Loss of epithelial Gq and G11 signaling inhibits TGFβ production but promotes IL-33–mediated macrophage polarization and emphysema

Signaling by Gq/11 is required for optimal TGFβ activation in the lung to prevent inflammation. Gq/11 signaling maintains healthy lungs Loss of signaling by the cytokine transforming growth factor–β (TGFβ) in mice results in emphysema-like symptoms, whereas excessive TGFβ signaling results in pulmonary fibrosis and ventilator-associated lung injury. G proteins of the Gq/11 and G12/13 families mediate the integrin-dependent activation and release of latent TGFβ from the epithelial cells. John et al. found that mice deficient in Gq/11, but not those deficient in G12/13, in lung epithelial cells had defective TGFβ activation and emphysema-like symptoms. In addition, the Gq/11-deficient mice had lung inflammation associated with increased amounts of the cytokine IL-33. However, the mice were protected from ventilator-induced injury. Together, these data suggest that Gq/11 signaling is required for optimal TGFβ activation in the lung and the prevention of inflammation. Heterotrimeric guanine nucleotide–binding protein (G protein) signaling links hundreds of G protein–coupled receptors with four G protein signaling pathways. Two of these, one mediated by Gq and G11 (Gq/11) and the other by G12 and G13 (G12/13), are implicated in the force-dependent activation of transforming growth factor–β (TGFβ) in lung epithelial cells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhanced TGFβ activation promotes acute lung injury and idiopathic pulmonary fibrosis. Therefore, precise control of alveolar TGFβ activation is essential for alveolar homeostasis. We investigated the involvement of the Gq/11 and G12/13 pathways in epithelial cells in generating active TGFβ and regulating alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation that was primarily caused by alternatively activated (M2-polarized) macrophages, enhanced matrix metalloproteinase 12 (MMP12) production, and age-related alveolar airspace enlargement consistent with emphysema. Mice with impaired Gq/11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and enhanced macrophage MMP12 synthesis but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in these alveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar Gq/11 signaling maintains alveolar homeostasis and likely independently increases TGFβ activation in response to the mechanical stress of the epithelium and decreases epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/11 signaling promotes inflammatory emphysema but protects against mechanically induced lung injury.


INTRODUCTION
Heterotrimeric guanine nucleotide-binding protein (G protein) signaling is a ubiquitous system that couples many hundreds of G proteincoupled receptors (GPCRs) to a diverse array of effector molecules. Despite the huge diversity of receptors and effectors, there are only four families of heterotrimeric G proteins: G s , G i /G o , G q /G 11 (G q/11 ), and G 12 /G 13 (G 12/13 ). Two of these families (G q/11 and G 12/13 ) mediate activation of the RhoA signaling pathways (1,2). Work by our group and others identified that G q/11 and G 12/13 signaling to RhoA and Rho kinase in epithelial cells is central to the a v b 6 integrin-mediated activation of transforming growth factor-b (TGFb) in vitro (3,4). However, the importance of heterotrimeric G protein signaling pathways in the activation of TGFb in the alveoli in vivo has not been described.
TGFb plays a central role in diverse physiological processes, including homeostasis, repair, and immunity. The essential role of TGFb in maintaining alveolar homeostasis was demonstrated in mice with defective TGFb signaling pathways. Mice deficient in Smad3 (5), a major intracellular signal transducer and transcriptional modulator of TGFb signaling, or the epithelial TGFb receptor subunit TGFbRII (6) develop age-related emphysema because of increased production of matrix metalloproteinase 12 (MMP12) by alveolar macrophages. Furthermore, overexpression of TGFb in the lung leads to pulmonary fibrosis (7), and increased TGFb abundance is associated with the development of acute and ventilator-associated lung injury (8,9). Dis-ruption of alveolar TGFb signaling also leads to human disease. Mutations in TGFb1 and its receptors, as well as reduced serum concentrations of TGFb1, are associated with the development of chronic obstructive pulmonary disease (COPD) (10)(11)(12). Furthermore, increased TGFb signaling in the alveoli is associated with conditions such as idiopathic pulmonary fibrosis (13). Therefore, precise control of alveolar TGFb activity is central to the homeostatic function of pulmonary alveoli. Activation of the latent TGFb complex is the ratelimiting step in TGFb biosynthesis, and it is mediated in cells in vitro by a number of distinct mechanisms, including physical changes such as low pH or oxidation, proteases (14)(15)(16), or through the direct physical interaction of the extracellular domain of integrins with the RGD-binding site in the latent TGFb-binding protein (17)(18)(19). Studies in mice suggest that in vivo, in development at least, most pulmonary TGFb1 activation is through both the a v b 6 and a v b 8 integrins (20,21). Furthermore, mice with no functional a v b 6 integrins develop MMP12dependent, age-related emphysema (22), consistent with observations from mice with defective TGFb signaling.
To investigate whether G protein signaling pathways were required to generate alveolar TGFb and control alveolar homeostasis, we generated mice that were deficient in both Ga q and Ga 11 (Ga q /Ga 11 ) or in both Ga 12 and Ga 13 (Ga 12 /Ga 13 ) surfactant protein C (SftpC)-positive cells, restricting the defect in signaling predominantly to type II alveolar (ATII) epithelium. We showed that alveolar epithelial G q/11 signaling was required not only to promote stretch-mediated production of TGFb by epithelial cells within the lung but also to suppress synthesis of the key epithelial alarmin interleukin-33 (IL-33) and inhibit the M2 polarization of alveolar macrophages. Loss of signaling through this pathway leads to emphysematous changes in the lungs because of the disruption of epithelial cell-mediated suppression of macrophage activation and the resulting widespread alveolar destruction, but protected against stretch-mediated lung injury.

