Protective Role of Rho Guanosine Diphosphate Dissociation Inhibitor, Ly-GDI, in Pulmonary Alveolitis

Growing evidences indicate that Ly-GDI, an inhibitory protein of Rho GTPases, plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly-GDI in inflammation remains largely unknown. In the current study, we found that Ly-GDI expression was significantly decreased in the IgG immune complex-injured lungs. To determine if Ly-GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly-GDI (Adeno-Ly-GDI). In vivo mouse lung expression of Ly-GDI resulted in a significant attenuation of IgG immune complex-induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly-GDI over-expression in the lungs produced significant less inflammatory mediators (TNF-α, IL-6, MCP-1, and MIP-1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno-GFP. Mechanically, IgG immune complex-induced NF-κB activity was markedly suppressed by Ly-GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly-GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF-κB activity.


Introduction
Ly-GDI, also known as D4-GDI, RhoGDI2, GDID4 or RhoGDIB, is a member of Rho guanidine dissociation inhibitor (RhoGDI) family that is composed of three members-RhoGDIα, RhoGDI2, and RhoGDI3 [1,2]. RhoGDI could keep the small signal proteins, RhoGTPases, in their cytosolic inactive state by interacting with the RhoGTPases' isoprenyl motif located in C-

IgG IC-Induced Acute Lung Injury
All procedures involving mice were approved by the Animal Care and Use Committee of Harvard Medical School (approval number 04584) which accepts as mandatory the PHS Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions and NIH Principles for the Utilization and Care of Vertebrate Animals Testing, Research, and Training. Eight-week old specific pathogen-free male C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were anesthetized intraperitoneally with ketamine HCl (100 μg/g), and IgG immune complexes-mediated acute lung injury was induced as described previously [21]. Briefly, mice received intratracheal injection of 0.24 mg rabbit anti-BSA IgG in a volume of 40 μl phosphate buffered (pH 7.4) saline (PBS). Immediately after airway administration of anti-BSA, 1 mg BSA dissolved in 200 μl PBS was injected intravenously. Negative control mice received airway injection of anti-BSA. Unless otherwise indicated, 4 h after IgG IC administration, mice were exsanguinated, the pulmonary circulation flushed via the pulmonary artery with 1 ml PBS and the lungs surgically dissected. Lungs were immediately frozen in liquid nitrogen.

Expression Vectors and Promoter Reporters
Full-length mouse Ly-GDI cDNA was amplified from total RNA of the mouse lung using reverse transcription-PCR (Invitrogen, Carlsbad, CA), sequenced, and inserted into pcDNA3.1 (+) (Invitrogen, Carlsbad, CA). Recombinant adenovirus containing mouse Ly-GDI (Adeno-Ly-GDI) was constructed by using BD Adeno-XTM Expression System 1 (BD Biosciences, Palo Alto, CA). To generate the virus, Adeno-Ly-GDI was digested with PacI and transfected to HEK-293 cells according to the manufacturer's instructions. Recombinant adenoviruses were purified by BD Adeno-X virus purification kit (BD Biosciences, Palo Alto, CA). The viral stocks were tittered using Adeno-X Rapid Titer Kit (D Biosciences, Palo Alto, CA). The mouse TNF-α and IL-6 luciferase constructs were kindly provided by Richard C. Schwartz (Michigan State University). NF-κB promoter reporter was obtained from Promega.

Intratracheal Injection of Adenovirus
50 μl of Adeno-GFP (BD Biosciences) or Adeno-Ly-GDI (5×10 8 pfu/mouse) was injected into mice through airway. 3 days later, lungs were harvested to determine expression of Ly-GDI, or mice were used for IgG IC-stimulated acute lung inflammation.
Bronchial Alveolar Lavage (BAL) Fluid Collection, Total and Differential White Blood Cell Counts, and Chemokine/Cytokine ELISAs

Permeability Index
Fours hours after intratracheally administration of IgG IC, mice were exsanguinated, and the thorax was opened. 1 ml of ice-cold PBS was injected via an intratracheal cannula. Cell-free supernatants were obtained by centrifuging bronchoalveolar lavage (BAL) fluids at 3000 rpm for 5 min. Mouse BSA level in BAL fluids was determined by ELISA (Bethyl Laboratories, Montgomery, TX), and the permeability index was expressed as the ratio of the albumin in the IgG IC-injured lungs versus that in the control-treated lungs of same type of mice.

