Senescence of Alveolar Type 2 Cells Drives Progressive Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is an insidious and fatal interstitial lung disease associated with declining pulmonary function. Accelerated aging, loss of epithelial progenitor cell function and/or numbers, and cellular senescence are implicated in the pathogenies of IPF.Objectives: We sought to investigate the role of alveolar type 2 (AT2) cellular senescence in initiation and/or progression of pulmonary fibrosis and therapeutic potential of targeting senescence-related pathways and senescent cells.Methods: Epithelial cells of 9 control donor proximal and distal lung tissues and 11 IPF fibrotic lung tissues were profiled by single-cell RNA sequencing to assesses the contribution of epithelial cells to the senescent cell fraction for IPF. A novel mouse model of conditional AT2 cell senescence was generated to study the role of cellular senescence in pulmonary fibrosis.Measurements and Main Results: We show that AT2 cells isolated from IPF lung tissue exhibit characteristic transcriptomic features of cellular senescence. We used conditional loss of Sin3a in adult mouse AT2 cells to initiate a program of p53-dependent cellular senescence, AT2 cell depletion, and spontaneous, progressive pulmonary fibrosis. We establish that senescence rather than loss of AT2 cells promotes progressive fibrosis and show that either genetic or pharmacologic interventions targeting p53 activation or senescence block fibrogenesis.Conclusions: Senescence of AT2 cells is sufficient to drive progressive pulmonary fibrosis. Early attenuation of senescence-related pathways and elimination of senescent cells are promising therapeutic approaches to prevent pulmonary fibrosis.

Sin3a regulates epithelial progenitor cell fate during lung development.

Mechanisms that regulate tissue-specific progenitors for maintenance and differentiation during development are poorly understood. Here, we demonstrate that the co-repressor protein Sin3a is crucial for lung endoderm development. Loss of Sin3a in mouse early foregut endoderm led to a specific and profound defect in lung development with lung buds failing to undergo branching morphogenesis and progressive atrophy of the proximal lung endoderm with complete epithelial loss at later stages of development. Consequently, neonatal pups died at birth due to respiratory insufficiency. Further analysis revealed that loss of Sin3a resulted in embryonic lung epithelial progenitor cells adopting a senescence-like state with permanent cell cycle arrest in G1 phase. This was mediated at least partially through upregulation of the cell cycle inhibitors Cdkn1a and Cdkn2c. At the same time, loss of endodermal Sin3a also disrupted cell differentiation of the mesoderm, suggesting aberrant epithelial-mesenchymal signaling. Together, these findings reveal that Sin3a is an essential regulator for early lung endoderm specification and differentiation.

Rare SOX2+ Airway Progenitor Cells Generate KRT5+ Cells that Repopulate Damaged Alveolar Parenchyma following Influenza Virus Infection.

Recent studies have implicated keratin 5 (KRT5)+ cells in repopulation of damaged lung tissue following severe H1N1 influenza virus infection. However, the origins of the cells repopulating the injured alveolar region remain controversial. We sought to determine the cellular dynamics of lung repair following influenza infection and define whether nascent KRT5+ cells repopulating alveolar epithelium were derived from pre-existing alveolar or airway progenitor cells. We found that the wound-healing response begins with proliferation of SOX2+ SCGB1A1- KRT5- progenitor cells in airways. These cells generate nascent KRT5+ cells as an early response to airway injury and yield progeny that colonize damaged alveolar parenchyma. Moreover, we show that local alveolar progenitors do not contribute to nascent KRT5+ cells after injury. Repopulation of injured airway and alveolar regions leads to proximalization of distal airways by pseudostratified epithelium and of alveoli by airway-derived epithelial cells that lack the normal characteristics of mature airway or alveolar epithelium.

Maternal exposure of rats to nicotine via infusion during gestation produces neurobehavioral deficits and elevated expression of glial fibrillary acidic protein in the cerebellum and CA1 subfield in the offspring at puberty.

Maternal smoking during pregnancy is known to be a significant contributor to developmental neurological health problems in the offspring. In animal studies, nicotine treatment via injection during gestation has been shown to produce episodic hypoxia in the developing fetus. Nicotine delivery via mini osmotic pump, while avoiding effects due to hypoxia-ischemia, it also provides a steady level of nicotine in the plasma. In the present study timed-pregnant Sprague-Dawley rats (300-350 g) were treated with nicotine (3.3 mg/kg, in bacteriostatic water via s.c. implantation of mini osmotic pump) from gestational days (GD) 4-20. Control animals were treated with bacteriostatic water via s.c. implantation of mini osmotic pump. Offspring on postnatal day (PND) 30 and 60, were evaluated for changes in the ligand binding for various types of nicotinic acetylcholine receptors and neuropathological alterations. Neurobehavioral evaluations for sensorimotor functions, beam-walk score, beam-walk time, incline plane and grip time response were carried out on PND 60 offspring. Beam-walk time and forepaw grip time showed significant impairments in both male and female offspring. Ligand binding densities for [3H]epibatidine, [3H]cytisine and [3H]alpha-bungarotoxin did not show any significant changes in nicotinic acetylcholine receptors subtypes in the cortex at PND 30 and 60. Histopathological evaluation using cresyl violet staining showed significant decrease in surviving Purkinje neurons in the cerebellum and a decrease in surviving neurons in the CA1 subfield of hippocampus on PND 30 and 60. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer of cerebellum and the CA1 subfield of hippocampus on PND 30 and 60 of both male and female offspring. These results indicate that maternal exposure to nicotine produces significant neurobehavioral deficits, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum and CA1 subfield of hippocampus of the offspring on PND 30 and 60. The results show that although 60-day-old male and female rat offspring of mothers exposed to nicotine during gestation did not differ from control in body weight gain or nicotinic acetylcholine receptors ligand binding, they exhibited significant sensorimotor deficits that were consistent with the neuropathological alterations seen in the brain. These neurobehavioral and pathological deficits indicate that maternal nicotine exposure may produce long-term adverse health effects in the offspring.