RESULTS
Crossing Gnaq fl/fl /Gna11 −/− or Gna12 −/− /Gna13 fl/fl mice with SftpC-Cre-positive animals generates offspring with deletion of Ga q or Ga 13 in type II alveolar epithelial cells To assess the role of the G q/11 and G 12/13 signaling pathways in alveolar epithelial cell function, we generated two strains of mice using Cre-loxP recombination. Mice heterozygous for the expression of Cre recombinase under the control of the SftpC promoter (SftpC +/− ) were crossed with mice either constitutively deficient in the gene encoding Ga 11 (Gna11 −/− ) and carrying floxed alleles of the gene encoding Ga q (Gnaq fl/fl ) (23) or constitutively deficient in the gene encoding Ga 12 (Gna12 −/− ) and carrying floxed alleles of the gene encoding Ga 13 (Gna13 fl/fl ) (24) to generate alveolar epithelial cell-specific, doubleknockout mice termed SftpC +/− ;Gnaq fl/fl ;Gna11 −/− and SftpC +/− ; Gna12 −/− ;Gna13 fl/fl , respectively. SftpC +/− ;Gnaq fl/fl ;Gna11 −/− progeny were born at the expected Mendelian ratios from regular matings of founder animals. In contrast, the litter sizes of mice in the SftpC +/− ; Gna12 −/− ;Gna13 fl/fl colony were smaller, and animals were not born at the expected Mendelian frequency (12.5% of each genotype), with only 6.8% of offspring being Cre-positive and carrying two null Gna12 alleles and two floxed Gna13 alleles.
Ga q /Ga 11  G q/11 -deficient mice have alveolar airspace enlargement and an obstructive lung defect Analysis of the lungs from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice (Fig. 1A) revealed that they were morphologically normal at 2 weeks of age, although by 4 weeks, there were widespread inflammatory infiltrates and architectural distortion within the lungs. There was enhanced inflammation and localized disruption of the alveolar architecture at 6 weeks, and this was maximal at 8 weeks of age. Mean linear intercept analysis confirmed that substantial increases in alveolar size were not detected in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice until they were 4 weeks of age (Fig. 1B) and that these animals subsequently showed a progressive and age-related increase in the size of the alveoli up to 8 weeks of age compared with age-matched Gna11 −/− control mice. Although there was no further increase in mean linear intercept beyond 8 weeks, alveolar size remained substantially greater in SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice than in Gna11 −/− mice up to 6 months of age (Fig. 1B). In contrast with that of SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice, mean linear intercept analysis of lung sections from Cre-positive heterozygous Ga q /Ga 11 colony mice and from SftpC +/− ;Gna12 −/− ;Gna13 fl/fl mice showed no evidence of increased alveolar airspace size ( fig. S2A) or inflammatory infiltrates ( fig. S2, B to D). Even at the later time points of 12 and 24 weeks of age ( fig. S2E), there was no evidence of inflammation or architectural distortion in the lungs of SftpC +/− ;Gna12 −/− ;Gna13 fl/fl mice, suggesting that G 12/13 signaling does not play a role in the postnatal development of alveoli or in alveolar homeostasis.
To confirm that the morphological changes had functional consequences, we measured lung function using invasive plethysmography at 8 weeks of age. SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice showed an obstructive defect as evidenced by a statistically significant reduction in the ratio of forced expiratory volume in the first 100 ms (FEV100) to forced vital capacity (FVC), that is, FEV100/FVC (Fig. 1C). A reduction in the FEV100/FVC ratio is a characteristic of the development of emphysema together with increased static lung volumes as reflected by statistically significantly increased total lung capacity Loss of alveolar epithelial G q/11 signaling leads to decreased activation of TGFb in the lung To determine whether TGFb signaling pathways were disrupted by the loss of Ga q and Ga 11 from epithelial cells, we measured the total TGFb1 concentrations in whole-lung homogenates and found them to be substantially decreased in SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice compared to those in Gna11 −/− control mice ( Fig. 2A). Analysis of supernatants collected from cultured lung slices detected the release of active TGFb from the Gna11 −/− lung slices, but no active TGFb1 was measured in the supernatants of SftpC +/− ;Gnaq fl/fl ;Gna11 −/− lung slices (Fig. 2B). To examine the role of Ga q and Ga 11 in TGFb activation in response to a contraction stimulus, we incubated lung slices with methacholine, which activates G q/11 signaling through muscarinic receptors. There was no substantial increase in the amount of active TGFb detected in the lung slice supernatants of either genotype (Fig.  2B); however, treatment of slices from Gna11 −/− mice with methacholine led to increased amounts of phosphorylated Smad2 (pSmad2) protein in lung slice lysates, which was not observed in the treated slices from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− animals (Fig. 2C).
To confirm that the reduction in total TGFb generation observed was the result of reduced alveolar TGFb activation in vivo, we used BAL leukocytes as reporter cells for the activation of TGFb in epithelial cells (Fig. 2D). Nuclear extracts of BAL leukocytes collected from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice had substantially less pSmad2 abundance than those from Gna11 −/− mice, thus confirming that the amount of active TGFb produced by the lung epithelium was reduced in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice (Fig. 2D). Furthermore, exogenous stimulation of BAL leukocytes from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice with TGFb for 1 hour stimulated a small, although statistically significant, increase in pSmad2 abundance, which was considerably blunted compared with that observed in cells from Gna11 −/− mice (Fig. 2D). RT-PCR analysis of the expression of two TGFb-responsive genes, Itgb6 (Fig. 2E) and Tsp1 (Fig. 2F), demonstrated that ATII cells from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice had statistically significantly lower amounts of both mRNAs than those from Gna11 −/− littermate controls.
Loss of G q/11 signaling leads to inflammatory cell recruitment and the M2 polarization of alveolar macrophages A characteristic feature of SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice was the appearance of enlarged and vacuolated cells within the alveolar airspaces (   the enlarged macrophages did not contain iNOS, suggesting that they had an M2 phenotype (Fig. 4F). Further analysis also demonstrated that macrophages from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− animals contained significantly more IL-10 than did those from Gna11 −/− mice (Fig. 4G), which is consistent with their polarization to an M2 phenotype.
