Following tissue damage by internal and external factors, tissue repair occurs through the dynamic interaction of a wide variety of cells; however, fibrosis can also occur as a result. To understand the complex cellular and molecular basis of fibrosis, it is necessary to analyze the cellular processes involved in vivo. This study aimed to develop an imaging system for visualizing the dynamics of immune cells in the lung using intravital two-photon microscopy and to elucidate the mechanisms involved in the pathogenesis of pulmonary fibrosis, a disorder for which there are few available treatments. By means of the intravital lung imaging system, we visualized cellular morphology and motility in the lung of bleomycin-induced pulmonary fibrosis in vivo. We identified a novel macrophage subset, which was not observed under homeostatic conditions, appearing adjacent to pulmonary blood vessels, followed by the development of fibrosis. To examine whether these macrophages are involved in the pathogenesis of fibrosis, we sorted these cells from fibrotic lungs and transferred them into the trachea of healthy recipient mice. Intravital imaging showed that the amount of collagen fibers significantly increased in the lung after adoptive transfer of cells, suggesting that they have the capacity to induce fibrosis. Furthermore, RNA-sequence analysis demonstrated that these macrophages expressed higher levels of several fibrotic markers, compared to other types of macrophages. We also found that myeloid cell-specific deletion of fibrotic markers alleviated pulmonary fibrosis in vivo. In conclusion, we established an intravital lung imaging system for visualizing fibrosis and identified a novel macrophage population in the lungs at the onset of fibrosis. This approach will yield compelling insights into the molecular mechanisms underlying fibrosis, which could also serve as the basis for developing novel anti-fibrotic therapies.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization due to pediatric viral respiratory tract infection, responsible for 200,000 infant deaths worldwide. There are no effective antiviral therapies or clinically approved RSV vaccines for infants. Therefore, understanding RSV-host interactions is crucial for developing new therapies. We have previously shown that RSV infection induces alveolar macrophage necroptosis through the activation of receptor-interacting protein kinase 3 (RIPK3), enhancing disease pathogenesis. Pharmacological inhibition or genetic deficiency of RIPK3 decreases RSV viral load and lung inflammation in mice. Dabrafenib is an FDA- and Health Canada-approved anticancer drug that selectively inhibits RIPK3 as an off-target effect, and lessens tissue injury in different disease models. Thus, we hypothesized that dabrafenib has antiviral effects against RSV by inhibiting RIPK3-mediated necroptosis. We assessed dabrafenib drug repurposing to treat RSV infection in vitro. To analyze the effect of dabrafenib on A549 cell viability, cells were treated with increasing concentrations of dabrafenib for different time points using MTT assay. Lactate dehydrogenase (LDH) release was measured as a marker of lytic cell death. A549 cells were infected with RSV-GFP and treated with dabrafenib either simultaneously, prophylactically, or therapeutically. Infection rate and fluorescence intensity were quantified by immunofluorescence. To understand the effect of dabrafenib on viral progeny release, the supernatant of infected A549 cells therapeutically treated with dabrafenib was used to perform a lysis plate titration assay in HEP-2 cells. We also tested the antiviral effects of dabrafenib using human primary nasal epithelial cells (HNECs) and quantified RSV replication by both immunofluorescence and qPCR. Proteomic analyses of epithelial cells infected with RSV and treated with dabrafenib was conducted using quantitative mass spectrometry. Dabrafenib did not alter A549 cell viability throughout 72h at all concentrations tested. Dabrafenib protected A549 cells from RSV-induced lytic cell death. Importantly, the prophylactic, therapeutic, and simultaneous treatments significantly decreased RSV infection rate and fluorescence intensity. Furthermore, therapeutic treatment with dabrafenib significantly reduced the release of RSV infectious progeny. Dabrafenib also reduced in 80% RSV viral load in HNECs. Dabrafenib profoundly altered the A549 cell proteome, inducing the upregulation of some proteins involved in antiviral response. Dabrafenib treatment significantly impairs RSV replication and protects respiratory epithelial cells from death. It also modulates the epithelial cell proteome by upregulating antiviral proteins. Repurposing dabrafenib may be a valuable therapeutic option against RSV infection.

Eosinophils (Eos) reside in multiple organs during homeostasis and respond rapidly to an inflammatory challenge. Although Eos share chemical staining properties, they also demonstrate phenotypic and functional plasticity that is not fully understood. Here, we used a murine model of allergic lung inflammation to characterize Eos subsets and determine their spatiotemporal and functional regulation during inflammation and its resolution in response to resolvin D2 (RvD2), a potent specialized pro-resolving mediator. Two Eos subsets were identified by CD101 expression: CD101low Eos, that were found in naïve lungs and during inflammation, and CD101high Eos that were present only during inflammation. CD101low and CD101high Eos had distinct anatomic localization as the first being found predominantly in the lung vascular niche and the second mainly located in bronchoalveolar space. CD101low Eos responded to allergen challenge by increasing their activation and migrating into the lung interstitium and bronchoalveolar space to then become CD101high Eos. This process was governed in part by IL-5. RvD2 reduced total number of Eos, specially CD101high Eos in bronchoalveolar lavage and changed their activation phenotype by at least two distinct mechanisms: decreasing IL-5-dependent recruitment of CD101low Eos and decreasing conversion of CD101low Eos to CD101high Eos. Collectively, these findings indicate that Eos are a heterogeneous pool of cells with distinct activation states and spatiotemporal regulation during resolution of inflammation and that RvD2 is a potent pro-resolving mediator for Eos recruitment and activation.