Periodic Reporting for period 1 - CFZEBRA (Balancing the immune response in cystic fibrosis: using zebrafish models of infection and inflammation to uncover new therapeutic approaches)
Periodo di rendicontazione: 2017-06-01 al 2019-05-31
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). In people with CF, the defect in CFTR gene causes a severe defect of mucociliary clearance with the development of thick mucus, which forms a protected niche for bacterial growth, resulting in chronic infection and high-intensity inflammation. These factors conspire to damage the lungs leading to extensive morbidity and early death. In addition to known mucociliary defects, several lines of evidence have shown that CFTR dysfunction causes intrinsic alterations in epithelial and innate immune cell responses to tissue injury and pathogens, creating a vicious circle of infection and dysregulated inflammation. Currently, there is no cure for CF and the evaluation of biological function of CFTR in available CF models suffers from several limitations, predominantly the evaluation of phenomena in a pre-existing inflammatory environment. Consequently, the mechanisms by which CFTR directly regulates innate immunity and how CF mutations contribute to infectious and inflammatory pathogenesis in CF have remained obscure. There is therefore a pressing need to develop models allowing direct observation of CFTR-dependent effects on innate immune responses in the absence of pre-existing inflammatory environment.
Zebrafish innate immunity is closely homologous to that of humans, while their optical transparency allows non-invasive, real-time monitoring of infectious and inflammatory processes in the whole organism. Importantly, zebrafish CFTR retains close sequence identity with human. Like mammals, zebrafish CFTR is expressed in epithelial surfaces and myeloid cells and plays an important role in homeostatic balance of fluid composition. The high degree of genetic and functional conservation between the zebrafish and mammalian CFTR and innate immune systems make zebrafish a clinically-relevant system to investigate CF immune-pathophysiology.
The overall objective of this project is to understand how CFTR dysfunction leads to abnormal inflammation and innate immunity in CF using CFTR-mutant zebrafish larvae as an innovative vertebrate model of inflammation and infection, combined with human CF stem cell approaches, in ways that are not possible in other models systems. To achieve this objective, the project is focus on 3 different research themes:
a) Generate and validate new models of CFTR dysfunction.
b) Define the host innate immune response to infection and sterile tissue injury in CF models.
c) Screen for anti-inflammatory / pro-resolution drugs with a potential role in re-balancing the immune responses.
The main goal of this action was to develop an innovative approach using new models in the search of strategies for mimicking the innate immune abnormalities of CF to find new avenues for increasing the understanding on deleterious immunity in CF and to deliver better therapeutic approaches to the clinic.
Zebrafish innate immunity is closely homologous to that of humans, while their optical transparency allows non-invasive, real-time monitoring of infectious and inflammatory processes in the whole organism. Importantly, zebrafish CFTR retains close sequence identity with human. Like mammals, zebrafish CFTR is expressed in epithelial surfaces and myeloid cells and plays an important role in homeostatic balance of fluid composition. The high degree of genetic and functional conservation between the zebrafish and mammalian CFTR and innate immune systems make zebrafish a clinically-relevant system to investigate CF immune-pathophysiology.
The overall objective of this project is to understand how CFTR dysfunction leads to abnormal inflammation and innate immunity in CF using CFTR-mutant zebrafish larvae as an innovative vertebrate model of inflammation and infection, combined with human CF stem cell approaches, in ways that are not possible in other models systems. To achieve this objective, the project is focus on 3 different research themes:
a) Generate and validate new models of CFTR dysfunction.
b) Define the host innate immune response to infection and sterile tissue injury in CF models.
c) Screen for anti-inflammatory / pro-resolution drugs with a potential role in re-balancing the immune responses.
The main goal of this action was to develop an innovative approach using new models in the search of strategies for mimicking the innate immune abnormalities of CF to find new avenues for increasing the understanding on deleterious immunity in CF and to deliver better therapeutic approaches to the clinic.
Chronic pulmonary infections accompanied by persistent neutrophil-dominated inflammation result in severe progressive lung destruction and are the leading causes of morbidity and mortality of CF patients. Our understanding of the mechanistic links between the CFTR mutations and the pathogenesis of infectious and inflammatory pulmonary disease in CF are far from complete.
>To assess the role of CFTR in regulating host innate immune potential in vivo, expression of cftr was knocked-out using CRISPR-Cas9 technology and/or knocked-down by morpholino-modified antisense oligonucleotides in various reporter transgenic lines.
>Exploiting these CFTR-depleted zebrafish models, we investigated the effects of CFTR dysfunction on host innate immune response to infection and tissue injury, and uncovered a number of altered inflammatory processes as critical mechanisms underlying infectious and inflammatory disease in CF.
CF zebrafish exhibit hyper-susceptibility to infection with CF pathogens
Among the deleterious bacteria found in CF airway, Mycobacterium abscessus has emerged as an important respiratory pathogen of major concern in CF centers worldwide. Nevertheless, the specific vulnerability of the CF population to M. abscessus, the potential link with CFTR dysfunction, and how these mycobacteria contribute to progression of lung disease remain unknown. Using CF zebrafish larvae we elucidated the role of CFTR in regulation of innate immunity to M. abscessus infections.
