IMPC: Role of airway macrophage Btk signalling in pulmonary aspergillosis

Pulmonary aspergillosis is a severe mould infection of the lung that is estimated to affect 7 to 8 million people globally, in the context of either weakened immune systems or chronic lung diseases. The most severe and rapidly fatal infections occur in individuals with very low numbers of neutrophils, a type of immune white cell. However, the vast majority of individuals developing chronic forms of pulmonary aspergillosis have normal numbers of neutrophils, but are found to have abnormal function of a different white cell called the macrophage.
Our studies have shown that when Aspergillus fumigatus, the causative agent of pulmonary aspergillosis, is engulfed by macrophages, it activates an immune pathway that is critically dependent upon the enzyme Bruton's tyrosine kinase (Btk). This is surprising, because this enzyme is usually thought of as specific to white cells that produce antibodies called B cells. Furthermore, clinical inhibitors of Btk such as Ibrutinib have become one of the main treatment for chronic lymphocytic leukaemia, and other autoimmune diseases.
Recently a number of reports have demonstrated that the use of Ibrutinib in combination with other immunosuppressive medicine confers a very high risk of pulmonary and invasive aspergillosis. Given our current understanding of immunity to aspergillosis, this strongly suggests that Ibrutinib impairs the innate immune system, of which macrophages and neutrophils play the major role. Given that macrophages are the first immune cells to come into contact with A. fumigatus in the lung, they play a critical role in immune signalling, and we have shown that this is dependent on Btk during A. fumiagtus infection, it is now our primary goal to study how deletion of the gene for Btk specifically in airway macrophages affects immune responses in pulmonary aspergillosis.
In order to achieve this, we propose to selective delete Btk in airway macrophages using a special form of cell-specific gene deletion calles Cre-Lox. To achieve this we will need to first breed mice with a specific mutation in the Btk gene flanks to another mouse that has expression of an enzyme just in airway macrophages that cuts out this Btk gene when the mice are crossed and the offspring inherit both genes. We will then infect these mice with A. fumigatus and study whether or not they are more likely to develop lethal forms of infection than normal mice. We will also study whether or not infection leads to impaired control of fungal growth in the lung, or abnormalities in either immune signalling or trafficking of immune cells into the lung during infection. Finally, we will use innovative microscopy techniques developed in the laboratory to specifically image individual macrophages in the lung infected with A. fumigatus in lung tissue sections, and whether they are more prone to die during infection.
These studies will give us comprehensive animal model information on what immune defects result when Btk is deleted in airway macrophages. The data generated will then be used to apply for a larger MRC grant to further describe the Btk molecular signalling pathway in macrophages, and how this is impaired in patients with X-linked agammaglobulinaemia, a disease associated with recurrent lung infections and mutations in Btk. These studies will ultimately reveal the importance of Btk in innate immunity and will have important ramifications for the development of novel therapies that selectively target these pathways in B cells without causing off target effects in macrophages.

Pulmonary aspergillosis is a severe mould lung infection that affects 7-8 million people globally. Whilst invasive disease is mainly a consequence of neutropaenia, chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis form the major burden of disease, accounting for 7-8 million cases globally. These individuals develop chronic and destructive mucosal infections in the absence of neutropaenia and have defects in macrophage responses to Aspergillus fumigatus challenge. We recently showed that Bruton's tyrposine kinase, a key mediator of B cell responses, is critical for macrophage responses to A. fumigatus. Furthermore, a number of studies have shown that patients with chronic lymphocytic leukaemia receiving the novel Btk inhibitor Ibrutinib are at very high risk of disseminated aspergillosis. Better understanding of the role of Btk in macrophage responses to A. fumigatus in the lung are now required.
I will exploit available Cre-Lox technology to delete Btk in CD11c airway macrophages. This will allow me to investigate the importance of macrophage Btk signalling in my murine models of pulmonary aspergillosis. I will undertake a survival analysis comparing CD11c-Btk mice to CD11c-Cre controls after intranasal challenge with 108 A. fumigatus conidia. I will then study control of fungal infection and immune responses by infecting CD11c-Btk mice to CD11c-Cre controls and then performing bronchoalveolar lavage at 24 hours post infection. Cytokine responses, fungal burden and neutrophil influx will be determined. I will exploit innovative precision cut lung slice protocols developed in my laboratory to directly visualise macrophage control of infection and cell death responses, and how this impacts control of infection in the lung, by confocal fluorescence microscopy.
These studies will comprehensively define the role of airway macrophage Btk responses in the lung, and form the basis for a follow on grant application.