers exist in a constitutively active conformation and can form homodimers that do not require a ligand for activation ( Fan et al . , 2008 ) . ErbBs achieve great signalling diversity: in part because of the individual biochemical properties of ligands and multiple homo-heterodimer combinations , and in part because they activate multiple components including those known to be critical in neutrophil cell survival such as PI3K , MAPK and GSK-3 , as well as phosphorylating the Bcl-2 protein Bad which inhibits its death-promoting activity ( Yarden and Sliwkowski , 2001 ) . A limitation of our study is the genetically intractability of human neutrophils , meaning we cannot exclude the possibility that the inhibitors are having off target effects in this system . Mammalian models of ErbB deletion are limited by profound abnormalities in utero and during development ( Britsch et al . , 1998; Dackor et al . , 2007; Gassmann et al . , 1995; Miettinen et al . , 1995; Riethmacher et al . , 1997 ) . For this reason , CRISPR/Cas9 was used to knockdown egfra and erbb2 in zebrafish , which confirmed a role for ErbBs in resolving inflammation . Targeting ErbBs genetically and pharmacologically reduces the number of neutrophils at the site of injury in zebrafish , which may reflect inhibition of a number of pathways that regulate neutrophil number in the tissue , including migration pathways ( Ellett et al . , 2015 ) . However , the increase in apoptotic neutrophil count at the site of injury with ErbB inhibitor treatment suggests ErbBs may be inducing anti-apoptotic signalling pathways within this inflammatory environment , which could at least in part be causing the phenotype . The reduced neutrophil count at the injury site may also be due to the increase in apoptotic neutrophils in the CHT , which may be preventing neutrophil migration to sites of injury . The unchanged whole body neutrophil number is potentially due to compensatory upregulation of neutrophil production within the CHT . Genetic deletion , but not pharmacological inhibition , of egfra and erbb2 significantly reduced whole body neutrophil number , which may reflect crispants being without egfra and erbb2 genes from a one-cell stage . Reduced neutrophils at the injury site of crispants may be explained by their reduced whole body neutrophil number , but potentially also defects in the migratory response of these neutrophils to a site of inflammation . Murine models of inflammatory disease , where tyrphostin AG825 was administered either at the time of inflammatory stimulus or once inflammation was established , show an impact on cell number , proinflammatory cytokine production and neutrophil apoptosis , further validating the use of ErbB inhibitors to reduce inflammation . In conclusion , we have identified a previously undefined role for ErbB RTKs in neutrophil survival pathways and a potential new use for ErbB inhibitors in accelerating inflammation resolution . These findings suggest the ErbB family of kinases may be novel targets for treatments of chronic inflammatory disease , and the potential for repurposing ErbB inhibitors currently in use for cancer may have significant clinical potential in a broader range of indications .
Our objectives for this study are to identify compounds that are able to resolve neutrophilic inflammation . To do this we performed unbiased chemical screens in both human neutrophils in vitro and zebrafish models of inflammation in vivo . Results were validated in murine models of peritoneal and airway inflammation and zebrafish tail injury models . Genetic evidence was obtained by CRISPR/Cas9 genetic editing in zebrafish . Neutrophils were isolated from peripheral blood of healthy subjects and COPD patients by dextran sedimentation and discontinuous plasma-Percoll gradient centrifugation , as previously described ( Haslett et al . , 1985; Ward et al . , 1999b ) in compliance with the guidelines of the South Sheffield Research Ethics Committee ( for young healthy subjects; reference number: STH13927 ) and the National Research Ethics Service ( NRES ) Committee Yorkshire and the Humber ( for COPD and age-matched healthy subjects; reference number: 10/H1016/25 ) . Informed consent was obtained after the nature and possible consequences of the study were explained . Mean age in years was 61 . 