Role and mechanisms of autophagy in lung metabolism and repair.

Mammalian lungs are metabolically active organs that frequently encounter environmental insults. Stress responses elicit protective autophagy in epithelial barrier cells and the supportive niche. Autophagy promotes the recycling of damaged intracellular organelles, denatured proteins, and other biological macromolecules for reuse as components required for lung cell survival. Autophagy, usually induced by metabolic defects, regulates cellular metabolism. Autophagy is a major adaptive response that protects cells and organisms from injury. Endogenous region-specific stem/progenitor cell populations are found in lung tissue, which are responsible for epithelial repair after lung damage. Additionally, glucose and fatty acid metabolism is altered in lung stem/progenitor cells in response to injury-related lung fibrosis. Autophagy deregulation has been observed to be involved in the development and progression of other respiratory diseases. This review explores the role and mechanisms of autophagy in regulating lung metabolism and epithelial repair.

LKB1 deficiency upregulates RELM-α to drive airway goblet cell metaplasia.

Targeting airway goblet cell metaplasia is a novel strategy that can potentially reduce the chronic obstructive pulmonary disease (COPD) symptoms. Tumor suppressor liver kinase B1 (LKB1) is an important regulator of the proliferation and differentiation of stem/progenitor cells. In this study, we report that LKB1 expression was downregulated in the lungs of patients with COPD and in those of cigarette smoke-exposed mice. Nkx2.1Cre; Lkb1f/f mice with conditional loss of Lkb1 in mouse lung epithelium displayed airway mucus hypersecretion and pulmonary macrophage infiltration. Single-cell transcriptomic analysis of the lung tissues from Nkx2.1Cre; Lkb1f/f mice further revealed that airway goblet cell differentiation was altered in the absence of LKB1. An organoid culture study demonstrated that Lkb1 deficiency in mouse airway (club) progenitor cells promoted the expression of FIZZ1/RELM-α, which drove airway goblet cell differentiation and pulmonary macrophage recruitment. Additionally, monocyte-derived macrophages in the lungs of Nkx2.1Cre; Lkb1f/f mice exhibited an alternatively activated M2 phenotype, while expressing RELM-α, which subsequently aggravated airway goblet cell metaplasia. Our findings suggest that the LKB1-mediated crosstalk between airway progenitor cells and macrophages regulates airway goblet cell metaplasia. Moreover, our data suggest that LKB1 agonists might serve as a potential therapeutic option to treat respiratory disorders associated with goblet cell metaplasia.

PM2.5 induces a senescent state in mouse AT2 cells.

PM2.5 is known to induce lung injury, but its toxic effects on lung regenerative machinery and the underlying mechanisms remain unknown. In this study, primary mouse alveolar type 2 (AT2) cells, considered stem cells in the gas-exchange barrier, were sorted using fluorescence-activated cell sorting. By developing microfluidic technology with constricted microchannels, we observed that both passage time and impedance opacities of mouse AT2 cells were reduced after PM2.5, indicating that PM2.5 induced a more deformable mechanical property and a higher membrane permeability. In vitro organoid cultures of primary mouse AT2 cells indicated that PM2.5 is able to impair the proliferative potential and self-renewal capacity of AT2 cells but does not affect AT1 differentiation. Furthermore, cell senescence biomarkers, p53 and γ-H2A.X at protein levels, P16ink4a and P21 at mRNA levels were increased in primary mouse AT2 cells after PM2.5 stimulations as shown by immunofluorescent staining and quantitative PCR analysis. Using several advanced single-cell technologies, this study sheds light on new mechanisms of the cytotoxic effects of atmospheric fine particulate matter on lung stem cell behavior.

Overexpression of bmp4, dazl, nanos3 and sycp2 in Hu Sheep Leydig Cells Using CRISPR/dcas9 System Promoted Male Germ Cell Related Gene Expression.

