Deletion of G protein-coupled receptor 116 enhances neutrophil function and aggravates lung injury in mice

Abstract

Acute lung injury (ALI) globally afflicts over 3 million individuals every year. It can eventually develop into acute respiratory distress syndrome (ARDS) with a high mortality of up to 40%. To date, ALI has been undertreated in terms of the feeble efficacy of clinical approaches and the lack of proven pharmacological targets. 1 G protein-coupled receptors (GPCRs), the promising targets of modern medicine, participate intensively in the regulation of human physiology and pathophysiology. Among them, Gpr116, which is expressed in alveolar epithelial cells and immune cells, has been reported to play a key role in maintaining alveolar homeostasis and inhibiting inflammation. 2 However, its role in the regulation of neutrophil function and ALI remains to be elucidated. In this study, we demonstrated the inhibitory role of Gpr116 on neutrophil function, which contributes to nonspecific lung injury in mice induced by lipopolysaccharide (LPS). In brief, LPS was intratracheally injected at a dose of 10 mg/kg in mice to induce ALI in vivo. Neutrophils isolated from the bone marrow of control and myeloid cell-specific Gpr116 knockout mice were stimulated with phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) in vitro. More details are available in the Supplementary materials. In clinical data, Gpr116 mRNA expression in blood samples was lower in patients with ARDS than in non-ARDS patients without statistical significance (Fig. 1A). Furthermore, we detected whether Gpr116 expression was altered in ALI mice. After administering LPS to C57BL/6J mice, we measured Gpr116 expression in lung tissue and BALF cells. Compared with that in control mice, the mRNA expression of Gpr116 in lung tissues and BALF cells of ALI mice decreased (Fig. 1B, C), along with similar changes in the protein expression of Gpr116 in the mouse lungs (Fig. S1). Besides, in the in vitro experiment, the mRNA expression of Gpr116 in bone marrow-derived neutrophils also decreased after PMA stimulation (Fig. S2). More significantly, Gpr116 expression in the representative immunofluorescence images was apparently downregulated and negatively correlated with the MPO-positive cells in the lungs of ALI mice compared to that of control mice (Fig. S3). These data indicate that Gpr116 expression is downregulated in lung injury, suggesting that it may play a role in the progression of ALI. Figure 1 The pathological effect of Gpr116 deletion in ALI mice and primary neutrophils. (A) Gpr116 mRNA levels in human blood samples based on the available GEO dataset (GSE 32707; t-tests). (B) Gpr116 mRNA levels in mouse lungs (n = 5; Student's t-test). (C) Gpr116 mRNA levels in BALF cells of mice (n = 5; Student's t-test). (D) Representative histopathological images (scale bars: 200 μm) and injury scores of mouse lungs (n = 5; one-way analysis of variance). (E) Total protein concentrations in mouse BALF (n = 3 for control and DNase I mice; n = 5 for LPS and DNase I + LPS mice; one-way analysis of variance). (F) Lung wet-to-dry (W/D) weight ratios (n = 5; one-way analysis of variance). (G) Survival curves of mice challenged with LPS (40 mg/kg, i.t.) (n = 5 for control mice; n = 8 for LPS mice; log-rank test). (H) Plasma concentrations of TNF-α and IL-6 (n = 5; one-way analysis of variance). (I) BALF concentrations of TNF-α and IL-6 (n = 5; one-way analysis of variance). (J) BALF concentrations of neutrophils quantified by flow cytometry (n = 5; one-way analysis of variance). (K) Plasma levels of cf-DNA (n = 5; one-way analysis of variance). (L) BALF levels of cf-DNA (n = 5; one-way analysis of variance). (M) Representative immunofluorescence images of NETs formation in mouse lungs (DAPI: blue; Cit-H3: red; MPO: green; DAPI + Cit-H3 + MPO + NETs are indicated by white arrowheads; scale bars: 50 μm). (N) TNF-α and IL-6 concentrations in the supernatants of bone marrow neutrophils from mice (n = 5; one-way analysis of variance). (O) cf-DNA concentrations in the supernatants of bone marrow neutrophils from mice (n = 5; one-way analysis of variance). (P) Representative immunofluorescence images of NETs formation in bone marrow-derived neutrophils (DAPI: blue; Cit-H3: red; MPO: green; the white arrow indicates DAPI  +  Cit-H3 + MPO  +  NETs; scale bars: 50 μm). Data are expressed as mean ± SEM of three repeated trials; ∗P < 0.05, ∗∗P < 0.01, #P < 0.05, ##P < 0.01. Figure 1 Accumulating evidence has shown that macrophages and neutrophils, the main effector cells of innate immunity, are recruited into the blood and migrate to alveolar capillaries along the gradient of chemoattractant. Both of them can release a variety of bactericidal mediators to induce diffuse damage of the blood-gas barrier and increased respiratory load, resulting in fatal hypoxemia. 