Notably, alveolar macrophages are not the only cell type which can engulf apoptotic cells12,27. lung fibrosis, confirming that AEC injury is sufficient to cause fibrosis. In the present study, we find that SPC-DTR mice develop increased activation of caspase 3/7 after initiation of diphtheria toxin treatment consistent with apoptosis within AECs. We also find evidence of efferocytosis, the uptake of apoptotic cells, by alveolar macrophages in this model. To determine the importance of efferocytosis in lung fibrosis, we treated cultured alveolar macrophages with apoptotic type II AECs and found that the uptake induced pro-fibrotic gene expression. We also found that the repetitive intrapulmonary administration of apoptotic type II AEC or MLE-12 cells induces lung fibrosis. Vegfa Finally, mice lacking a key efferocytosis receptor, CD36, developed attenuated fibrosis in response to apoptotic MLE-12 cells. Collectively, these studies support a novel mechanism linking AEC apoptosis with macrophage 8-Dehydrocholesterol pro-fibrotic activation via efferocytosis and reveal previously unrecognized therapeutic targets. Introduction Progressive alveolar fibrosis is usually a serious complication of certain systemic inflammatory disorders, inorganic and organic dust exposures, drug toxicity and main diseases of the lung including idiopathic pulmonary fibrosis (IPF)1C5. Mounting evidence implicates defects in the type II alveolar epithelial cell (AEC) in disease pathogenesis6. For example, histopathologic abnormalities of the epithelium including apoptosis are observed in tissue sections from IPF patients and in animal models of pulmonary fibrosis7C9. Furthermore, mutations in type II AEC genes including surfactant proteins A and C are linked to familial disease10. Finally, transgenic animal experiments from our laboratory confirm that targeted injury to the type II alveolar epithelium is sufficient to 8-Dehydrocholesterol initiate lung scarring11. Despite the substantial evidence linking type II AEC injury/death to the development of fibrosis, the pathways that translate an epithelial insult into lung collagen accumulation have not been well-characterized. Possible mechanisms by which damage to the alveolar epithelium lead to fibrosis have focused on either loss of anti-fibrotic functions supplied by healthy cells or an up-regulation of pro-fibrotic factors from the hurt AECs. An alternative mechanism supported by emerging evidence suggests that the apoptotic AECs can directly trigger progressive fibrosis by inducing a response in neighboring cells. Cellular apoptosis terminates with fragmentation resulting in formation of vesicles termed apoptotic body. Apoptotic body are characterized in part by the appearance of phosphatidylserine around the outer leaflet of the lipid bilayer which serves as a acknowledgement signal for phagocytic cells to ingest the cellular debris. Apoptotic cells and body modulate cell behavior as they undergo phagocytosis in a process known as efferocytosis12. For example, in models of acute lung injury, efferocytosis of apoptotic neutrophils has emerged as a key pathway in regulating macrophage function and restoring homeostasis by promoting release of anti-inflammatory cytokines13. The ingestion of apoptotic neutrophils is usually well analyzed and involves protein receptors expressed on the surface of the ingesting cells and the apoptotic body12. Of notice, there is considerable overlap between anti-inflammatory and pro-fibrotic pathways as exemplified by one statement that found the anti-inflammatory effects of macrophages which experienced ingested apoptotic cells resulted from your increased expression of TGF1 (a well-established pro-fibrotic cytokine)13,14. Further evidence linking apoptotic cells with lung fibrosis comes from a report in which the administration of a single dose of lavaged alveolar cells (presumably macrophages) induced to undergo apoptosis caused a fibrotic response in mice15. Although much less is known about the fate of apoptotic AECs and whether their uptake by macrophages might be an important inciting event in fibrosis, we hypothesized that this efferocytosis of apoptotic type II AECs would significantly contribute to the initiation of fibrosis following lung injury. To test this hypothesis, we employed a transgenic model of fibrosis in which mice engineered to 8-Dehydrocholesterol express the diphtheria toxin receptor (DTR) on their type II AECs are treated with repeated doses of diphtheria toxin (DT)11. We also directly administered repeated doses of apoptotic AECs into the lungs of healthy mice. We found that targeted epithelial injury led to.
