It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

Acute lower respiratory tract infections are a leading cause of morbidity and mortality world-wide. Streptococcus pneumoniae (pneumococcus) is the most common bacterial cause of community-acquired pneumonia. Recovery from pneumococcal pneumonia results in the formation of resident memory CD4+ T cells, which act on lung epithelial cells to accelerate immune responses. Alveolar macrophages (AMs) are tissue-resident macrophages localized in the air spaces, where they orchestrate the lung anti-microbial responses. We hypothesized that recovery from pneumococcal pneumonia results in remodeling of the pool of alveolar macrophages, which act in concordance with other immune cells to protect the lungs from future infections. Although AM numbers were unchanged in experienced lungs, their surface phenotype showed significant changes, most prominently an increased MHC-II and a decreased SiglecF. This experienced AM phenotype was regionally-localized and long-lasting. Experienced AMs also exhibited extensive remodeling on the metabolomics and transcriptional level. Experienced AMs demonstrated significant increases in phosphocreatine and its metabolite precursors. The transcriptional analyses also revealed extensive changes. At baseline, experienced AMs exhibited a significant reduction in cell cycle activity and mRNA processing compared to naïve mice. During acute pneumonia, experienced AMs exhibited significant increases in immune signaling and energy metabolism. Moreover, transcriptional data also revealed strong but imperfect enrichment of a signature previously associated with IFN𝛾 signaling and marrow-derived AMs. IFN𝛾 gain and loss of functions experiments corroborated transcriptional data and revealed an essential role for IFN𝛾 in directly driving the AM MHC-II remodeling. Several immune cells produced IFN𝛾, with neutrophils being the most prominent source after the 1st pneumococcal challenge but other cells predominating after the 2nd pneumococcal challenge. CD4+ T cell depletion studies demonstrated that AMs' experienced phenotype was independent of CD4+ T cells. In contrast to naïve mice, lineage-tracing studies demonstrated that marrow-derived AMs predominately constitute the experienced AM pool. Upon experience, both embryonic AMs and marrow-derived AMs demonstrated similar remodeling for both SiglecF and MHC-II on their surfaces. While all AM similarly remodeled independent of their origin, marrow-derived AMs in experienced lungs displayed some differences from their embryonic counterparts, being less phagocytic. In conclusion, recovery from pneumococcal pneumonia remodels the pool of alveolar macrophages to acquire adaptive characteristics. This remodeling involves a combination of recruitment of new cells and trained immunity via IFN𝛾 signaling.

Idiopathic pulmonary fibrosis is a chronic and fatal disease that causes the stiffening of lung tissue and gradual lung function decline. Currently, there are no effectives therapies for this disease. Fibrotic lungs are characterized by accumulation of smooth muscle α actin- (SMA) expressing myofibroblasts and excessive deposition of a collagen rich extracellular matrix. The differentiation of lung fibroblasts into myofibroblasts is stimulated by numerous growth factors, including transforming growth factor β (TGFβ), and potentiated by a stiff mechanical environment. Our laboratory has identified a secreted matrix protein, aortic carboxypeptidase-like protein (ACLP), which is upregulated in idiopathic pulmonary fibrosis. Additionally, ACLP knockout mice are protected from experimentally induced fibrosis. This led to the hypothesis that ACLP promotes the fibroblast to myofibroblast transition, and the goal of this research was to characterize the mechanism of ACLP action. ACLP expression preceded SMA and collagen type I expression in rapidly differentiating primary mouse lung myofibroblasts. In gain of function studies, recombinant ACLP induced SMA and collagen I expression in both primary differentiating myofibroblasts as well as IMR90 human lung fibroblasts. ACLP knockdown by siRNA slowed myofibroblast differentiation and partially reverted fully differentiated myofibroblasts into fibroblasts. Because of the similarities among ACLP targets and TGFβ targets, it was hypothesized that ACLP stimulates TGFβ signaling. In lung fibroblasts, ACLP induced Smad3 phosphorylation and nuclear translocation, a feature of TGFβ signaling. The effects of ACLP on myofibroblast differentiation were dependent on TGFβ receptor (TβR) kinase activity and ACLP interacted directly with T&betaR II to promote myofibroblast differentiation. A recombinant TβR II Fc chimera was used to inhibit ACLP-induced SMA expression, but this reagent had no effect on ACLP-induced collagen type I expression, which suggests a differential regulation of SMA and collagen by ACLP. Additionally, ACLP modulated changes in differentiation between cells grown on softer versus stiffer matrices. Using recombinant fragments of the ACLP protein, the N-terminal thrombospondin repeat domain was found to be necessary and sufficient to promote myofibroblast differentiation. Taken together, these studies identified a novel mechanism of ACLP action in fibroblasts and may lead to new therapeutic strategies to treat fibrotic disease.