We showed that CF zebrafish exhibit increased susceptibility to M. abscessus infection, resulting in increased mortality and higher bacterial loads, in part due to an inability to generate effective oxidative responses in professional phagocytes. (Bernut et al, 2019, Cell Rep 26:1828-40.e4)
Loss of CFTR function leads an overactive neutrophilic inflammation to tissue injury
It is commonly assumed that the CF-related lung pathology is primarily an infectious disorder: susceptibility to invading pathogens results from airway mucus obstruction and collapse of mucociliary clearance and that the resultant persistent infection drives chronic inflammatory lung damage. However, in CF airways, there is an abnormal inflammatory phenotype often present in absence of detectable infection, raising the question of what drives non-infectious inflammation in CF. Using CF zebrafish, we investigated the effects of CFTR dysfunction on host innate immune response to sterile tissue injury.
We showed that sterile injury leads to high-intensive inflammation, typified by increased then sustained accumulation of neutrophils at wounds: 1) excessive epithelial ROS release drives increased neutrophil recruitment towards wounds; 2) reduction of neutrophil apoptosis and impaired retrograde migration of neutrophils resulting in delayed resolution of inflammation. 3) Therefore, the increased number of neutrophils that mobilized in an uncontrolled manner at wound sites causes persistent inflammation, severe tissue damages, and abnormal tissue repair, which all can be prevented by pharmacological modulation of neutrophilic response using the anti-inflammatory molecule, Tanshinone IIA. (Bernut et al, 2018, J Cyst Fibros June, Vol 17, Sup 3, PS8-9; manuscript in preparation)
>To assess the role of CFTR in regulating host innate immune potential in vivo, expression of cftr was knocked-out using CRISPR-Cas9 technology and/or knocked-down by morpholino-modified antisense oligonucleotides in various reporter transgenic lines.
>Exploiting these CFTR-depleted zebrafish models, we investigated the effects of CFTR dysfunction on host innate immune response to infection and tissue injury, and uncovered a number of altered inflammatory processes as critical mechanisms underlying infectious and inflammatory disease in CF.
CF zebrafish exhibit hyper-susceptibility to infection with CF pathogens
Among the deleterious bacteria found in CF airway, Mycobacterium abscessus has emerged as an important respiratory pathogen of major concern in CF centers worldwide. Nevertheless, the specific vulnerability of the CF population to M. abscessus, the potential link with CFTR dysfunction, and how these mycobacteria contribute to progression of lung disease remain unknown. Using CF zebrafish larvae we elucidated the role of CFTR in regulation of innate immunity to M. abscessus infections.
We showed that CF zebrafish exhibit increased susceptibility to M. abscessus infection, resulting in increased mortality and higher bacterial loads, in part due to an inability to generate effective oxidative responses in professional phagocytes. (Bernut et al, 2019, Cell Rep 26:1828-40.e4)
Loss of CFTR function leads an overactive neutrophilic inflammation to tissue injury
It is commonly assumed that the CF-related lung pathology is primarily an infectious disorder: susceptibility to invading pathogens results from airway mucus obstruction and collapse of mucociliary clearance and that the resultant persistent infection drives chronic inflammatory lung damage. However, in CF airways, there is an abnormal inflammatory phenotype often present in absence of detectable infection, raising the question of what drives non-infectious inflammation in CF. Using CF zebrafish, we investigated the effects of CFTR dysfunction on host innate immune response to sterile tissue injury.
We showed that sterile injury leads to high-intensive inflammation, typified by increased then sustained accumulation of neutrophils at wounds: 1) excessive epithelial ROS release drives increased neutrophil recruitment towards wounds; 2) reduction of neutrophil apoptosis and impaired retrograde migration of neutrophils resulting in delayed resolution of inflammation. 3) Therefore, the increased number of neutrophils that mobilized in an uncontrolled manner at wound sites causes persistent inflammation, severe tissue damages, and abnormal tissue repair, which all can be prevented by pharmacological modulation of neutrophilic response using the anti-inflammatory molecule, Tanshinone IIA. (Bernut et al, 2018, J Cyst Fibros June, Vol 17, Sup 3, PS8-9; manuscript in preparation)
CF is a genetic disease resulting from mutations in CFTR and causes premature death by progressive respiratory failure, itself caused by lung destruction from a vicious circle of repetitive infection and excessive inflammation. Thus far, our understanding of the mechanistic links between the CFTR mutations and the pathogenesis of infectious and inflammatory pulmonary disease in CF is far from complete. To overcome this, we established and exploited new CF models to recapitulate aspects of CF immune-pathogenesis and provide a more comprehensive delineation of the cellular basis linking CFTR deficiency with infectious and inflammatory pathogenesis of the CF airways. We show that CF mutations cause primary alterations in epithelial and innate immune cell responses to both pathogens and tissue injury creating a vicious circle of infection, dysregulated inflammation and failed tissue repair and identify new therapeutic molecules active in a CFTR-deficient context to restore innate immune potential. This will help guide future therapies aimed at correcting the innate susceptibility of CF patients to infective and inflammatory lung disease, with consequent improvement of their life quality and expectancy.