Male germ cells directly affect the reproduction of males; however, their accurate isolation and culture in vitro is extremely challenging, hindering the study of germ cell development and function. CRISPR/dcas9, as an efficient gene reprogramming system, has been verified to promote the transdifferentiation of pluripotent stem cells into male germ cells by editing target genes. In our research, we explored the expression pattern of the germ cell related genes bmp4, dazl,nanos3 and sycp2 in Hu sheep testicular development and constructed the overexpression model using the CRISPR/dcas9 system. The results indicated that four genes showed more expression in testis tissue than in other tissues, and that bmp4, dazl and sycp2 present higher expression levels in nine-month-old sheep testes than in three-month-olds, while nanos3 expressed the opposite trend (p < 0.05). In addition, the expression of four potential genes in spermatogenic cells was slightly different, but they were all expressed in sheep Leydig cells. To verify the potential roles of the four genes in the process of inducing differentiation of male germ cells, we performed cell transfection in vitro. We found that the expression of the germ cell related genes Prdm1, Prdm14, Mvh and Sox17 were significantly increased after the overexpression of the four genes in Leydig cells, and the co-transfection effect was the most significant (p < 0.05). Our results illustrate the crucial functions of bmp4, dazl, nanos3 and sycp2 in Hu sheep testis development and verified the effectiveness of the overexpression model that was constructed using the CRISPR/dcas9 system, which provided a basis for further male germ cell differentiation in vitro.

Comparative Transcriptomic Analysis of Hu Sheep Pituitary Gland Prolificacy at the Follicular and Luteal Phases.

The pituitary gland directly regulates the reproduction of domestic animals. Research has increasingly focused on the potential regulatory mechanism of non-coding RNA in pituitary development. Little is known about the differential expression pattern of lncRNAs in Hu sheep, a famous sheep breed with high fecundity, and its role in the pituitary gland between the follicular phase and luteal phase. Herein, to identify the transcriptomic differences of the sheep pituitary gland during the estrus cycle, RNA sequencing (RNA-Seq) was performed. The results showed that 3529 lncRNAs and 16,651 mRNAs were identified in the pituitary gland. Among of them, 144 differentially expressed (DE) lncRNA transcripts and 557 DE mRNA transcripts were screened in the follicular and luteal phases. Moreover, GO and KEGG analyses demonstrated that 39 downregulated and 22 upregulated genes interacted with pituitary functions and reproduction. Lastly, the interaction of the candidate lncRNA XR_001039544.4 and its targeted gene LHB were validated in sheep pituitary cells in vitro. LncRNA XR_001039544.4 and LHB showed high expression levels in the luteal phase in Hu sheep. LncRNA XR_001039544.4 is mainly located in the cytoplasm, as determined by FISH analysis, indicating that XR_001039544.4 might act as competing endogenous RNAs for miRNAs to regulate LHB. LncRNA XR_001039544.4 knockdown significantly inhibited LH secretion and cell proliferation. LncRNA XR_001039544.4 may regulate the secretion of LH in the luteal-phase pituitary gland via affecting cell proliferation. Taken together, these findings provided genome-wide lncRNA- and mRNA-expression profiles for the sheep pituitary gland between the follicular and luteal phases, thereby contributing to the elucidation of the molecular mechanisms of pituitary function.

Macrophages in Lung Injury, Repair, and Fibrosis.

Fibrosis progression in the lung commonly results in impaired functional gas exchange, respiratory failure, or even death. In addition to the aberrant activation and differentiation of lung fibroblasts, persistent alveolar injury and incomplete repair are the driving factors of lung fibrotic response. Macrophages are activated and polarized in response to lipopolysaccharide- or bleomycin-induced lung injury. The classically activated macrophage (M1) and alternatively activated macrophage (M2) have been extensively investigated in lung injury, repair, and fibrosis. In the present review, we summarized the current data on monocyte-derived macrophages that are recruited to the lung, as well as alveolar resident macrophages and their polarization, pyroptosis, and phagocytosis in acute lung injury (ALI). Additionally, we described how macrophages interact with lung epithelial cells during lung repair. Finally, we emphasized the role of macrophage polarization in the pulmonary fibrotic response, and elucidated the potential benefits of targeting macrophage in alleviating pulmonary fibrosis.