Disruption of alveolar epithelial G q/11 signaling leads to increased IL-33 production To determine the effect of disrupting alveolar epithelial G q/11 and G 12/13 signaling on global gene networks, we performed Affymetrix GeneChip analysis on mRNA isolated from alveolar epithelial cells from  S4, A to C). Although the amount of the soluble IL-33 receptor ST2 was not substantially increased in lung tissue homogenates ( fig. S4D), it was markedly increased in the BALF from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice compared with that in the BALF from Gna11 −/− mice, suggesting that IL-33 was secreted into the extracellular compartment (Fig. 5F). The concentrations of IL-33 in the lungs of mice deficient in Itgb6 (which encodes the b 6 integrin subunit) were not substantially different from those in the lungs of wild-type mice (Fig. 5G), suggesting that a v b 6 integrin-mediated activation of TGFb was not required to suppress IL-33 production.
The increased IL-33 production from G q/11 -deficient epithelial cells promotes the polarization of alveolar macrophages to an M2 phenotype To assess the role of IL-33 in M2 macrophage polarization, we cultured alveolar macrophages from Gna11 −/− mice in BALF from SftpC +/− ; Gnaq fl/fl ;Gna11 −/− mice, which resulted in a substantial increase in Il10 mRNA abundance (Fig. 6A). Concomitant treatment with exogenous TGFb had no effect on Il10 mRNA abundance; however, the induction of Il10 mRNA expression was completely inhibited by an anti-IL-33 blocking antibody (Fig. 6A). Similarly, exposure of Gna11 −/− macrophages to BALF from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice resulted in a trend toward increased Arginase 1 (Arg1; M2a/c) and Sphk1 mRNA abundances (characteristic of M2 macrophages), although these differences were not statistically significant (Fig. 6B). Neutralization of IL-33 in the BALF from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice blocked the increase in Arg1 and Sphk1 mRNA abundances in the alveolar macrophages (Fig. 6B). Furthermore, culturing alveolar macrophages from Gna11 −/− mice in the BALF from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice for 24 hours led to the increased abundance of the M2a macrophage marker RELMa, as measured by immunofluorescence, which was substantially inhibited in the presence of an anti-IL-33 blocking antibody (Fig. 6, C and D). Together, these data suggest that the M2 macrophage polarization in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice is driven by the increased production of IL-33 by alveolar epithelial cells.
G q/11 signaling in alveolar epithelial cells transduces stretch-mediated generation of TGFb in the lungs To assess the role of alveolar epithelial G q/11 signaling in stretchmediated TGFb generation, we exposed Gna11 −/− mice and SftpC +/− ; Gnaq fl/fl ;Gna11 −/− mice to high-pressure ventilation to induce ventilatorinduced lung injury (VILI). Mice of each genotype were ventilated for 60 min to a standardized plateau pressure (Gna11 −/− mice, 37.93 ± 0.37 cm H 2 O; SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice, 37.83 ± 0.38 cm H 2 O) that was designed to impart an equivalent degree of mechanical stress and stretch to each set of lungs. This period of high-pressure ventilation [tidal volume (V T )] was followed by 3 hours of noninjurious ventilation. Gna11 −/− mice showed statistically significant increases in peak inspiratory pressure (Fig. 7A), plateau pressure (Fig. 7B), and lung elastance (Fig. 7C) after exposure to high V T , which is indicative of the development of lung injury. In contrast, SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice were protected from the development of physiological responses associated with VILI (Fig. 7, A to C). There was no statistically significant difference in lung resistance between the two genotypes of mice (Fig. 7D).
Protection from the development of VILI in SftpC +/− ;Gnaq fl/fl ; Gna11 −/− mice was linked to the inability of these animals to generate TGFb1 in response to mechanical stretch (Fig. 7E). Lung TGFb1 concentrations were substantially increased in Gna11 −/− animals exposed to short-term, high-stretch ventilation compared with those in animals exposed to only noninjurious low-pressure ventilation for 4 hours. These stretch-induced increases in TGFb1 were completely abrogated in SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice. Although IL-33 concentrations were statistically significantly greater in the lungs of SftpC +/− ;Gnaq fl/fl ; Gna11 −/− mice than in those of their Gna11 −/− littermate controls, IL-33 production in the lungs was not dependent on alveolar epithelial stretch (Fig. 7F), suggesting that G q/11 signaling has nonoverlapping effects on the TGFb and IL-33 signaling pathways.

DISCUSSION
The aims of this study were to investigate the role of two key G protein signaling pathways, those mediated by G q/11 and G 12/13 , in the regulation of alveolar epithelial homeostasis and to understand the pathological consequences of disrupting these pathways within the alveoli. Because of the functional redundancy of closely related G proteins S C I E N C E S I G N A L I N G | R E S E A R C H A R T I C L E and the induction of compensatory processes, it was necessary to perform studies in animals in which pairs of G proteins were deleted. Embryonic lethality has been reported in both G q/11 (23) and G 12/13 (25) knockout animals; therefore, a cell-targeted approach was required in which pairs of G proteins were deleted only in SftpC-positive epithelial cells within the lung. Comparisons were made with Gna11 −/− or Gna12 −/− Cre-negative littermate control mice, which previously had no reported lung abnormalities.