7 ± 2 . 3 ( n = 10 ) and 66 . 0 ± 3 . 6 ( n = 7 ) for COPD and age-matched healthy subjects respectively . Ultrapure neutrophils , for Kinexus antibody array experiments , were obtained by immunomagnetic negative selection as previously described ( Sabroe et al . , 2002 ) . Neutrophils were cultured ( 2 . 5 × 106/ml ) in RPMI 1640 ( Gibco , Invitrogen Ltd ) supplemented with 10% FCS 1% penicillin-streptomycin , in the presence or absence of the following reagents: GMCSF ( PeproTech , Inc ) , N6-MB-cAMP ( Biolog ) , anti-ErbB3 blocking antibody , Tyrphostin AG825 ( both Sigma-Aldrich ) , CP-724714 ( AdooQ Bioscience ) , Erbstatin analog ( Cayman Chemicals ) , Pyocyanin ( Usher et al . , 2002 ) or compounds from PKIS ( Published Kinase Inhibitor Set 1 , GlaxoSmithKline ) at concentrations as indicated . In vitro screening of PKIS in neutrophil apoptosis assays . PKIS consists of 367 small molecule protein kinase inhibitors and is profiled with respect to target specificity ( Elkins et al . , 2016 ) . In primary screen experiments , neutrophils ( from five independent donors over 5 days ) were incubated with each compound at 62 µM for 6 hr . Apoptosis was measured by flow cytometry ( Attune , Invitrogen ) . Secondary screening was performed with selected compounds that accelerated neutrophil apoptosis greater than twofold in the primary screen . Compounds were incubated with neutrophils at 10 µM for 6 hr and apoptosis assessed by Attune flow cytometry . Neutrophil apoptosis was assessed by light microscopy and by flow cytometry . Briefly , for the assessment of apoptosis by light microscopy based on well-characterised morphological changes , neutrophils were cytocentrifuged , fixed with methanol , stained with Reastain Quick-Diff ( Gentaur ) , and then apoptotic and non-apoptotic neutrophils were counted with an inverted , oil immersion microscope ( Nikon Eclipse TE300 , Japan ) at 100X magnification ( Savill et al . , 1989 ) . To assess apoptosis by flow cytometry , neutrophils were stained with PE conjugated Annexin-V ( BD Pharminogen ) and TO-PRO-3 ( Thermofisher Scientific ) ( Savill et al . , 1989; Vermes et al . , 1995; Ward et al . , 1999a ) and sample acquisition was performed by an Attune flow cytometer ( Life Technologies ) and data analysed by FlowJo ( FlowJo LLC ) . Neutrophils were incubated with N6-MB-cAMP [100 µM] for 30 and 60 min or lysed immediately following isolation ( t0 ) . Cells were lysed in PBS containing Triton-X , 1 µM PMSF and protease inhibitor cocktail and following 2 min on ice were centrifuged at 10 , 000 RPM to remove insoluble material . Lysates ( containing protein at 6 mg/mL ) from four donors were pooled prior to Kinex antibody microarray analysis ( Kinexus Bioinformatics ) ( Zhang and Pelech , 2012 ) . Lysates are subjected to 812 antibodies including phospho-site specific antibodies to specifically measure phosphorylation of the target protein . Fluorescent signals from the array were corrected to background and log2 transformed and a Z score calculated by subtracting the overall average intensity of all spots within a sample , from the raw intensity for each spot , and dividing it by the standard deviations ( SD ) of all the measured intensities within each sample ( Cheadle et al . , 2003 ) . Z ratio values are further calculated by taking the difference between the averages of the Z scores and dividing by the SD of all differences of the comparison ( e . g , 30 min treated samples versus 0 min control ) . A Z ratio of ±1 . 5 is considered to be a significant change from control . Whole cell lysates were prepared by resuspending human neutrophils ( 5 × 106 ) in 50 µl hypotonic lysis buffer ( 1 mM PMSF , 50 mM NaF , 10 mM Sodium orthovanadate , protease inhibitors cocktail in water ) , and by boiling with 50 µl 2X SDS buffer ( 0 . 1M 1 , 4-Dithio-DL-threitol , 4% SDS , 20% Glycerol , 0 . 0625M Tris-HCl pH6 . 8% and 0 . 