https://koasas.kaist.ac.kr/bitstream/10203/278042/1/000565918500008.pdf

Inflammatory Signals Induce AT2 Cell-Derived Damage-Associated Transient Progenitors that Mediate Alveolar Regeneration.

The respiratory system undergoes a diversity of structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify the heterogeneity of pulmonary cell types and dynamic changes in gene expression mediating adaptation to respiration, here we perform single cell RNA analyses of mouse lung on postnatal day 1. Using an iterative cell type identification strategy we unbiasedly identify the heterogeneity of murine pulmonary cell types. We identify distinct populations of epithelial, endothelial, mesenchymal, and immune cells, each containing distinct subpopulations. Furthermore we compare temporal changes in RNA expression patterns before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, including activation of cell stress and the unfolded protein response during perinatal adaptation of the lung. The present data provide a single cell view of the adaptation to air breathing after birth.

/pdf/single-cell-rna-analysis-identifies-cellular-heterogeneity-4fxre1rjrd.pdf

Single Cell RNA Analysis Identifies Cellular Heterogeneity and Adaptive Responses of the Lung at Birth

Epithelial cell dysfunction has emerged as a central component of the pathophysiology of diffuse parenchymal diseases including idiopathic pulmonary fibrosis (IPF). Alveolar type 2 (AT2) cells represent a metabolically active lung cell population important for surfactant biosynthesis and alveolar homeostasis. AT2 cells and other distal lung epithelia, like all eukaryotic cells, contain an elegant quality control network to respond to intrinsic metabolic and biosynthetic challenges imparted by mutant protein conformers, dysfunctional subcellular organelles, and dysregulated telomeres. Failed AT2 quality control components (the ubiquitin-proteasome system, unfolded protein response, macroautophagy, mitophagy, and telomere maintenance) result in diverse cellular endophenotypes and molecular signatures including ER stress, defective autophagy, mitochondrial dysfunction, apoptosis, inflammatory cell recruitment, profibrotic signaling, and altered progenitor function that ultimately converge to drive downstream fibrotic remodeling in the IPF lung. As this complex network becomes increasingly better understood, opportunities will emerge to identify targets and therapeutic strategies for IPF.

/pdf/contributions-of-alveolar-epithelial-cell-quality-control-to-55zmg7abrf.pdf

Contributions of alveolar epithelial cell quality control to pulmonary fibrosis

Calreticulin (CALR) has anti-tumor effects by increasing dendritic cell maturation and tumor antigen presentation. However, whether CALR affects macrophages and modulates progression of acute respiratory distress syndrome/acute lung injury (ARDS/ALI) remains unknown. In this study, we discovered that CALR protein was highly expressed in the mice with LPS-induced ALI and CALR expression level was positively correlated to the severity of ALI. Commercial anti-CALR antibody (aCALR) can neutralize recombinant CALR (rCALR) and suppress the expression of TNF-alpha and IL-6 in the rCALR-treated macrophages. Blocking CALR activity by intraperitoneal (i.p.) administration of aCALR significantly suppressed ALI, accompanied with lower total cell counts, neutrophil and T cell infiltration in bronchoalveolar lavage (BAL) and lung tissues. The expression of CXCL15, IL-6, IL-1beta, TNF-alpha, and CALR were significantly reduced, in association with more polarization of Siglec F+CD206+M2 subtype macrophages in the aCALR-treated mice. Pre-depletion of circulating monocytes did not abolish the aCALR-mediated suppression of ALI. Further analysis in bone marrow-derived macrophages (BMDMs) showed that aCALR suppressed the expression of CD80, IL-6, IL-1beta, IL-18, NLRP3, and p-p38 MAPK; but enhanced the expression of CD206 and IL-10. In addition, we observed more expression and phosphorylation of STAT6 in the aCALR-treated BMDM. Lack of STAT6 resulted in comparable and slightly higher expression of CALR, TNF-alpha and IL-6 in the aCALR-treated STAT6-/- BMDMs than the untreated cells. Therefore, we conclude that CALR is a novel biomarker in the evaluation of ALI. Blocking CALR activity by aCALR effectively suppressed ALI independent of circulating monocytes. Siglec F+CD206+M2 subtype macrophages and p38 MAPK/STAT6 signaling pathway played important role in the immune regulation of aCALR. Blocking CALR activity is a promising therapeutic approach in the treatment of ARDS/ALI.

Calreticulin Blockade Attenuates Murine Acute Lung Injury by Inducing Polarization of M2 Subtype Macrophages.