We did not find any phenotype associated with the alveolar deletion of both Gna12 and Gna13 with respect to lung development. Similarly, we did not find any phenotype associated with Gna11 −/− animals, although we cannot exclude a role for these G proteins in response to lung injury. However, we did show that deletion of both Ga q and Ga 11 in SftpC-positive ATII cells led to a phenotype of im-paired TGFb signaling and enhanced IL-33 production. This resulted in M2 macrophage polarization and increased expression of Mmp12 by macrophages, which resulted in changes within the lungs, including inflammation and alveolar airspace enlargement consistent with emphysema. Although we cannot completely exclude the possibility that the observed phenotype was primarily because of the alveolar deletion of Gnaq, we believe that this is unlikely for a number of reasons. First, most genetic models have not demonstrated any evidence of defects in the absence of Ga q alone. In the few cases in which Ga q deficiency promotes abnormalities, such as defective platelet activation (26) and the development of cerebellar ataxia (27), this is because Ga 11 is not found in platelets (26) or is present at relatively low abundance in Purkinje cells (28). Second, these observations are consistent with biochemical data showing that GPCRs do not distinguish between Ga q and Ga 11 (29)(30)(31)(32) and that both G proteins regulate the same effectors (33,34). Therefore, it is likely that the observed phenotype is the result of deletion of both the Ga q and Ga 11 signaling molecules.
We determined that impaired G q/11 signaling in alveolar epithelial cells led to two nonredundant, nonoverlapping consequences in the alveoli. First, we observed reduced stretch-mediated TGFb1 generation in the alveoli as measured by reduced TGFb1 release in vivo, in addition to decreased amounts of active TGFb and pSmad2, which were measured ex vivo in methacholine-treated lung slices from SftpC +/− ;Gnaq fl/fl ; Gna11 −/− mice. This deficiency in TGFb production in the alveoli ultimately resulted in altered macrophage functioning because of a reduction in Tgfbr1 expression and an increase in Mmp12 expression in alveolar macrophages. Second, we identified increased synthesis of IL-33 by alveolar epithelial cells, which promoted the polarization of alveolar macrophages toward an M2 phenotype in ex vivo experiments. These data confirm our previous in vitro findings that suggest that G q/11 signaling is crucial for mediating the activation of TGFb in alveolar epithelial cells (4), and highlight a hitherto unknown role for and IL-33 (F) in the lungs of the indicated mice exposed to normal ventilation alone (4 hours) or high stretch for 1 hour followed by 3 hours of normal ventilation. Statistical analysis was performed by repeated-measures ANOVA (A to D) or by one-way ANOVA with Dunn-Šidák-corrected post hoc comparisons (E and F). ****P < 0.0001. Data are means ± SEM (A to D) or calculated mean values (E and F) from four to nine mice per group. alveolar G q/11 signaling in suppressing IL-33 production and maintaining alveolar macrophage homeostasis. It is likely that the G q/11dependent generation of TGFb and suppression of IL-33 production is a fundamental pathway in the epithelium. Previous studies of mice deficient in Rac1 (a downstream effector of G q/11 signaling), specifically in airway epithelial cells, demonstrated enhanced inflammatory responses, impaired TGFb production, and exaggerated IL-33-dependent responses (35), with which our data are consistent. Moreover, the intestinal helminth Heligmosomoides polygyrus secretes proteins that induce TGFb signaling (36) and inhibit IL-33 (37), thus mimicking the effect of epithelial G q/11 signaling.
Previous in vivo studies that have attenuated TGFb signaling in the lung, either through whole-body deletion of Smad3 (5) or a v b 6 integrins (22,38) or through epithelial cell-specific deletion of TGFbRII (6), have shown mild pulmonary inflammation, epithelial airspace enlargement, and enhanced Mmp12 expression, consistent with our findings with the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice. However, the degree of inflammation and airspace destruction observed in the lungs of SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice was considerably worse at an earlier stage of development than that reported in the previous studies. It is possible that the combination of increased expression of Mmp9 and Mmp12 within the BAL leukocytes of the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice may contribute to the exaggerated response seen in these animals, because both effects may play a role in the development of emphysema (22,39). However, we believe that the severe phenotype most likely results from the combined effects of a failure of TGFb activation on alveolar epithelial cells and an increase in their production of IL-33. IL-33 is associated with enhanced pulmonary inflammation and increased generation of ST2 and IL-10 (40) in addition to promoting the polarization of macrophages toward an alternatively activated (M2) state (41,42). Increased epithelial IL-33 has also previously been linked to the development of COPD (43).
These data demonstrate that SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice have reduced amounts of TGFb in their lungs, which, in contrast with Gna11 −/− mice, could not be increased in response to epithelial cell stretch after high-pressure ventilation in vivo or methacholineinduced contraction ex vivo. This is consistent with a failure of a v b 6 integrin-mediated activation of TGFb, which requires force generation through RhoA-induced intracellular cytoskeletal reorganization (4,44,45). The phenotype of impaired a v b 6 integrin-mediated TGFb activation is further supported by the reduced abundance of pSmad2 in alveolar macrophages and the reduced abundance of itgb6 mRNA in alveolar epithelial cells from SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice. Our previous findings suggested a critical role for Ga q in the regulation of a v b 6 integrin-mediated TGFb activation independently of the abundance of a v b 6 (4). However, TGFb signaling increases the epithelial cell abundance of a v b 6 integrin by inducing itgb6 expression (46), which has led to the proposal of a TGFb-a v b 6 integrin positive feed-forward loop (47). These data support this hypothesis, because sustained reduction of alveolar TGFb activation in the SftpC +/− ; Gnaq fl/fl ;Gna11 −/− mice led to reduced itgb6 expression in alveolar epithelial cells.
Itgb6 −/− mice are protected from the development of VILI (9), and our data demonstrate that SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice are also protected from the deleterious effects of high-pressure ventilation, suggesting that G q/11 signaling pathways are crucial for epithelial mechanotransduced signals that promote TGFb activation. Furthermore, we observed that airspace enlargement in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice developed postnatally, when spontaneous ventilation began, and was not apparent until the mice were 4 weeks of age. Therefore, we propose the hypothesis that spontaneous ventilation leads to cyclical alveolar stretch and the release of GPCR ligands that act through the G q/11 pathway to promote alveolar TGFb activation, suppress macrophage Mmp12 expression, and prevent the subsequent development of emphysema. However, further evidence is required to determine whether the stretch-induced production of TGFb is directly linked to TGFb activation by the a v b 6 integrin.
