Cigarette smoke (CS) is associated with chronic obstructive pulmonary disease (COPD) and cancer. However, the underlying pathological mechanisms are not well understood. We recently reported that ...mice exposed to long-term intermittent CS for 3 months developed more severe emphysema and higher incidence of adenocarcinoma than mice exposed to long-term continuous CS for 3 months and long-term continuous CS exposure activated alveolar stem cell proliferation. However, the influence of variations in the CS exposure pattern in alveolar stem cell in unknown. Here, we exposed mice to 3 weeks of continuous or intermittent CS to identify whether different CS exposure patterns would result in differential effects on stem cells and the mechanisms underlying these potential differences.
Female mice expressing GFP in alveolar type 2 (AT2) cells, which are stem cells of the alveolar compartment, were exposed to mainstream CS via nasal inhalation. AT2 cells were collected based on their GFP expression by flow cytometry and co-cultured with fibroblasts in stem cell 3D organoid/colony-forming assays. We compared gene expression profiles of continuous and intermittent CS-exposed AT2 cells using microarray analysis and performed a functional assessment of a differentially expressed gene to confirm its involvement in the process using activator and inhibitor studies.
AT2 cells sorted from intermittent CS-exposed mice formed significantly more colonies compared to those from continuous CS-exposed mice, and both CS-exposed groups formed significantly more colonies when compared to air-exposed cells. Comparative microarray analysis revealed the upregulation of genes related to fatty acid oxidation (FAO) pathways in AT2 cells from intermittent CS-exposed mice. Treatment of intermittent CS-exposed mice with etomoxir, an inhibitor of the FAO regulator Cpt1a, for 5 weeks resulted in a significant suppression of the efficiency of AT2 cell colony formation. In vitro treatment of naïve AT2 cells with a FAO activator and inhibitor further confirmed the relationship between FAO and AT2 stem cell function.
Alveolar stem cell function was more strongly activated by intermittent CS exposure than by continuous CS exposure. We provide evidence that AT2 stem cells respond to intermittent CS exposure by activating stem cell proliferation via the activation of FAO.
•TAMs dominate the immune response to the FGF9-induced adenocarcinoma.•These TAMs are enriched for the alternatively activated (M2) macrophage subtype.•These TAMs performed an immune suppressive ...function and promoted tumor growth.•These TAMs induced fibroblast proliferation and angiogenesis.•These TAMs overexpress Tgf-β, Vegf, Fgf2, Fgf10, Fgfr2 and matrix metalloproteinases.
Tumor-associated macrophages (TAMs) are known to promote tumorigenesis but the mechanism(s) remain elusive. We have developed a mouse model of lung cancer that is initiated through an inducible overexpression of fibroblast growth factor 9 (FGF9) in type-2 pneumocytes. Expression of FGF9 in adult lungs resulted in a rapid development of multiple adenocarcinoma-like tumor nodules, and is associated with an intense immunological reaction. The purpose of this study is to characterize the immune response to the FGF9-induced lung adenocarcinoma and to determine the contribution of TAMs to growth and survival of these tumors.
We used flow cytometry, immunostaining, RT-PCR and in vitro culture system on various cell populations isolated from the FGF9-induced adenocarcinoma mouse lungs.
Immunostaining demonstrated that the majority of the inflammatory cells recruited to FGF9-induced lung tumors were macrophages. These TAMs were enriched for the alternatively activated (M2) macrophage subtype. TAMs performed a significantly high immune suppressive function on T-cells and displayed high levels of arginase-1 expression and activity. The growth and colony forming potential of tumor cells was induced by co-culture with TAMs. Additionally, TAMs were shown to promote fibroblast proliferation and angiogenesis. TAMs had high expression of Tgf-β, Vegf, Fgf2, Fgf10, Fgfr2 and several matrix metalloproteinases; factors that play multiple roles in supporting tumor growth, immune protection, fibroblast activation and angiogenesis.
Our results provide evidence that the Fgf9-induced lung adenocarcinoma is associated with recruitment and activation of M2-biased TAMs, which provided multiple means of support to the tumor. This model represents an excellent means to further study the complex interactions between TAMs, their related chemokines, and progression of lung adenocarcinoma, and adds further evidence to support the importance of TAMs in tumorigenesis.