3 Therefore, we bred myeloid cell-specific Gpr116 knockout mice using conditional CRE/LoxP deletion approaches (Fig. S4, S5) to further verify the role of Gpr116 in immune regulation in ALI. As shown by H&E staining, compared to flox mice, Gpr116 cKO mice were more sensitive to LPS challenge and had a higher lung injury score (Fig. 1D). In addition, the pathological damage of ALI also involves rupture of alveolar capillary membranes and leakage of protein-rich fluid in the alveoli, leading to pulmonary edema, which could be verified by the protein levels in BALF and lung W/D ratios. Here, we observed a dramatic increase of protein level in the BALF of Gpr116 cKO mice compared to flox mice (Fig. 1E). Likewise, the W/D ratio of lung tissues was also greater in cKO mice than that in flox mice (Fig. 1F). Furthermore, deletion of Gpr116 reduced mouse survival rate after LPS stimulation (40 mg/kg, i.t.) (Fig. 1G). In addition, we investigated the effect of Gpr116 on LPS-induced inflammation. Compared with flox mice, BALF and plasma concentrations of TNF-α and IL-6 in cKO mice increased after LPS injection, indicating that Gpr116 may inhibit pulmonary and systemic inflammation, although not all these discrepancies were statistically significant (Fig. 1I, H). These results are consistent with the reported role of Gpr116 in immune regulation, for example, Gpr116 suppresses inflammatory responses of alveolar macrophages via NF-κB signaling. 2 In fact, lung is thought to be main reservoir of neutrophils, and neutrophil functions involving recruitment, reactive oxygen species (ROS) production, and NETs release are closely associated with the pathogenesis of ALI. Nevertheless, the role of Gpr116 in neutrophil function remained to be illuminated. Here, we observed a dramatic increase in neutrophil lung infiltration in Gpr116 cKO mice compared to flox mice (Fig. 1J; Fig. S7). It has been reported that NETs, the new mechanisms of neutrophil immunity, are large, extracellular, web-like structures composed of cytosolic and granular proteins that are assembled on a scaffold of decondensed chromatin. Excessive NETs formation, not only a pathological result but also a pathogenic factor, plays an important role in ALI. 4 , 5 Therefore, we speculated that the protective effect of Gpr116 on ALI may be achieved by regulating the release of NETs. Subsequently, PicoGreen assays, Western blotting and immunofluorescence staining were performed to detect the marker levels of NETs involving cf-DNA, citrullinated histone H3 (Cit-H3), and MPO. As expected, we found that Gpr116 deletion dramatically increased the release of NETs in ALI mice (Fig. 1K–M; Fig. S8–9). ROS production in mouse lungs was also detected to further explore the regulatory effect of Gpr116 on neutrophil function. As Figure S6 shows, Gpr116 deficiency dramatically increased the production of ROS in ALI mouse lungs. DNase I is a specific inhibitor of NETs and can reduce NETs-associated citrullinated histones and minimize immune cell recruitment. To further confirm the inhibitory effects of Gpr116 on NETs release, we eliminated the formation of NETs by DNase I pretreatment before LPS stimulation. It was observed that specific inhibition of NETs reversed the aggravation of lung injury induced by Gpr116 deletion, as indicated by the reduced lung injury score (Fig. 1D), as well as the decreased protein levels in BALF and lung W/D ratios (Fig. 1E, F). Furthermore, the ELISA assays illustrated that the exacerbation of pulmonary and systemic inflammatory responses following Gpr116 deletion in ALI mice was abrogated by DNase I preintervention, as evidenced by the decreased TNF-α and IL-6 levels in BALF (Fig. 1I) and plasma (Fig. 1H), respectively. Taken together, these data suggest that Gpr116 may protect mice against lung injury by inhibiting neutrophil recruitment, NETs release, ROS production and inflammatory response. Rather, we cannot exclude the pathogenic role of macrophages during the progression of ALI. Therefore, we carried out magnetic isolation (Fig. S10) of neutrophils from bone marrow of control and conditional CRE/LoxP deletion of Gpr116 mice to further investigate the molecular mechanism of protective role of Gpr116. Consistent with the in vivo data, the NETs release of neutrophils from Gpr116 cKO mice increased notably after PMA challenge (Fig. 1O, P). Besides, the elevation of TNF-α and IL-6 levels in neutrophil supernatant induced by Gpr116 deletion was remarkable (Fig. 1N). These results elucidate the suppressive role of Gpr116 in neutrophil function, which potentially contributes to diffuse damage of blood-air barrier in an ALI mouse model. In conclusion, our data delineate that GPR116 can inhibit neutrophilic lung infiltration, NETs release, ROS production and inflammatory response to alleviate lung injury and mortality in ALI mice, which extends the therapeutic potential of GPR116. Further studies are needed to clarify the underlying mechanism of the regulation of GPR116 in neutrophil function to provide compelling evidence that GPR116 is a promising therapeutic target in alleviating ALI. Author contributions T Wang and JJ Bian designed the research; T Wang, Y Wang, and Q Xiang performed the research and analyzed the data; SW Lin, PP Jin, J Wang, and N Li offered technical support; T Wang, JF Wang and JJ Bian drafted and revised the manuscript. Conflict of interests The authors declare no conflict of interests. Funding This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China (No. 81871579) and the Military Medical Science and Technology Youth Cultivate Program (China) (No. 19QNP018). Ethics declaration All experimental procedures were approved by the Laboratory Animal Ethics Committee of the Naval Medical University and were performed according to the relevant guidelines. Availability of data and materials The datasets used in this study are available from the corresponding author on reasonable request.