We suggest that G q/11 signaling has nonoverlapping effects on TGFb activation and IL-33 production. Although previous studies have suggested that IL-33 is a mechanically stimulated cytokine (48), we found no evidence that IL-33 production in the lungs was regulated by exposure to epithelial cell stretch. Therefore, it is likely that the enhanced IL-33 generation observed in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice was driven by transcriptional signals rather than by mechanotransduction. TGFb limits the production of IL-33 by macrophages (49); therefore, it is possible that the observed increase in IL-33 abundance in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice was a result of reduced TGFb activity in the alveoli. However, we do not favor this hypothesis because, in contrast to the findings in the SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice, we did not observe any change in IL-33 concentrations in the lungs of Itgb6 −/− mice.
In summary, our findings suggest that G q/11 signaling is required for two nonoverlapping signaling pathways: the mechanotransduced generation of TGFb and the transcriptional suppression of IL-33, both of which appear to be essential for the maintenance of alveolar macrophage homeostasis. Enhanced IL-33 production promotes the polarization of the alveolar macrophages to an M2 phenotype, whereas the failure of epithelial cells to generate TGFb led to impaired Tgfbr1 expression in alveolar macrophages and the loss of TGFb-mediated suppression of MMP12 production by macrophages. Loss of the homeostatic control of alveolar macrophages results in widespread alveolar destruction and airspace enlargement associated with the development of emphysema. These data confirm the central role of G q/11 signaling in stretch-mediated alveolar TGFb generation in vivo, reveal a hitherto unknown role for G q/11 signaling in the regulation of IL-33 by epithelial cells, and highlight a molecular pathway that is required to prevent ventilation-associated alveolar disease.

Study design
All animal experiments were designed to test a specific hypothesis or objective, with data being recorded and reported in accordance with the guidelines from the Fund for The Replacement of Animals in Medical Experiments (FRAME) and Animal Research: Reporting of In Vivo Experiments (ARRIVE). Phenotyping experiments were performed blind to genotype to minimize bias and with a factorial design to minimize confounding variables. Animals were assigned a six-digit identity code at weaning and were genotyped before analysis, but data collection was performed by researchers blinded to genotype. Initial studies were performed to establish the baseline phenotype of the mice and the variability of observed biochemical, morphological, and physiological parameters. For VILI and lung function experiments, mice were assigned to experimental groups based on genotype; however, experimental procedures, data collection, and analysis were performed by an investigator blinded to the allocation sequence and genotype. Studies were powered to detect a 1 SD difference in endpoints using a power calculation assuming an 80% power at the 5% significance level, resulting in group sizes of five to eight mice per group based on initial phenotyping data. Sample sizes were predetermined for in vitro studies with isolated primary cells, including RNA analysis and gene array studies, and on the basis of large expected effect sizes, sampling required three to six mice of each genotype.

Experimental animals
Mice were housed under specific pathogen-free conditions, and all animal experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and approved by the Animal Welfare and Ethical Review Committee at the University of Nottingham. The generation of floxed alleles of the genes encoding Ga q (Gnaq) and Ga 13 (Gna13) and of the null alleles for the genes encoding Ga 11 (Gna11) and Ga 12 (Gna12) has been described previously (23,24). Mice constitutively deficient in Gna11 and containing floxed alleles of Gnaq or mice constitutively deficient in Gna12 and containing floxed alleles of Gna13 were crossed with mice expressing Cre recombinase under the control of the SftpC promoter (SftpC-Cre) obtained from B. Hogan (Duke University) (50). Mice were genotyped from DNA isolated from ear notch biopsies by PCR analysis with allele-specific primers (table S1) and analyzed by electrophoresis on ethidium bromide-stained agarose gels as previously described (23,24). The genetic background of the mice was predominantly C57BL6 (at least a sixth-generation backcross for Ga q /Ga 11 and Ga 12 /Ga 13 mice and at least a fourth-generation backcross for SftpC-Cre mice).

Quantitative morphometry and mean linear intercept
Mice were terminally anesthetized with Euthatal (Merial Animal Health), and the lungs and trachea were exposed. The lungs were perfused with phosphate-buffered saline (PBS) containing heparin (40 U/ml) through the left ventricle to remove all the blood before the trachea was cannulated. Lungs were insufflated in situ with 10% neutral-buffered formalin at a constant pressure of 20 cm H 2 O, removed, and paraffin-embedded for the preparation of histological sections. Lung morphology was assessed in H&E-stained 5-mm tissue sections. For each set of lungs, eight random fields were photographed across all lobes with a Nikon Eclipse 90i microscope at ×10 magnification and with NIS-Elements Software v3.2. Images were overlaid with a 100-mm grid, and the mean linear intercept [defined as the linear sum of the lengths, in micrometers, of all lines in all frames counted divided by the number of intercepts (defined as an alveolar septum intersecting with a counting line)] was calculated with a method adapted from Dunnill (51).

