Essential regulation of lung surfactant homeostasis by the orphan G protein-coupled receptor GPR116

Abstract

GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

Figure 1. Gpr116 is expressed in lung ECs and ATII cells and is important for maintaining lung surfactant homeostasis

(A) Survival analysis of Gpr116 WT and KO mice. (B) An accumulation of eosinophilic, SP-A+ material was observed in the alveolar space of 6-month-old lungs of KO mice by H&E and SP-A staining. (C) The accumulated lung material stained positive with Oil Red O dye indicative of a buildup of neutral lipids in the alveolar space. Scale bar in (B) and (C) is 50μm. (D) Immunoblotting for SP-A, SP-B, SP-C, and SP-D in KO versus WT whole lung lysates. (E) Immunohistochemical staining of normal human lung. GPR116 protein (red) can be found in both ECs and ATII cells (arrows in inset) whereas alveolar macrophages were negative (yellow arrowheads). Scale bar is 100μm. (F) Immunofluorescence staining of normal human lung. GPR116 protein (red) co-localizes with SP-B (Green). Scale bar is 5μm. (G) QPCR analysis was used to assess Gpr116 expression in unfractionated lung tissue, lung ECs, and ATII cells and alveolar macrophages (MØ). Lineage specific markers were used to verify enrichment of isolated macrophages (Emr1, encoding F4/80 antigen), ECs (Cdh5), and ATII cells (Sftpa). Error bars represent SD. See also Figures S1 and S2.

Figure 2. Foamy macrophages result from changes in the alveolar microenvironment and are not caused by GPR116 loss in macrophages

(A) Clarity of BALF was evaluated in Gpr116 WT and KO mice. (B) Phosphatidylcholine (PC) levels were quantified in KO versus WT BALF at 3 and 16 months. (p< 0.01). Error bars represent SD. (C) H&E staining of BALF from WT and KO mice. Approximately half of the macrophages in the KO were enlarged and foamy and a smaller fraction (~3%) was binucleate (inset). Scale bar is 50μm. (D) Ultrastructural analysis of surfactant in the alveolar space of Gpr116 KO lungs. Scale bar is 10μm. (E) Ultrastructural analysis of macrophages with surfactant-laden phagosomes. Macrophages contained rod-shaped electron-dense inclusions (arrows) similar to those in Sftpa transgenic mice (Elhalwagi et al., 1999), possibly Ym1 crystals, as well as electron-transparent cholesterol clefts (arrowheads). Scale bar is 2μm. (F) SP-A immunohistochemistry performed on lungs following bone marrow transplantation. The transfer of Gpr116 WT BM into KO mice failed to prevent the accumulation of SP-A+ material in the alveolar space. Scale bar is 100μm (G) Immunofluorescence performed on BALF following bone marrow transplantation. An accumulation of enlarged, occasionally binucleate, macrophages was observed 6 months following the transfer of WT BM into the KO host. GFP expression of BALF cells confirms their origin from GFP-positive WT donor mice. To visualize all BM cells, membranes were labeled with CellMask Orange (CMO, red) and nuclei with DAPI (blue). Scale bar is 10μm. See also Figure S3.

Figure 3. GPR116 expression in ATII cells is important for lung surfactant homeostasis

(A) Surfactant accumulation in the alveolar space was assessed following constitutive SFTPC-Cre mediated deletion (B) Surfactant accumulation in the alveolar space was assessed following doxycyclineinducible SFTPC-Cre mediated deletion. Scale bar in (A) and (B) is 50μm. (C) Immunoblotting for SP-A, SP-B, SP-C, and SP-D in BALF derived from Gpr116^−/flox conditional KO mice containing the indicated transgenes. (D) Phosphatidylcholine (PC) and total cholesterol levels were quantified in WT versus KO BALF at 1 year of age (n=3, p<0.01). Error bars represent SD. See also Figure S4.

Figure 4. Gpr116 disruption leads to a defect in surfactant uptake by ATII cells

(A) GPR116-GFP expression is localized to the cell surface and LB-like structures in A549 cells. Scale bar is 5μm. (B) Immunofluorescence staining was used to evaluate co-localization of GPR116 (green) with ABCA3 (red) in A549 cells. The merged image shows partial co-localization (yellow) in lamellar body-like structures. In this experiment cells were permeabilized to detect ABCA3 which resulted in reduced staining of GPR116 at the cell surface. Scale bar is 10μm. (C) QPCR analysis of purified ATII cells for SP-A, SP-B, SP-C and SP-D at 6 weeks. (n=3). Error bars represent SD. (D) Immunoblotting of SP-A and SP-B in whole lung lysates or isolated ATII cells derived from 6-week-old Gpr116 KO versus WT mice. β-actin was used as a loading control. (E) Metabolic labeling of SP-A protein in ATII cells derived from Gpr116 WT and KO lungs. (F) Immunoprecipitation (IP) of SP-A with rabbit anti-SPA antibodies. Note the efficient IP of SP-A from Gpr116 KO BALF required butanol (BuOH) extraction. (G) Total protein and total glycerophospholipid (GPL) levels were quantified in BALF from 6-week old WT or KO mice (n=5). (H) Total PC, PE, PG, PI and PS levels were quantified in BALF from 6-week old WT or KO mice (n=5). (I) The relative distribution of GPLs were quantified in BALF from 6-week old WT or KO mice (n=5). (J) The individual PC species were quantified in BALF from 6-week old WT or KO mice (n=5). The PC species are labeled according to the total number of carbons in their fatty acid (FA) chains followed by the number of double bonds in the FA chains. (K) Purified human SP-A was mixed with surfactant from WT or KO mice and uptake into isolated ATII cells was monitored by Immunoblotting with mouse anti-human SP-A antibodies. β-actin was used as a loading control. (L) Uptake of rhodamine-phosphatidylethanolamine (rhodamine-PE) labeled WT or KO surfactant was evaluated in A549 cells. The control represents rhodamine-PE alone. Scale bar is 100μm. (M) Uptake of rhodamine-PE labeled WT or KO surfactant was evaluated in ATII-enriched cell fractions from SPC-GFP transgenic mice. In this particular experiment, GFP-positive ATII cells (green) were not sorted by flow cytometry but a partial enrichment of ATII cells was achieved by magnetic depletion of hematopoietic cells and endothelial cells. Scale bar is 5μm. Error bars represent SD (C) or SEM (G–J). *p<0.05, **p<0.01, ***p<0.001 See also Tables S2 and S3.