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.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1 −/− and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1 −/− mice develop airway inflammation in acute and chronic airway inflammation models. Peli1 −/− animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

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23Neutrophilic inflammation with prolonged neutrophil survival is common to many 24 inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There 25 are few specific therapies that reverse neutrophilic inflammation, but uncovering 26 mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. 27Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model 28 identified ErbBs as common targets of compounds that accelerated inflammation resolution. 65unbiased screening approaches in vitro and in vivo, we here identify inhibitors of the ErbB 66 family of receptor tyrosine kinases (RTKs) as potential therapeutic drivers of inflammation 67 resolution. The ErbB family consist of four RTKs with structural homology to the human 68 epidermal growth factor receptor (EGFR/ErbB1/Her-1). In an in vivo zebrafish model of 69 inflammation, we show that inhibition of ErbBs, pharmacologically and genetically, reduced 70 the number of neutrophils at the site of injury. Furthermore, ErbB inhibitors reduced 71 inflammation in a murine peritonitis model and promoted neutrophil apoptosis and clearance 72 by macrophages in the mouse lung. This study reveals an opportunity for the use of ErbB73 inhibitors as a treatment for chronic neutrophilic inflammatory disease.74 75 Results 76 Identifying kinases regulating the resolution of neutrophilic inflammation in vivo 77Using a well-characterised transgenic zebrafish inflammation model (Henry, Loynes, Whyte, & Renshaw, 2013;Renshaw et al., 2006), we adopted a chemical genetics approach, which 79 has great potential for accelerated drug discovery (Jones & Bunnage, 2017). We initiated 80 inflammation by controlled tissue injury of the zebrafish tail fin and screened a library of 81 kinase inhibitors in order to establish which kinases could be exploited to enhance 82 inflammation resolution in vivo (Fig. S1A). We quantified the ability of a library of 367 publicly 83 available kinase inhibitors (PKIS) (Elkins et al., 2016) to reduce neutrophil number at the site 84 of injury during the resolution phase of inflammation. The screen identified 16 hit compounds 85 which reduced neutrophil number at the site of injury in the zebrafish model (Fig. 1A). For 86 each compound the degree of kinase inhibition had been established (Elkins et al., 2016) 78 87( Fig. 1A). A number of kinases were inhibited by the 16 compounds, with Abelson murine 88 leukaemia viral homolog 1 (ABL1), Platelet-derived growth factor receptor (PDGFR) α, 89PDGFRβ, p38α and ErbB4 being the top five most frequently targeted kinases overall. In 90 addition to frequency of target, we also interrogated selectivity of compound. The most 91 selective compounds, i.e. those that strongly inhibited individual kinases or kinase families, 92 targeted the kinases YES, ABL1, p38 and the ErbB family. Apoptosis is an important 93 mechanism contributing to inflammation resolution; we therefore sought to identify kinases 94 common to both inflammation resolution...