Lung function measurements by forced pulmonary maneuvers
Eight-week-old mice were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and medetomidine hydrochloride (1 mg/kg) to maintain spontaneous breathing under anesthesia. Mice were tracheostomized, placed in a body plethysmograph, and connected to a computer-controlled ventilator (Forced Pulmonary Maneuver System, Buxco Research Systems). An average breathing frequency of 120 breaths/min was imposed on the anesthetized animal by pressurecontrolled ventilation until a regular breathing pattern and complete expiration at each breathing cycle were obtained. To measure FRC, ventilation was stopped at the end of expiration through the immediate closure of a valve located proximally to the endotracheal tube. Pressure changes at the mouth and in the body box after spontaneous breathing maneuvers against a closed valve were recorded to calculate the FRC (Boyle's law). To measure the TLC, RV, inspiratory capacity, and vital capacity, the quasi-static pressure volume maneuver was performed. In this maneuver, the lungs are inflated to a standard pressure of +30 cm H 2 O, which was followed by a slow exhalation until a negative pressure of −30 cm H 2 O was reached. The quasi-static compliance was defined as the volume/pressure ratio at 50% of the expiration (Cchord50). For the fast flow volume maneuver, lungs were first inflated to +30 cm H 2 O (TLC) and, immediately afterward, connected to a highly negative pressure to enforce expiration until the RV reached −30 cm H 2 O. Forced expiratory flows, times of expiration and inspiration, and forced expiratory volumes (FEV100 and FEV200) were recorded during this maneuver. Suboptimal maneuvers were rejected, and for each test in every single mouse, at least three acceptable maneuvers were conducted to obtain a reliable mean for all numeric parameters.

Ventilator-induced lung injury
Mice were anesthetized [ketamine (80 mg/kg)/xylazine (8 mg/kg)] and surgically instrumented for ventilation as described in detail previously (52) by an experimenter blinded to genotype. Briefly, animals were tracheostomized and connected to a custom-made ventilator/ pulmonary function testing system. The left carotid artery was cannulated to enable continual monitoring of arterial blood pressure, removal of samples for blood gas analysis at predetermined intervals, and infusion of fluids [heparin (10 U/ml) in 0.9% NaCl, 0.3 ml/hour]. During surgery and the subsequent stabilization period, mice were ventilated with a noninjurious strategy [V T of 8 to 9 ml/kg; 3 cm H 2 O positive end expiratory pressure (PEEP); 120 breaths/min] using 100% O 2 . Lung volume history was standardized by sustained inflation, and baseline respiratory mechanics were evaluated by the end-inflation occlusion technique. After baseline measurements were made, V T was increased to produce stretch-induced lung injury. Specifically, ventilation was standardized with a plateau pressure of 37.5 to 38.5 cm H 2 O, which was designed to ensure that mice of different genotypes were exposed to an equivalent degree of mechanical stress to stretch the lungs. Additionally, PEEP was set to 0, respiratory rate was 80 breaths/min, and inspired gas was changed to 96% O 2 /4% CO 2 to prevent hypocapnia. Animals were ventilated with this constant tidal volume for 60 min or until airway plateau pressure had increased by 15%, whichever occurred first. Ventilation was then returned to match the baseline "prestretch" strategy and maintained for a further 3 hours. Sustained inflation maneuvers were performed every 30 min during this "noninjurious" ventilation period to reduce the development of atelectasis. Anesthesia was maintained by bolus intraperitoneal administration of ketamine (40 mg/kg)/xylazine (4 mg/kg) every 20 to 25 min. Animals were terminated by overdose of anesthetic, followed by exsanguination. The lungs were lavaged with 750 ml of ice-cold PBS containing phosphatase inhibitor cocktail (Calbiochem), and the recovered fluid was centrifuged at 210g for 5 min at 4°C. Aliquots of supernatant and the lavage cell pellet were frozen at −80°C. After lavage, the chest wall was opened, and the right lung was tied off at the hilum, removed, and snap-frozen in liquid N 2 . Finally, the left lung was inflated with 4% paraformaldehyde at 20 cm H 2 O before being embedded in paraffin for histological analysis.
Precision-cut lung slices Mice were terminally anesthetized as described earlier and exsanguinated, and then their lungs and trachea were exposed. The lungs were perfused with PBS containing heparin (200 U/ml) through the left ventricle to remove all the blood. The trachea was cannulated, and the lungs were filled with 1.2 ml of 1% low-melting point agarose prewarmed to 42°C, which was followed by 0.3 ml of air. The lungs were cooled by covering them with ice-cold PBS to enable the agarose to set and were carefully removed, and the lobes were dissected. Lung slices (150 mm thick) were prepared from each lung lobe with a VT1200S Vibratome (Leica). Lung slices (two per well) were cultured overnight in serum-free Dulbecco's modified Eagle's medium (DMEM) containing penicillin and streptomycin before being stimulated. To induce contraction of the lung slices, they were repeatedly stimulated for 10 min with 100 mM methacholine at 1-hour intervals for 8 hours. After they were stimulated, the lung slices were washed in PBS and homogenized in protein lysis buffer [20 mM tris-HCl (pH 7.4), 137 nM NaCl, 2 nM EDTA, 25 mM b-glycerophosphate, 1 nM Na 3 VO 4 , 1% Triton X-100, 10% glycerol]. This buffer was supplemented with leupeptin, phenylmethylsulfonyl fluoride, a protease inhibitor (protease inhibitor mixture, Roche Applied Science), dithiothreitol, and a phosphatase inhibitor (PhosSTOP, Roche Applied Science).

Western blotting
The amounts of Ga q or IL-33 in whole-cell extracts of ATII cells were determined by Western blotting analysis. Protein samples (30 mg per lane) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane. After blocking for 1 hour (in tris-buffered saline, 5% nonfat milk, 0.1% Tween 20), the membrane was incubated either overnight at 4°C with monoclonal anti-Ga q antibody in blocking buffer or for 48 hours with anti-IL-33 antibody (Nessy-1). After being washed [with PBS (pH 7.4), 0.3% Tween 20], the membrane was incubated at room temperature in blocking buffer with the appropriate HRP-conjugated secondary antibody for 1 or 2 hours for the detection of Ga q or IL-33, respectively. The membrane was incubated with enhanced chemiluminescence (ECL) Western blotting detection reagent and visualized by exposure to Hyperfilm ECL. To quantify the differential production of IL-33 in the alveolar epithelial cells, densitometry was performed with Adobe Photoshop CC2014 software, and the mean pixel densities were calculated. Densitometry data are presented as the ratio of IL-33 abundance to that of GAPDH in cells independently isolated from three mice per group.