Many of the signaling pathways and growth factors involved with pulmonary fibrosis are well-described but little is known about why the fibroproliferative process progresses over time. Ventilation-induced lung injury occurs as a result of epithelial stress and strain from atelectrauma and volutrauma, respectively, both of which occur in the setting of alveolar collapse. We suggest that ventilation-induced lung injury may be a unifying process explaining perpetuation of the fibrosis on the basis that (a) alveolar collapse has been repeatedly described in patients with pulmonary fibrosis, thereby providing a setting in which ventilation-induced lung injury can occur, and (b) patients with pulmonary fibrosis, many of the risk factors and genetic variants associated with pulmonary fibrosis as well as most of models used to study pulmonary fibrosis are all associated with surfactant abnormalities and/or with alveolar type 2 cell injury that would predispose to surfactant abnormalities, thereby increasing surface tension and causing alveolar collapse. The purpose of this Perspective is to review the literature documenting that alveolar collapse is an important aspect of the pathology and pathophysiology of pulmonary fibrosis, to summarize the abnormalities in surfactant and/or in type 2 cells that occur in patients with pulmonary fibrosis, and in association with the risk factors, genetic variations and animal models of pulmonary fibrosis and to estimate the strain resulting from alveolar collapse that could result in ventilation-induced lung injury.

/pdf/is-progression-of-pulmonary-fibrosis-due-to-ventilation-385xnlrilk.pdf

Is Progression of Pulmonary Fibrosis due to Ventilation-induced Lung Injury?

Adaptation to air breathing after birth is dependent upon the synthesis and secretion of pulmonary surfactant by alveolar type 2 (AT2) cells. Surfactant, a complex mixture of phospholipids and proteins, is secreted into the alveolus, where it reduces collapsing forces at the air-liquid interface to maintain lung volumes during the ventilatory cycle. ABCA3, an ATP-dependent Walker domain containing transport protein, is required for surfactant synthesis and lung function at birth. Mutations in ABCA3 cause severe surfactant deficiency and respiratory failure in newborn infants. We conditionally deleted the Abca3 gene in AT2 cells in the mature mouse lung. Loss of ABCA3 caused alveolar cell injury and respiratory failure. ABCA3-related lung dysfunction was associated with surfactant deficiency, inflammation, and alveolar-capillary leak. Extensive but incomplete deletion of ABCA3 caused alveolar injury and inflammation, and it initiated proliferation of progenitor cells, restoring ABCA3 expression, lung structure, and function. M2-like macrophages were recruited to sites of AT2 cell proliferation during the regenerative process and were present in lung tissue from patients with severe lung disease caused by mutations in ABCA3. The remarkable and selective regeneration of ABCA3-sufficient AT2 progenitor cells provides plausible approaches for future correction of ABCA3 and other genetic disorders associated with surfactant deficiency and acute interstitial lung disease.

/pdf/alveolar-injury-and-regeneration-following-deletion-of-abca3-1vdg39k73x.pdf

Alveolar injury and regeneration following deletion of ABCA3

Efficient Transduction of Alveolar Type 2 Cells with Adeno-associated Virus for the Study of Lung Regeneration.

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation. Grant supportThis work was supported in part by HL131634 (JPB) from the National Heart, Lung and Blood Institute of the National Institutes of Health.

/pdf/activation-of-gpr116-adgrf5-by-its-tethered-agonist-requires-3i91upv32w.pdf

Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane region

The primary cilium is a ubiquitous, microtubule-based cellular organelle. Primary cilia dysfunction results in a group of disorders termed ciliopathies. C2 domain containing 3 centriole elongation regulator (C2cd3), encodes a centriolar protein essential for ciliogenesis. Mutations in human C2CD3 are associated with the human ciliopathy Oral-Facial-Digital syndrome type 14 (OFD14). In order to better understand the etiology of ciliopathies including OFD14, we generated numerous murine models targeting C2cd3. Initial analysis revealed several tissue-specific isoforms of C2cd3, and while the loss of C2cd3 has previously been reported to result in exencephaly, tight mesencephalic flexure, pericardial edema, abnormal heart looping and a twisted body axis, further analysis revealed that genetic background may also contribute to phenotypic variation. Additional analyses of a conditional allelic series targeting C-terminal PKC-C2 domains or the N-terminal C2CD3N-C2 domain of C2cd3 revealed a variable degree of phenotypic severity, suggesting that while the N-terminal C2CD3N-C2 domain was critical for early embryonic development as a whole, there was also a craniofacial specific role for the C2CD3N-C2 domains. Together, through generation of novel models and evaluation of C2cd3 expression, these data provide valuable insight into mechanisms of pathology for craniofacial ciliopathies that can be further explored in the future.

/pdf/centriolar-protein-c2cd3-is-required-for-craniofacial-3rsw8rxpot.pdf

Kari M. Brown

Papers

Alveolar injury and regeneration following deletion of ABCA3

Efficient Transduction of Alveolar Type 2 Cells with Adeno-associated Virus for the Study of Lung Regeneration.

Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane region

Centriolar Protein C2cd3 Is Required for Craniofacial Development.