High fat diet (HFD) decreases the lifespan of mice, and is a risk factor for several human diseases. Here, we investigated the effects of a HFD on lung epithelial and stem cells and its interaction ...with aging. Young and old mice were fed with either a standard diet (SD) or a HFD then their trachea and lung were examined for histological changes, inflammation, and mitochondrial function. Their stem cell function was examined using the in vitro organoid/colony forming efficiency (CFE) assay. Aging reduced the number of tracheal basal and alveolar type-2 (AT2) cells. HFD significantly increased the number of AT2 cells. Aging also caused a significant increase in lung inflammation, and HFD caused a similar increase, in young mice. Aging reduced mitochondrial mass and function, and increased reactive oxygen species. In young mice, HFD caused mitochondrial changes similar to the aging-induced changes. Organoid culture of tracheal and lung epithelial cells collected from both young and old HFD-fed mice showed higher CFE compared to SD-fed mice. Switching the HFD to low calorie/fat diet (LCD) efficiently reversed several of the HFD-induced effects. Thus, HFD induces several histological, inflammatory, and functional changes in the lung, and exacerbates the aging-induced lung inflammation and mitochondrial deterioration. LCD can reverse many of the HFD-induced effects.
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•High fat diet (HFD) caused an increase in the number of alveolar type-2 cells.•Both aging and HFD caused an increase in inflammatory cell infiltration of the lung.•Both aging and HFD impaired various aspects of mitochondrial function.•HFD promoted in vitro colony formation by lung stem cells, while aging inhibited it.•Switching the diet to a low FD reversed several of the HFD-induced effects.
Lung cancer and chronic obstructive pulmonary disease are leading causes of morbidity and mortality worldwide, and cigarette smoking is a main risk factor for both. The presence of emphysema, an ...irreversible lung disease, further raises the risk of lung cancer in patients with chronic obstructive pulmonary disease. The mechanisms involved in smoke-induced tumorigenesis and emphysema are not fully understood, attributable to a lack of appropriate animal models. Here, we optimized a model of cigarette smoke (CS)-induced lung cancer and emphysema in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen. We investigated whether variations in CS exposure patterns with the same total amount and duration of exposure affect tumorigenesis and/or development of emphysema. Continuous CS exposure for 3 months significantly suppressed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced development of adenomas and adenocarcinomas; however, emphysema independently developed during this period. Surprisingly, intermittent CS exposure increased the severity of emphysema and resulted in a higher incidence of adenocarcinomas. Furthermore, intermittent CS exposure elicited a marked increase in M2-polarized macrophages within and near the developed tumors. By employing a CS exposure protocol with repeated cycles of cessation and relapse, we provide evidence that intermittent CS exposure enhances tumorigenesis and emphysema progression more than that of continuous CS exposure.
Helper T (Th) and regulatory T (Treg) cell differentiation programs promote the eradication of pathogens, while minimizing adverse immune reactions. Here, we found that Nr4a family of nuclear ...receptors supports Treg cell induction and represses Th1 and Th2 cell differentiation from naive CD4+ T cells. Nr4a factors are transiently induced in CD4+ T cells immediately after antigen stimulation, thereby mediating epigenetic changes. In differentiating Treg cells, Nr4a factors mainly upregulated the early responsive genes in the Treg cell-specifying gene set, either directly or in cooperation with Ets family transcription factors. In contrast, Nr4a factors repressed AP-1 activity by interrupting a positive feedback loop for Batf factor expression, thus suppressing Th2 cell-associated genes. In an allergic airway inflammation model, Nr4a factors suppressed the pathogenesis, mediating oral tolerance. Lastly, pharmacological activation of an engineered Nr4a molecule prevented allergic airway inflammation, indicating that Nr4a factors may be novel therapeutic targets for inflammatory diseases.
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•Among “Treg signature genes”, Nr4a factors mainly induce early responsive ones•Nr4a activate target genes directly or by supporting Ets factors' function•Nr4a factors repress Th2-driving positive feedback loop for Batf factor expression•Pharmacological activation of Nr4a factors' activity prevented airway inflammation
Biological Sciences; Immunology; Cell Biology
Sphingolipids play a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, little is known about the precise roles of sphingosine-1-phosphate (S1P), a bioactive ...sphingolipid metabolite, and its receptor modulation in COPD. In this study, we demonstrated that the S1P receptor modulator ONO-4641 induced the expansion of lung CD11b
Gr-1
cells and lymphocytopenia in naive mice. ONO-4641-expanded CD11b
Gr-1
cells showed higher arginase-1 activity, decreased T cell proliferation, and lower IFN-γ production in CD3
T cells, similar to the features of myeloid-derived suppressor cells. ONO-4641 treatment decreased airspace enlargement in elastase-induced and cigarette smoke-induced emphysema models and attenuated emphysema exacerbation induced by post-elastase pneumococcal infection, which was also associated with an increased number of lung CD11b
Gr-1
cells. Adoptive transfer of ONO-4641-expanded CD11b
Gr-1
cells protected against elastase-induced emphysema. Lymphocytopenia observed in these models likely contributed to beneficial ONO-4641 effects. Thus, ONO-4641 attenuated murine pulmonary emphysema by expanding lung CD11b
Gr-1
cell populations and inducing lymphocytopenia. The S1P receptor might be a promising target for strategies aimed at ameliorating pulmonary emphysema progression.
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