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.
Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Our previous research identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo (Rahman et al., 2019). Here we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration to an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting the drug may have therapeutic benefits in acute inflammatory settings Phosphoproteomics analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.
IntroductionDespite epidemiological associations between community acquired pneumonia (CAP) and myocardial infarction, mechanisms that modify cardiovascular disease during CAP are not well defined. In particular, largely due to a lack of relevant experimental models, the effect of pneumonia on atherosclerotic plaques is unclear. We describe the development of a murine model of the commonest cause of CAP, Streptococcus pneumoniae pneumonia, on a background of established atherosclerosis. We go on to use our model to investigate the effects of pneumococcal pneumonia on atherosclerosis.MethodsC57BL/6J and ApoE-/- mice were fed a high fat diet to promote atherosclerotic plaque formation. Mice were then infected with a range of S. pneumoniae serotypes (1, 4 or 14) with the aim of establishing a model to study atherosclerotic plaque evolution after pneumonia and bacteremia. Laser capture microdissection of plaque macrophages enabled transcriptomic analysis.ResultsIntratracheal instillation of S. pneumoniae in mice fed a cholate containing diet resulted in low survival rates following infection, suggestive of increased susceptibility to severe infection. Optimization steps resulted in a final model of male ApoE-/- mice fed a Western diet then infected by intranasal instillation of serotype 4 (TIGR4) S. pneumoniae followed by antibiotic administration. This protocol resulted in high rates of bacteremia (88.9%) and survival (88.5%). Pneumonia resulted in increased aortic sinus plaque macrophage content 2 weeks post pneumonia but not at 8 weeks, and no difference in plaque burden or other plaque vulnerability markers were found at either time point. Microarray and qPCR analysis of plaque macrophages identified downregulation of two E3 ubiquitin ligases, Huwe1 and Itch, following pneumonia. Treatment with atorvastatin failed to alter plaque macrophage content or other plaque features.DiscussionWithout antibiotics, ApoE-/- mice fed a high fat diet were highly susceptible to mortality following S. pneumoniae infection. The major infection associated change in plaque morphology was an early increase in plaque macrophages. Our results also hint at a role for the ubiquitin proteasome system in the response to pneumococcal infection in the plaque microenvironment.
Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo. Here, we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration, and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.
Background Despite epidemiological associations between community acquired pneumonia (CAP) and myocardial infarction, mechanisms that modify cardiovascular disease during CAP are not well defined. In particular, largely due to a lack of relevant experimental models, the effect of pneumonia on atherosclerotic plaques is unclear. We describe the development of a murine model of the commonest cause of CAP, Streptococcus pneumoniae pneumonia, on a background of established atherosclerosis. We go on to use our model to investigate the effects of pneumococcal pneumonia on atherosclerosis. Methods C57BL/6J and ApoE−/− mice were fed a high fat diet to promote atherosclerotic plaque formation. Mice were then infected with a range of S. pneumoniae serotypes (1, 4 or 14) with the aim of establishing a model to study atherosclerotic plaque evolution after pneumonia and bacteraemia. Laser capture microdissection of plaque macrophages enabled transcriptomic analysis. Results Intratracheal instillation of S. pneumoniae in mice fed a cholate containing diet resulted in low survival rates following infection, confirming increased susceptibility to severe infection. Optimization steps resulted in a final model of male ApoE−/− mice fed a Western diet then infected by intranasal instillation of serotype 4 (TIGR4) S. pneumoniae followed by antibiotic administration. This protocol resulted in high rates of bacteremia (88.9%) and survival (88.5%). Pneumonia resulted in increased aortic sinus plaque macrophage content 2 weeks post pneumonia but not at 8 weeks, and no difference in plaque burden or other plaque vulnerability markers were found at either time point. Microarray and qPCR analysis of plaque macrophages identified downregulation of two E3 ubiquitin ligases, Huwe1 and Itch, following pneumonia. Treatment with atorvastatin failed to alter plaque macrophage content or other plaque features. Conclusions Without antibiotics, ApoE−/− mice fed a high fat diet were highly susceptible to mortality following S. pneumoniae infection. The major infection associated change in plaque morphology was an early increase in plaque macrophages. Our results also hint at a role for the ubiquitin proteasome system in the response to pneumococcal infection in the plaque microenvironment.
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