Increased expression of glial fibrillary acidic protein in cerebellum and hippocampus: differential effects on neonatal brain regional acetylcholinesterase following maternal exposure to combined chlorpyrifos and nicotine.

Cigarette smoking and environmental exposure to chlorpyrifos during pregnancy could lead to developmental toxicity in the offspring. In the present study, pregnant female Sprague-Dawley rats (300-350 g) were treated daily with nicotine (1 mg/kg, sc) or chlorpyrifos (0.1 mg/kg, dermal) or a combination of nicotine and chlorpyrifos from gestational days (GD) 4-20. Control animals were treated with saline and ethanol. Male offspring from the mothers treated with nicotine alone gained significantly less weight on postnatal day (PND) 30 as compared to control. On PND 7, there was a significant increase in brain acetylcholinesterase (AChE) activity in pups from nicotine- and chlorpyrifos-treated dams, whereas plasma butyrylcholinesterase (BChE) activity was significantly elevated in pups of mothers treated with either chlorpyrifos alone or pesticide combined with nicotine. On PND 30 there was a significant increase in AChE activity in brainstem and cerebellum in all treated male pups. In female pups on PND 30 there was a significant rise in AChE activity in brainstem of chlorpyrifos alone and in cerebellum of the combination nicotine and chlorpyrifos group. Histopathological evaluation demonstrated an increased neuronal cell death in the cerebellum granular cell layer of female offspring from nicotine or combined nicotine with chlorpyrifos group. A rise in glial fibrillary acidic protein (GFAP) immunostaining was observed in the CA1 subfield of hippocampus and cerebellum on PND 30 in female and male offspring of mothers treated with either nicotine or nicotine in combination with chlorpyrifos, but to a lesser extent in males. Data suggest that maternal exposure to nicotine and chlorpyrifos, alone or in combination, produces differential alterations in brain regional AChE activity and expression of GFAP in cerebellum and hippocampus in offspring on PND 30.

Maternal exposure to nicotine and chlorpyrifos, alone and in combination, leads to persistently elevated expression of glial fibrillary acidic protein in the cerebellum of the offspring in late puberty.

We previously showed that maternal exposure to nicotine, alone or in combination with chlorpyrifos, caused an increase in glial fibrillary acidic protein (GFAP) immunostaining in the CA1 subfield of hippocampus and cerebellum in postnatal day (PND) 30 offspring. In the present study, PND 60 offspring were evaluated for histopathological and cholinergic effects following maternal exposure to nicotine and chlorpyrifos, alone and in combination. Timed-pregnant Sprague-Dawley rats (300-350 g) were treated daily with nicotine (1 mg/kg, s.c., in normal saline) or chlorpyrifos (0.1 mg/kg, dermal, in ethanol) or a combination of nicotine and chlorpyrifos from gestational days (GD) 4 to 20. Control animals were treated with saline and ethanol. On PND 60, the offspring were evaluated for cholinergic changes and pathological effects. Plasma butyrylcholinesterase (BChE) activity in the female offspring from chlorpyrifos treated mothers showed a significant increase (approximately 183% of control). Male offspring from mothers treated with either chlorpyrifos or nicotine alone showed a significant increase in the acetylcholinesterase (AChE) activity in the brainstem while female offspring from mothers treated with either nicotine or a combination of nicotine and chlorpyrifos showed a significant increase (approximately 134 and 126% of control, respectively) in AChE activity in the brainstem. No significant changes were observed in the ligand binding densities for alpha4beta2 and alpha7 nicotinic acetylcholine receptors in the cortex. Histopathological evaluation using cresyl violet staining showed a significant decrease in surviving Purkinje neurons in the cerebellum of the offspring from nicotine treated mothers. An increase in GFAP immunostaining in cerebellar white matter was observed in the offspring from the mothers treated with nicotine. These results suggest that maternal exposure to real-life levels of nicotine and/or chlorpyrifos causes differential regulation of brainstem AChE activity. Also, nicotine caused a decrease in the surviving neurons and an increased expression of GFAP in cerebellar white matter of the offspring on PND 60. These changes can lead to long-term neurological adverse health effects later in life.