The current study demonstrates that constitutive overexpression of Fizz1 in lung epithelial cells increases the percentage of bone marrow derived hematopoietic cells in the lung. Our findings support earlier work where Fizz1 was overexpressed in the lung using a recombinant adeno-associated viral (AAV) vector, and bone marrow derived cells of hematopoietic lineage were recruited to the smooth muscle layer of pulmonary vessels . In the current study we further characterized the changes in the lung cell population with Fizz1 overexpression using FACS analysis and demonstrated that Fizz1 induced recruitment was associated with a higher percentage of cells staining for both CD11c and F4/80, which are markers associated with dendritic cells. This is the first study to demonstrate a direct role for Fizz1 produced in lung epithelial cells in vivo in the recruitment of dendritic cells into the lung and further supports a role for the Fizz family as a chemoattractant for bone marrow derived cells. Recently, Fizz2 has also been shown to directly cause chemoattraction of bone-marrow-derived dendritic cells, and lung recruitment of bone-marrow-derived cells including dendritic cells was impaired in Fizz2 knockout mice .
Our study demonstrated that epithelial overexpression of Fizz1 without a concomitant lung injury was not associated with changes in lung architecture or lung fibrosis. The role of Fizz1 in vivo in altering pulmonary fibrosis is unclear. Fizz1 overexpression with adenoviral vectors increased thickening and collagen surrounding pulmonary arteries . However, Fizz1 knockout mice challenged with the parasitic eggs of S. Mansoni eggs displayed heightened pulmonary fibrosis suggesting that Fizz1 is a negative regulator of parasite induced pro-fibrotic Th2 cytokines and in this model Fizz1 is anti-fibrotic . Another recent study using Fizz1 KO mice demonstrates that Fizz1 was largely dispensable during Aspergilus fumigatous allergen-induced production of Th2 cytokines, eosinophilia and airway remodeling .
The migration of myeloid cells from the bone marrow to the lung has been shown to regulate inflammation and the pathogen burden in the lung. Data over the past decades also supports a role for bone marrow derived fibrocytes in contributing to the cellular accumulation of mesenchymal cells and extracellular matrix . Although, myeloid cells have been shown to migrate into the lung, the secretory mediators involved in the migration and maturation to different cell subsets, along with their functional role remains an area of active investigation. The present study does not establish whether Fizz1 chemoattraction of dendritic cells is due to Fizz1 alone or Fizz1 regulation or interaction with other chemoattractant molecules. The myeloid cell subsets that populate in the lung include dendritic cells, alveolar macrophages and interstitial macrophages. Notably, findings of this study demonstrate that Fizz1 overexpression increased dendritic cells in the lung without altering lung architecture. Dendritic cells are critical members of adaptive immunity in the lung involved in antigen processing and mediating immunity responses. Lung dendritic cells have been shown to accumulate in human fibrotic lung disease and bleomycin-induced fibrosis, but have not been shown to independently and directly cause lung remodeling. The present study shows that Fizz1 mediated attraction of dendritic cells was not adequate to influence lung fibrosis. Fizz1 overexpression has no effect on CD45 + CD11b + cells in the lung during bleomycin-induced injury. Future studies are warranted to identify the roles of Fizz1-recruited dendritic cells in immunity against bacterial and viral pathogens and also infections associated injury and remodeling in the lung.
As we were not able to demonstrate direct remodeling from Fizz1 overexpression, we examined if Fizz1 overexpression altered fibrosis in known inflammatory models. Fizz1 was overexpressed in the lung prior to challenge with bleomycin or silica. Both models induce inflammatory changes leading to fibrosis; however the inflammatory responses are different with bleomycin resulting in an oxidative injury associated with neutrophil influx, while silica leads to macrophage-related activation and formation of inflammatory nodules. With both challenges, overexpression of Fizz1 did not alter the degree of lung remodeling. In the bleomycin injury, expression of Fizz1 did cause a greater loss of body weight. Although the mode of action is not known, these data further support the concept that lung epithelial cell produced Fizz1 has systemic effects consistent with effect on systemic recruitment of bone marrow derived cells. While the precise mode of activity is not known, Fizz1 has been shown to alter IL-13 induced chemokine levels, inhibit nerve growth factor neuronal survival, and cause bone marrow cell migration in a transwell assay through interactions with Bruton’s tyrosine kinase. Thus Fizz1 may act directly or indirectly by influences on cytokine or growth factor induced regulation.
In spite of an increase in Fizz1 transcripts induced by bleomycin in the lung, the results of our study do not support a role for Fizz1 produced in the lung epithelium as a direct cause of lung fibrosis. In the present study, single instillations of bleomycin and silica were used which cause a slowly resolving inflammation leading to diffused fibrosis or nodular fibrosis. In hypoxic injury, the injury is continuous and leads to pulmonary hypertension. Fizz1 may act in a localized fashion to increase fibrotic responses around pulmonary arterioles during chronic hypoxia but not to increase granuloma formation around S. mansoni parasites or retained silica particles. In addition, the differences in the activation of immune pathways, cellular sites of Fizz1 production such as macrophages and eosinophils, and the amount of Fizz1 produced may be responsible for observed differences between studies. Lack of observable histological and hydroxyproline differences in bleomycin or silica-induced lung remodeling in Fizz1 overexpressing mice could be due to doses of bleomycin or silica. While the doses used in this study have been used in previous studies, the tested dose may mask or not be a sufficient dose to show Fizz1 effects. Alternatively, other members of the Fizz family may be involved in pulmonary fibrosis. When challenged with bleomycin, Fizz2 knockout mice show a decrease in the absolute number of total BAL cells, cytokine gene expression and extracellular matrix deposition than control mice .