004% Bromophenol blue ) . Protein samples were separated by SDS-polyacrylamide gel electrophoresis , and electrotransfer onto PVDF ( polyvenylidene difluoride ) membranes was performed by semi-dry blotting method . Membranes were then blocked with 5% skimmed milk in TBS-tween and probed against antibodies to p-AKT , AKT ( both Cell signalling Technology ) , Mcl-1 ( Santa Cruz Biotechnology ) , ErbB2 ( New England Biolabs ) , p38 or β-actin ( loading controls , StressMarq Biosciences Inc or Sigma respectively ) , followed by HRP-conjugated secondary antibodies and detection with chemiluminescent substrate solution ECL2 ( GE Healthcare ) . The neutrophil-specific , GFP-expressing transgenic zebrafish line , Tg ( mpx:GFP ) i114 , ( referred to as mpx:GFP ) ( Renshaw et al . , 2006 ) was raised and maintained according to standard protocols ( Nüsslein-Volhard and Dahm , 2002 ) in UK Home Office approved aquaria in the Bateson Centre at the University of Sheffield , according to institutional guidelines . Adult fish are maintained in 14 hr light and 10 hr dark cycle at 28°C . PKIS screening: Tail fin transection was performed on mpx:GFP zebrafish larvae at 3 days post-fertilisation ( dpf ) ( Elks et al . , 2011; Renshaw et al . , 2006 ) . At 6 hr post-injury ( hpi ) , larvae that had mounted a good inflammatory response , as defined by recruitment of >15 neutrophils to the injury site , were arrayed at a density of 3 larvae per well and incubated with PKIS compounds at a final concentration of 25 µM or vehicle control for a further 6 hr . At 12 hpi , the plate was scanned using prototype PhenoSight equipment ( Ash Biotech ) . Images were scored manually as described previously ( Robertson et al . , 2014 ) . In brief , each well of three larvae was assigned a score between 0–3 , corresponding to the number of larvae within the well with a reduced number of neutrophils at the site of injury . Kinase inhibitors which reduced green fluorescence at the injury site to an extent that their mean score was ≥1 . 5 were regarded as hit compounds . ErbB inhibition studies: Briefly , two dpf mpx:GFP larvae were treated with Tyrphostin AG825 [10 µM] for 16 hr before undergoing tailfin transection ( Elks et al . , 2011; Renshaw et al . , 2006 ) . The number of neutrophils at the site of injury was determined at 4 and 8 hpi by counting GFP-positive neutrophils by fluorescent microscopy . To enumerate neutrophils across the whole body , uninjured larvae were treated with Tyrphostin AG825 [10 µM] for 24 hr and then mounted in 0 . 8% low-melting point agarose ( Sigma-Aldrich ) followed by imaging by fluorescence microscopy ( Nikon Eclipse TE2000-U ) at 4X magnification , followed by manual counting . Larvae from each experimental group were pooled into 1 . 5 mL eppendorf tubes . TSA signal amplification of GFP-labelled neutrophils ( driven by endogenous peroxidase activity ) was carried out using TSA Plus Fluorescein System ( Perkin Elmer ) . Larvae were fixed overnight in 4% paraformaldehyde at 4°C after which they were subjected to proteinase K digestion . Larvae were post-fixed in 4% paraformaldehyde , before subsequent TUNEL staining for apoptosis using ApopTag Red In Situ Apoptosis Detection Kit ( Millipore ) . Larvae were then mounted in low-melting point agarose and images acquired and analysed using UltraVIEWVoX spinning disc confocal laser imaging system with Volocity 6 . 3 software ( Perkin Elmer ) . Apoptotic neutrophil count was determined firstly by identifying cells with co-localisation of the TSA and TUNEL stains , then confirmed by accounting for apoptotic neutrophil morphology . Transient dual knockdown of egfra and erbb2 was induced using a Cas9 nuclease ( New England Biolabs ) in combination with transactivating RNA ( tracr ) and synthetic guide RNAs specific to zebrafish egfra and erbb2 genes ( Merck ) . The non-targetting control in these experiments was a guide RNA targetted towards tyrosinase , a gene involved in pigment formation and therefore easy to identify when mutated , and which is used by others in the field as a CRISPR/Cas9 control ( O’Connor et al . , 2019; Varshney et al . , 2016 ) . We have previously shown that this guide does not influence neutrophilic inflammation in the zebrafish ( Evans et al . , 2019; Isles et al . , 2019 ) . Guide RNAs were designed using the online tool CHOPCHOP ( https://chopchop . cbu . uib . no/ ) with the following sequences: efgra: TGAATCTCGGAGCGCGCAGGAGG; erbb2: AACGCTTTGGACCTACACGTGGG; tyrosinase: GGACUGGAGGACUUCUGGGG . Each guide RNA was resuspended to 20 μM in nuclease-free water with 10 mM Tris-HCl ( pH8 ) . Guide RNA [20 μM] , tracr [20 μM] and Cas9 protein [20 μM] were combined ( in a 1:1:1 ratio ) . 