RNA isolation and quantitative RT-PCR Total RNA was extracted from lung samples, purified ATII cells, and BAL cells by homogenization in TRIzol B (Invitrogen) and was pro-cessed according to the manufacturer's instructions. Samples were reverse-transcribed into complementary DNA (cDNA) with Moloney murine leukemia virus reverse transcriptase (for BAL cell pellets; Promega) or SuperScript II Reverse Transcriptase (for lung samples and ATII cells; Invitrogen). cDNA was subjected to quantitative RT-PCR analysis with gene-specific primers (table S1). Amplification was performed with MXPro3000 (Stratagene) with KAPA SYBR FastTaq (Anachem) on the following program: initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 10 s. Amplification of a single DNA product was confirmed by melting curve analysis. Data were analyzed relative to the abundances of the housekeeping genes hypoxanthine phosphoribosyltransferase 1 (Hprt) (for BAL samples) or glucuronidase b (Gusb) (for whole-lung and ATII samples) and expressed as the fold change in mRNA abundance using the DDC t equation as described previously (53). Primers sequences for quantitative RT-PCR are listed in table S2.
BAL and cytospin analysis Animals were terminally anesthetized as described earlier, their tracheae were cannulated, and their lungs were washed with eight sequential 0.5-ml aliquots of cold PBS or PBS containing 1× phosphatase inhibitor solution (Active Motif). Aliquots were pooled and centrifuged, and the cell pellets were resuspended in PBS or PBS containing 1× phosphatase inhibitor solution (Active Motif) for the analysis of pSmad2 and macrophage culture experiments. Cells were counted with Scepter 2.0 automated cell counter (Millipore), and cell concentrates were cytospun onto glass slides and stained with Diff-Quick (Dade Diagnostics). Cell subsets were counted under objective magnification of ×40 (200 cells per slide). Designation of alveolar macrophages as "normal" or "enlarged" was made visually based on cell size. The enlarged macrophages observed only in SftpC +/− ;Gnaq fl/fl ; Gna11 −/− mice were identified after cytospin analysis by their increased cytoplasmic surface area when compared with alveolar macrophages from Gna11 −/− , Gna12 −/− , or SftpC +/− ;Gna12 −/− ;Gna13 fl/fl animals.
Culture of BAL cells BAL cells were collected into serum-free DMEM (Lonza) containing glutamine, penicillin, and streptomycin and pelleted by centrifugation at 150g for 5 min at 4°C. BALF was collected, and BAL cells were pooled from Gna11 −/− mice (n = 10 to 24 mice) and SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice (n = 3 to 5). For crossover experiments in which the effect of secreted factors in BALF on gene expression was examined, 2.5 × 10 5 BAL cells from Gna11 −/− mice or SftpC +/− ;Gnaq fl/fl ;Gna11 −/− mice were resuspended in 1 ml of the appropriate BALF and then were plated into 12-well plates for RNA endpoint analysis or in 250 ml of BALF and then were plated into 8-well chamber slides for immunofluorescence staining. Cells were incubated with BALF for 24 hours at 37°C, nonadherent cells were removed by washing, and the adherent macrophages were lysed by the addition of TRIzol B before RNA was isolated or were fixed in ice-cold 100% methanol for 10 min before immunofluorescence staining was performed. For cytokine stimulation or blocking experiments, serum-free DMEM or BALF was mixed with TGFb (2 ng/ml), anti-TGFb antibody (5 mg/ml; 1D11), or anti-IL-33 antibody (1.5 mg/ml; Bondy-1-1) and incubated with BAL cells in culture for 24 hours.
Immunostaining and image analysis Formalin-fixed lung tissue sections (5 mm thick) were deparaffinized and rehydrated before antigen retrieval by being microwaved in 10 mM S C I E N C E S I G N A L I N G | R E S E A R C H A R T I C L E citric acid buffer (pH 6.0). Endogenous peroxidase activity in the sections was blocked with methanol containing 3% H 2 O 2 for 15 min at room temperature. Nonspecific binding was blocked with 5% normal serum before the sections were incubated overnight with primary antibody at 4°C, which was followed by a 30-to 60-min incubation with an appropriately labeled secondary antibody at room temperature. All sections were visualized with diaminobenzidine and counterstained with hematoxylin and mounted in DPX (Sigma-Aldrich) for immunohistochemical analysis or were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen) for 5 min and mounted with ProLong Gold Antifade (Invitrogen) for immunofluorescence analysis. To stain isolated alveolar macrophages, methanol-fixed cells were permeabilized with 0.1% Triton X-100 for 10 min before being blocked in 5% normal serum, incubated overnight with primary antibody at 4°C, and then incubated for 30 to 60 min with an appropriately fluorescently labeled secondary antibody at room temperature. All antibody incubations were performed in staining buffer containing PBS, 5% serum, and 0.1% bovine serum albumin. Slides were washed in PBS (Sigma-Aldrich). Images were captured with a Nikon 90i (for immunohistochemistry) or a Nikon Ti confocal microscope (for immunofluorescence) with NIS-Elements Software v3.2. To quantify the staining of isolated alveolar macrophages, individual cells were identified with NIS-Elements Software v3.2, and the total numbers of pixels for both antibodystained and negative control cells were calculated. Pixel counts were determined in 40 to 100 individual cells from images captured in 5 to 20 randomly selected fields of view. The average number of pixels per cell in the unstained sections (negative control) was subtracted from the values calculated for antibody-stained sections to eliminate autofluorescence, and the data were presented as the number of pixels per cell.

