Thyroid hormone receptor repression is linked to type I pneumocyte-associated respiratory distress syndrome

Abstract

Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of type I pneumocytes, the cells that mediate gas exchange, is poorly understood. In contrast, the glucocorticoid receptor and its cognate ligand have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear co-repressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) in C57BL/6 mice (SMRTmRID) produces a previously unidentified respiratory distress syndrome caused by prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which, in turn, seems to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify TR as a second nuclear receptor involved in lung development, specifically type I pneumocyte differentiation, and suggest a possible new type of therapeutic option in the treatment of RDS that is unresponsive to glucocorticoids.

Figure 1. SMRT^mRID mice die postnatally due to acute respiratory failure

a) A representative newborn SMRT litter, with two SMRT^mRID pups in the middle. Arrows indicate the visible lungs. b) A representative picture of blood from newborn WT and SMRT^mRID littermates. c) Comparison of the lungs from newborn WT and SMRT^mRID littermates in PBS. d) Microscopic images of H&E stained lung tissue from newborn SMRT littermates and the quantification of alveoli sizes. Scale bars, upper panel, 200 µm. Scale bars, lower panel, 50 µm.

Figure 2. Lungs of SMRT^mRID mice lack mature type I pneumocytes

a) High magnification light microscopic images of H&E stained lung tissue from newborn WT and SMRT^mRID littermates. Arrowheads indicate some of the type II pneumocyte-like cells. Scale bars, 20 µm. b) Electron microscopic images of lung tissue from newborn WT and SMRT^mRID littermates. Scale bars, 5 µm. c) Immunostaining of type I pneumocyte markers (T1α and Cav1) in E18.5 WT and SMRT^mRID littermate lungs. Scale bars, 50 µm. d) Expression of type I and II pneumocyte markers in E17.5 SMRT littermate lungs. Gene expression in each individual embryo was analyzed. * p<0.05; *** p<0.001.

Figure 3. Anti-thyroid drugs rescue SMRT^mRID animals and restore type I pneumocyte development

a) A representative PTU treated newborn SMRT litter. Arrows indicate the visible lungs. b) A representative picture of the blood from PTU treated newborn WT and SMRT^mRID littermates. c) Comparison of the lungs from PTU treated newborn WT and SMRT^mRID littermates in PBS. d) Microscopic images of H&E stained lung tissue from SMRT littermates (E18.5 and P0). Scale bars, 50 µm. e) Electron microscopic images of lung tissue from PTU treated newborn WT and SMRT^mRID littermates. Scale bars, 5 µm.

Figure 4. Klf2 is reduced in SMRT^mRID lungs and activates type I pneumocyte gene program

a) Expression of Klf2 in E17.5 and E18.5 SMRT littermate lungs. Gene expression in each individual embryo was analyzed. * p<0.05; ** p<0.01. b) Immunostaining of Klf2 in E18.5 and P0 WT and SMRT^mRID littermate lungs. Scale bars, 50 µm. c) Adenoviral-mediated Klf2 expression induces type I pneumocyte marker expression in MLE12 cells. The MOI of adenovirus used is indicated. * p<0.05; *** p<0.001. d) Klf2 binds to the promoter region of type I pneumocyte marker genes in a ChIP assay. * p<0.05; ** p<0.01. e) Klf2, but not NKX2.1, directly activates T1α, Aqp5 and Cav1 promoters. Transient transfections were done in triplicate wells in CV-1 cells and repeated at least 3 times. Luciferase values normalized to β-gal were shown. ** p<0.01.

Figure 5. Klf2 is essential for type I pneumocyte and normal lung development in vivo

a) A representative picture showing a highly Klf2 chimeric mouse (#33, in the middle) and its sinking lung in PBS compared to its littermates. The lungs of its littermates are clearly visible and indicated by arrows. b) Expression of Klf2 and type I pneumocyte markers in highly Klf2 chimeric mice and their respective littermates. c) Immunostaining of Klf2 and type I pneumocyte markers in highly Klf2 chimeric mice and their respective littermates. Scale bars, 50 µm. d) H&E staining of lungs from highly Klf2 chimeric mice and their respective littermates. Scale bars, 100 µm. e) A working model proposing that GR and TR regulate distinct pathways of pneumocyte development.