0 . 5 μL phenol red was added to each injection solution for visualisation . A graticule was used to calibrate glass capillary needles to dispense 0 . 5 nL of injection solution , and 1 nL was injected into the yolk sac of single-cell stage mpx:GFP embryos . Tail injury assays were carried out at two dpf as described above . High-resolution melt curve analysis was used to determine the rate of egfra and erbb2 mutation in larvae at two dpf . Genomic DNA was collected from individual larvae in both the control and experimental groups , by adding 90 μL 50 mM NaOH to each larvae in a 96-well qPCR plate and incubating at 95°C for 20 min . 10 μL Tris-HCl ( pH 8 ) was then added as a buffer . Master mixes containing either egfra or erbb2 primers ( Integrated DNA Technologies ) ( sequences in table below ) were made up , with each well containing: 0 . 5 μL 10 μM forward primer , 0 . 5 μL 10 μM reverse primer , 5 μL 2X DyNAmo Flash SYBR Green ( Thermo Scientific ) , 3 μL milliQ water . One μL genomic DNA was added to each master mix in a 96-well qPCR plate . Melt curve analysis was performed and analysed with Bio-Rad Precision Melt Analysis software . Mutation rate was calculated by determining the percentage of egfra erbb2 larvae that showed a different melt-curve profile to the genomic DNA collected from tyrosinase fish ( based on 95% confidence intervals ) . The average mutation rate in our experiments was 97 . 5% and 87 . 9% for egfra and erbb2 , respectively . Primer sequences used for high-resolution melt curve analysis . GeneForward primer sequenceReverse primer sequenceProduct sizeegfraCCAGCGGTTCGGTTTATTCAGCGTCTTCGCGTATTCTTGAGG100erbb2ACAAAGAGCCCAAAAACAGGTTTATCCTTCAGTGCATACCCAGA93 All work involving animals was performed in accordance with the Animal ( Scientific procedures ) Act 1986 . Protocols were produced in line with PREPARE guidelines and FRAME recommendations and were reviewed by the University of Sheffield’s Animal Welfare Committee . C57BL/6 mice ( female , 9–10 weeks old ) were nebulised with LPS ( 3 mg per group of 8 mice ) ( Pseudomonas aeruginosa , Sigma-Aldrich ) and immediately injected intraperitoneally ( i . p . ) with either Tyrphostin AG825 ( Tocris Bioscience ) at 20 mg/Kg in 10% DMSO v/v in vegetable oil ( eight mice , treatment group ) or an equivalent volume of 10% DMSO v/v in vegetable oil ( eight mice , control group ) ( Kedrin et al . , 2009; Roos et al . , 2014 ) . After 48 hr the mice were sacrificed by terminal anaesthesia by i . p . pentobarbitone and subjected to bronchoalveolar lavage ( BAL , 4 × 1 mL of saline ) . BAL samples were microcentrifuged and the cellular fraction counted by a hemocytometer and cytocentrifuged . Neutrophil apoptosis and macrophage efferocytosis of apoptotic neutrophils was quantified by oil immersion light microscopy ( Nikon Eclipse TE300 , Japan ) . C57BL/6 mice were i . p . injected with 1 mg zymosan ( Sigma-Aldrich ) and 4 hr later injected i . p with 20 mg/Kg Tyrphostin AG825 in 10% DMSO v/v in vegetable oil ( five mice , treatment group ) or an equivalent volume of 10% DMSO v/v in vegetable oil ( five mice , control group ) . At 20 hr the mice were subjected to terminal gaseous anaesthesia ( isoflurane ) followed by a cardiac puncture and peritoneal lavage ( 4 × 1 mL of saline ) . WBC , neutrophils and macrophages were enumerated in blood by an automated haematology analyser ( KX-21N , Sysmex , Milton Keynes , UK ) . Lavage samples were microcentrifuged and the cellular fraction subjected to flow cytometry and cytocentrifuged for light microscopy . IL-6 , KC ( Duoset ELISA kits , R and D systems ) and IgM ( Thermofisher Scientific ) in cell free lavage were measured by ELISA as per manufacturer’s instructions . Data were analysed using GraphPad Prism 8 ( GraphPad Software , San Diego , CA ) using one-way or two-way ANOVA ( with appropriate post-test detailed in the Figure legends ) for all in vitro data and appropriate in vivo experiments . Non-parametric tests ( Mann-Whitney U-test or Kruskal-Wallis test ) were used for selected in vivo experiments with non-Gaussian distribution . Data are expressed as mean ± SEM ( standard error of mean ) , and significance was accepted at p<0 . 05 .
Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions , including chronic obstructive pulmonary disease ( COPD ) . There are few specific therapies that reverse neutrophilic inflammation , but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets . Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution . The ErbB inhibitors gefitinib , CP-724714 , erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils , including neutrophils from people with COPD . Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo . Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice , and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model . Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish . Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease .
Chronic obstructive pulmonary disease ( or COPD ) is a serious condition that causes the lungs to become inflamed for long periods of time , leading to permanent damage of the airways . Immune cells known as neutrophils promote inflammation after an injury , or during an infection , to aid the healing process . However , if they are active for too long , they may also cause tissue damage and drive inflammatory diseases including COPD . To limit damage to the body , neutrophils usually have a very short lifespan and die by a regulated process known as apoptosis . Finding ways to stimulate apoptosis in neutrophils may be key to developing better treatments for inflammatory diseases . Cells contain many enzymes known as kinases that control apoptosis and other cell processes . Drugs that inhibit specific kinases are effective treatments for some types of cancer and other conditions , and new kinase-inhibiting drugs are currently being developed . However , it remains unclear which kinases regulate apoptosis in neutrophils or which kinase-inhibiting drugs may have the potential to treat COPD and other inflammatory diseases . To address these questions , Rahman et al . tested over 350 kinase-inhibiting drugs to identify ones that promote apoptosis in neutrophils . The experiments showed that human neutrophils treated with drugs that inhibit the ErbB family of kinases died by apoptosis more quickly than untreated neutrophils . Next , Rahman et al . used zebrafish with injured tail fins as models to study inflammation . Zebrafish treated with one of these drugs – known as Tyrphostin AG825 – had lower levels of inflammation and their neutrophils underwent apoptosis more frequently than untreated zebrafish . Since drugs can have off-target effects , Rahman et al . went on to show using gene-editing technology that reducing the activity of two genes that encode ErbB kinases in zebrafish also decreased the levels of inflammation in the fish . Further experiments used mice that develop inflammation in the lungs similar to COPD in humans . As expected , neutrophils in the lungs of mice treated with Tyrphostin AG825 underwent apoptosis more frequently than those in untreated mice . These dead neutrophils were effectively cleared by other immune cells called macrophages , which also helps limit damage caused by neutrophils . Together , these findings show that Tyrphostin AG825 and other drugs that inhibit ErbB kinases help to reduce inflammation by promoting the death of neutrophils . Since several of these drugs are already used to treat human cancers , it may be possible in the future to repurpose them for use in people with COPD and other long-term inflammatory diseases . Determining whether this is possible is an aim for future studies .