The Orphan G Protein-Coupled Receptor GPR116 Regulates Pulmonary Surfactant Pool Size

Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Both quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. While the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathway(s) that senses and regulates endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein-coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels one day following birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116Δexon17, were generated. Gpr116Δexon17 mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12- fold) that is further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116Δexon17 mice was associated with increased SatPC secretion at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia and alveolar destruction at 20 weeks. Through microarray analyses, we demonstrate that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116Δexon17 type II cells. Collectively, these data suggest that GPR116 functions as molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion from alveolar type II cells

TGFBI functions similar to periostin but is uniquely dispensable during cardiac injury.

Extracellular matrix production and accumulation stabilize the heart under normal conditions as well as form a protective scar after myocardial infarction injury, although excessive extracellular matrix accumulation with long-standing heart disease is pathological. In the current study we investigate the role of the matricellular protein, transforming growth factor beta-induced (TGFBI), which is induced in various forms of heart disease. Additionally, we sought to understand whether TGFBI is functionally redundant to its closely related family member periostin, which is also induced in the diseased heart. Surgical models of myocardial infarction and cardiac pressure overload were used in mice with genetic loss of Postn and/or Tgfbi to examine the roles of these genes during the fibrotic response. Additionally, cardiac-specific TGFBI transgenic mice were generated and analyzed. We observed that deletion of Tgfbi did not alter cardiac disease after myocardial infarction in contrast to greater ventricular wall rupture in Postn gene-deleted mice. Moreover, Tgfbi and Postn double gene-deleted mice showed a similar post-myocardial infarction disease phenotype as Postn-deleted mice. Over-expression of TGFBI in the hearts of mice had a similar effect as previously shown in mice with periostin over-expression. Thus, TGFBI and periostin act similarly in the heart in affecting fibrosis and disease responsiveness, although TGFBI is not seemingly necessary in the heart after myocardial infarction injury and is fully compensated by the more prominently expressed effector periostin

Cardiac-specific over-expression of TGFBI.

<p>(A) Schematic representation of the double transgenic (DTG) system used to generate cardiac-specific, doxycycline inducible expression of TGFBI. (B) Western blot analysis of TGFBI in hearts of DTG mice. GAPDH was used as a control. (C) Immunohistochemistry of heart histological sections at 6 months of age from WT or TGFBI DTG mice. Sections were imaged for the proteins or markers shown. Scale = 10 μm. (D) Heart-weight (HW) normalized to body-weight (BW) in WT and DTG mice at 6 months of age. *p<0.05 vs WT using an unpaired Student’s T-test. (E) Percent fractional shortening (FS%) in WT and DTG mice at 6 months of age. *p<0.05 vs WT using an unpaired Student’s T-test. (F) Left ventricular dimension in diastole (LVED) in WT and DTG mice at 6 months of age as measured by echocardiography. (G) Percent fibrosis measured from Masson’s trichrome-stained cardiac histological sections from WT and DTG mice at 1 year of age. (H) Assessment of FS% in the indicated groups of mice over 12 weeks after sham or TAC surgery. (I) HW normalized to BW in the indicated groups of mice after 12 weeks after TAC. *p<0.05 vs corresponding sham group. (J) Assessment of lung-weight (LW) normalized to BW in the indicated groups of mice after 12 weeks of TAC. *p<0.05 vs corresponding sham group analyzed by parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

Loss of <i>Tgfbi</i> does not alter disease progression after MI.

<p>(A) Percent survival in the groups of mice shown over 7 days following MI surgery. No mortality was observed in sham-operated WT or <i>Tgfbi</i>^<i>-/-</i> mice. No differences in survival between WT and <i>Tgfbi</i>^<i>-/-</i> after MI was detected using the log rank Mantel-Cox statistical test. (B) Western blot analysis for TGFBI and periostin from isolated infarct areas of the hearts of the indicated groups of mice 7 days after MI. GAPDH was used as a loading control. (C and D) Quantification of protein levels normalized to GAPDH using densitometry for periostin in WT and <i>Tgfbi</i>^<i>-/-</i>-operated hearts and for TGFBI in WT and <i>Postn</i>^<i>-/-</i>-operated hearts. *p<0.05 vs WT using an unpaired Student’s T-test. (E) Percent survival in the indicated groups of mice over 7 days after sham or MI surgery. p<0.05 vs WT MI-operated animals using the log rank Mantel-Cox test (F) Percent ventricular fractional shortening (FS%) as measured by echocardiography in the indicated genotypes of mice 8 weeks after MI. *p<0.05 vs sham. (G) Left ventricular end diastolic dimension (LVED) as measured by echocardiography in the indicated groups of mice 8 weeks after MI. *p<0.05 vs sham using a parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

TGFBI and periostin are induced in the heart after injury.

<p>(A) Western blot analysis of periostin and TGFBI in isolated adult cardiomyocytes and fibroblasts. Sarcomeric α-actin was used as a control for cardiomyocyte purity and GAPDH was used as a loading control. (B) Quantitative real time PCR for <i>Postn</i> and <i>Tgfbi</i> from 1 week sham or MI-operated hearts. mRNA levels were normalized to 18s ribosomal RNA. *p<0.05 for both genes in MI-operated animals compared to sham animals using an unpaired Student’s T-test. n = 3 animals. (C) Western blot analysis for periostin and TGFBI in the infarcted areas isolated from hearts 24 hours, 7 and 14 days after MI surgery. GAPDH was used as a loading control. Each lane corresponds to protein from one mouse. (D and E) Quantification of TGFBI (D) and periostin (E) protein levels from the conditions shown in panel C except that 3 hearts were analyzed in total each. (F) Immunohistochemistry for the indicated markers/proteins on sham or MI-operated hearts after 1 week. Images were taken at 400x magnification. Scale = 20 μm.</p

Loss of <i>Tgfbi</i> does not appreciably alter disease progression after long-term TAC.

<p>(A) Percent ventricular fractional shortening as measured by echocardiography 12 weeks after TAC in the indicated groups. *p<0.05 vs sham-operated controls. (B) Heart-weight normalized to body-weight in the indicated groups of mice after 12 weeks of TAC. *p<0.05 vs sham-operated controls. (C) Cardiomyocyte cross-sectional area from ventricular histological sections in the indicated groups of mice 12 weeks after MI. *p<0.05 vs sham-operated controls. Four to six hearts were processed from each group, with at least 3 fields analyzed per histological section. (D) Lung-weight normalized to body-weight in the indicated groups of mice 12 weeks after TAC. *p<0.05 vs sham-operated controls. (E) Ventricular fibrosis from Masson's trichrome-stained histological images in the indicated genotypes of mice after 12 weeks of TAC. *p<0.05 vs sham-operated controls. Four to six hearts were processed from each group, with at least 3 fields analyzed per histological section. (F) Representative Masson’s trichrome-stained histological heart sections from sham and TAC hearts after 12 weeks of MI. Images were taken at 100x magnification. Scale bar = 100 μm. All statistical analyses were performed using a parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

Single-cell multiomic profiling of human lungs reveals cell-type-specific and age-dynamic control of SARS-CoV2 host genes.

Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis-regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20, a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate interpretation of risk loci for lung diseases