Project description:A single-cell transcriptional analysis was performed on lung immune cellsisolated from influenza PR8 infected mice at 14 days post injury. The goal is to investigate the potential signaling pathway that regulates dysplastic KRT5 cells expansion. The left lung lobe from influenza infected mice were dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all CD45+ cells. We found that the regulation of actin cytoskeleton, focal adhesion, Hippo signaling pathway are highly activated in dysplastic KRT5+ cells, suggesting that these signaling pathway may play a role in the expansion of dysplastic KRT5 cells in influenza infected lungs.
Project description:A single-cell transcriptional analysis was performed on lung epithelial cells isolated from influenza PR8 infected mice at 14 days post injury. The goal is to investigate the potential signaling pathway that regulates dysplastic KRT5 cells expansion. The left lung lobe from influenza infected mice were dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all Epcam+ cells. We found that the regulation of actin cytoskeleton, focal adhesion, Hippo signaling pathway are highly activated in dysplastic KRT5+ cells, suggesting that these signaling pathway may play a role in the expansion of dysplastic KRT5 cells in influenza infected lungs.
Project description:Chronic obstructive pulmonary disease (COPD) is characterized by inflammation and emphysema, leading to progressive alveolar destruction. Currently, no therapies effectively regenerate the alveolar epithelium. Here, we developed and characterized a feeder- and serum-free primary adult human organoid model to investigate how inflammatory conditions influence alveolar regeneration. Using Wnt activation and heregulin-β, we achieved long-term expansion of progenitor cells, while AT2 maturation protocols enhanced surfactant production, including the formation of tubular myelin. Introducing a LATS inhibitor to the expansion conditions induced an AT1 differentiation program without eliminating AT2 cells. Single-cell RNA-sequencing revealed multipotent progenitor-like populations, reflecting a state of heightened plasticity associated with regeneration. To model the effects of inflammation, we exposed these organoids to cytokines elevated in COPD. Notably, interferon-gamma (IFN-γ) exerted distinct effects on AT1 and AT2 cells: while it was cytotoxic to AT1 cells, it promoted growth in regenerating AT2 cells in a dose- and time-dependent manner. These findings underscore the nuanced influence of pro-inflammatory cytokines on alveolar regeneration. Our organoid model provides a reductionist platform for mechanistic studies in human cells, aiming to identify therapies that prevent alveolar destruction and improve alveolar regeneration in COPD
Project description:Macrophage activation syndrome (MAS) is a life-threatening cytokine storm syndrome complicating systemic juvenile idiopathic arthritis (SJIA) and driven by IFN-gamma. SJIA and MAS are also associated with an unexplained emerging inflammatory lung disease (SJIA-LD), with our recent work supporting pulmonary activation of IFN-gamma pathways as a pathologic link between SJIA-LD and MAS. Our objective was to mechanistically define the novel observation of pulmonary inflammation in the TLR9 mouse model of MAS. In acute MAS, lungs exhibit mild but diffuse CD4-predominant, perivascular interstitial inflammation with elevated IFN-gamma, IFN-induced chemokines, and alveolar macrophage expression of IFN-gamma-induced genes. Single-cell RNA-sequencing confirmed IFN-driven transcriptional changes across immune and parenchymal lung cell types. Resolution of MAS was associated with increased alveolar macrophage and interstitial lymphocytic infiltration. alveolar macrophage microarrays confirmed IFN-gamma-induced proinflammatory polarization during acute MAS, which switches towards an anti-inflammatory phenotype during MAS resolution. Interestingly, recurrent MAS led to increased alveolar inflammation and lung injury, and reset alveolar macrophagepolarization towards a proinflammatory state. Furthermore, in mice bearing macrophages insensitive to IFN-gamma, both systemic feature of MAS and pulmonary inflammation were attenuated. These findings demonstrate that experimental MAS induces IFN-gamma-driven pulmonary inflammation replicating key features of SJIA-LD, and provides a model system for testing novel treatments directed towards SJIA-LD.
Project description:In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production is observed, often mediated by the pro-inflammatory cytokine IFN-γ. IL-10 inhibits IFN-γ secretion, largely by its effects on macrophages and dendritic cells, but, paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work we used different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also found that IL-10, unexpectedly, induces IFN-γ production by all T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening new perspectives for the design of IL-10-based immunotherapies.
Project description:In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production is observed, often mediated by the pro-inflammatory cytokine IFN-γ. IL-10 inhibits IFN-γ secretion, largely by its effects on macrophages and dendritic cells, but, paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work we used different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also found that IL-10, unexpectedly, induces IFN-γ production by all T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening new perspectives for the design of IL-10-based immunotherapies.
Project description:Influenza A Virus (IAV) triggers an exuberant host response that promotes acute lung injury. However, the determinants of the pathological host response to IAV remain incompletely understood. In the current study, we identified interferon (IFN)-γ-regulated subset of monocytes, CCR2+ monocytes, as a driver of lung damage during IAV pathogenesis. IFN-γ regulated the recruitment and inflammatory phenotype of CCR2+ monocytes, and CCR2 (CCR2-/-) and IFN-γ (IFN-γ-/-) deficient mice exhibited reduced lung inflammation, pathology, and increased resistance against bacterial co-infection by Streptococcus pneumoniae (Spn). Adoptive transfer of WT (IFN-γR1+), but not IFN-γR1 deficient (IFN-γR1-) CCR2+ monocytes, restored the wild-type (WT)-like pathological phenotype of lung damage in IAV-infected CCR2-/- mice. The CD8+ T cells were the most significant source of IFN-γ in IAV-infected lungs. Collectively, our data highlight that IFN-γ regulates CCR2+ monocyte-mediated lung pathology during IAV pathogenesis.
Project description:Severe respiratory viral infection leads to extensive damage to the alveolar epithelium and also induce a robust immune response. How immune response impacts alveolar regeneration, especially how immune microenvironment interacts with lung stem/progenitor cells is poorly understood. Here, we find that dysplastic Krt5+ basal-like cells, which emerge after severe viral infection, preferably recruit and maintain the tissue residence of both CD4+ effector T cells and CD8+ T cells in a CXCR3- and Integrin α4/β2-dependent manner after viral clearance. Persistent CD4+ and CD8+ T cells impair alveolar regeneration mediated by airway secretory cells, thus inhibiting lung functional repair. Mechanically, CD4+ and CD8+ T cells function by secreting IFNγ rather than direct interactions with epithelial cells. Importantly, anti-IFNγ treatment promotes alveolar regeneration and functional recovery in vivo. Overall, our study reveals the pathogenetic role of Krt5+ pods in lung regeneration, providing new insights into how dysplastic repair impairs functional regeneration of the alveolar epithelium via interactions with immune cells.