Isolation of ATII epithelial cells
Mice were terminally anesthetized as described earlier and exsanguinated, and their lungs and trachea were exposed. The lungs were perfused with PBS containing heparin (200 U/ml) through the left ventricle to remove all the blood. The trachea was cannulated, and BAL was performed with eight sequential 0.5-ml aliquots of cold PBS to remove leukocytes. The lungs were filled with 1 ml of dispase (BD Pharmingen) and allowed to collapse naturally before the instillation of 0.5 ml of 1% low-melting point agarose (Promega) prewarmed to 42°C. The lungs were then covered in ice for 2 min to enable the agarose to set before they were removed. The lungs were incubated at room temperature for 45 min in 2 ml of dispase and then washed briefly in ice-cold PBS before being transferred to petri dishes containing DMEM with 10% fetal calf serum, glutamine, and deoxyribonuclease 1 (100 U/ml; Sigma). Lung tissue was carefully separated from the large airways, and blood vessels before the cell suspension were dissociated. The crude cell suspension was sequentially filtered through 70-and 40-mm tissue sieves (BD) and then plated into plastic petri dishes coated with mouse immunoglobulin G (IgG) (1.5 mg of IgG per plate). Nonadherent cells were collected after a 1-hour incubation at 37°C and were pelleted by centrifugation at 150g for 5 min. Contaminating leukocytes were removed by negative sorting with magnetic beads labeled with sheep anti-rat CD16/32 and CD45 antibodies (Dynabeads, Dynal) for 1 hour at 4°C according to the manufacturer's instructions. The remaining cells were resuspended in Bronchial Epithelial Growth Medium (BEGM; Lonza) without hydrocortisone and containing keratinocyte growth factor (10 mg/ml; R&D Systems), and the purity of the ATII cell suspension was assessed by modified Papanicolaou staining of cell cytospins as previously described (54). The cells were plated onto collagen-coated 24-well plates, and ATII cells were grown to confluence. Cell purity was routinely >75%, with the major contaminating cells being fibroblasts. For experiments in which ATII cells were to be used for mRNA analysis, an additional purification step was added after the completion of the standard protocol in which epithelial cells in the cell suspension were enriched by positive sorting with sheep anti-rat magnetic beads labeled with an anti-E-cadherin antibody (Dynabeads, Dynal). After washing to remove the bead-free cells, the mixture of beads and ATII cells was immediately mixed with TRIzol B reagent.
ELISA/Luminex analysis Total TGFb1 concentrations in murine lung homogenates were measured after acid activation of samples with a murine TGFb1 Quantikine ELISA kit (R&D Systems). Concentrations of active TGFb secreted into lung slice supernatants were also determined with this ELISA kit, but without acid activation. The concentrations of soluble ST2 were measured in lung homogenates and BALF with a mouse ST2/IL-1 R4 Quantikine ELISA kit (R&D Systems), whereas the concentrations of IL-33, IL-4, IL-10, and IL-13 were assessed with customized mouse Magnetic Luminex Screening Assay (R&D Systems). Additional IL-33 analysis was performed with a mouse IL-33 ELISA kit (R&D Systems). All assays were performed according to the manufacturer's instructions.
Measurement of pSmad2 by ELISA Nuclear protein was prepared from BAL cells with Nuclear Extract Kit (Active Motif) according to the manufacturer's protocol. To analyze lung slices, total protein extracts were generated according to the manufacturer's instructions (Cell Signaling Technology). The amounts of pSmad2 were measured in 10 mg of nuclear protein (BAL cells) or 10 mg of total protein (lung slices) with solid-phase PathScan Phospho-Smad2 (Ser 465/467 ) Sandwich ELISA Kit (Cell Signaling Technology) according to the manufacturer's instructions.

Microarray analysis
Total RNA was assessed quantitatively using NanoDrop and qualitatively (RNA integrity number > 7) by BioAnalyzer (Agilent). After the quantity and quality of RNA were assessed, 10 ng of total RNA was used to prepare microarray hybridization probes with Epistem's RNA-Amp RNA amplification kits. After fragmentation, the biotinylated complementary RNA was hybridized to GeneChip Murine MOE430 2.0 microarrays (Epistem). After hybridization, the microarrays were washed and scanned with GeneChip Scanner 7G. Microarray data were background-corrected, log 2 -transformed, and normalized with robust multiarray average (RMA), followed by quantile normalization, and the RMA data from all the arrays that passed quality control were analyzed with Partek Genomics Suite version 6.5. Principal components analysis was used to interrogate the effects of all the tracked and recorded experimental technical factors (dates, yields, randomization order, etc.) on overall cohort data structure. Multivariate ANOVA was applied within Partek Genomics Suite software for each pairwise comparison of groups using the method of moments. To further rank or prioritize genes that might be useful as candidate biomarkers, the degree of change in RNA abundance, or fold change, was used to select genes that differed in expression statistically significantly by one-way ANOVA (unadjusted P value of <0.05) between compared groups and also exhibited a twofold or greater change in expression. Further selection was performed by establishing overlaps between treatment groups. Further investigation of biological relevance was performed with Ingenuity IPA software.

Statistical analysis
All statistical analyses were performed with GraphPad Prism 6 software. Data were assessed for normality. Where differences between two groups were measured, the pooled mean values were compared with unpaired t tests. Comparisons between more than two groups were made by one-way ANOVA with Bonferroni or Dunn-Šidák multiple testing corrections applied to post hoc, pairwise comparisons. Comparisons between groups over multiple time points were made by repeated-measures ANOVA. Corrected P values <0.05 were considered to be statistically significant.

SUPPLEMENTARY MATERIALS
www.sciencesignaling.org/cgi/content/full/9/451/ra104/DC1 Fig. S1. Characterization of alveolar epithelial cell-specific, G protein-deficient mouse colonies. Fig. S2. Histological analysis of littermate control mice. Fig. S3. Loss of epithelial Ga q /Ga 11 signaling leads to altered lung function. Fig. S4. Effect of the deficiency in epithelial G q/11 signaling on the concentrations of T H 2 cytokines and ST2 in the lung. Table S1. Sequences of genotyping primers. Table S2. Sequences of RT-PCR primers. Data File S1. Affymetrix gene array data.