Histological Assay
Fours hours after airway deposition of IgG IC, 0.6 ml of 10% buffered (pH 7.2) formalin was injected intratracheally. The lungs were fixed in a 10% buffered formalin solution for morphological assay by tissue sectioning and staining with hematoxylin and eosin (H&E).

Luciferase Assay
Transient transfections were performed with 2×10 5 cells plated in 12-well plates by using 0.5 μg of DNA and 1.5 μl of Fugene 1 6 Transfection Reagent in 48.5 μl of Opti-MEM I medium (Invitrogen, CA). 48 h after transfection, the cells were either incubated with or without 100 μg/ml IgG IC for 4 h. Cell lysates were used to measure luciferase activity by using the Dual-Luciferase Reporter Assay System (Promega, WI).

RNA Isolation and Detection of mRNA by Real-Time PCR
Total RNAs were extracted from mouse lungs with Trizol (Invitrogen, CA) according to the manufacturer's procedure. After isolation, 2 μg of total RNA was subjected to reverse transcription by using the Superscript II RNase H -Reverse Transcriptase (Invitrogen, CA). The Ly-GDI mRNA level was investigated by real time PCR. PCR was performed with primers for Ly-GDI: 5' primer, 5'-GGA CTG GCA TGA GAG TGG AT -3' and 3' primer, 5'-AGG TGA GGT GGT CCT GTT TG -3'. Following reverse transcription, the cDNA was amplified and quantified using a Sequence Detection System (SDS 7300) and a PCR universal protocol as follows: AmpliTaq Gold activation at 95°C for 15 s and, annealing/extension at 60°C for 1 min. The fluorescence of the double-stranded products accumulated was monitored in real time. The relative mRNA levels were normalized to levels of GAPDH mRNA in the same sample.

Western Blot Analysis
Mouse lungs were lysed in cold RIPA buffer. Samples containing 100 μg protein were electrophoresed in a 12% polyacrylamide gel and then transferred to a PVDF membrane. Membranes were incubated with rabbit anti-Flag antibody (Abcam, MA), goat anti-Ly-GDI antibody (Santa Cruz, CA), and rabbit anti-GAPDH antibody (Cell Signaling, MA), respectively. After 3 washes in TBST, the membranes were incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare, Piscataway, NJ). The membrane was developed by enhanced chemiluminescence technique according to the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL).

Statistical Analysis
All values were expressed as the mean ± S. E. M. Data sets were analyzed using Student's t test or one-way ANOVA, with individual group means being compared with the Student-Newman-Keuls multiple comparison test.

Adenovirus-Mediated Ly-GDI Expression in the Lung Attenuates IgG IC-Induced Lung Injury
A recent report shows that Ly-GDI expression significantly inhibits FcγR-mediated phagocytosis [1], however, its role in inflammation mediated by FcγR activation is unknown. We determined Ly-GDI expression in IgG IC-injured lung. As shown in Fig 1, lung Ly-GDI expression assessed by real-time PCR ( Fig 1A) and Western blot ( Fig 1B) was significantly decreased 4 h after IgG IC deposition in the lung. To evaluate the role of Ly-GDI in FcγR-mediated acute lung inflammation, we constructed recombinant adenovirus which could express Ly-GDI with a Flag tag in its N-terminal region (Adeno-Ly-GDI). In vitro experiment demonstrated that Ly-GDI expression was dramatically elevated in HEK293 cells infected by Adeno-Ly-GDI when compared with cells incubated with control virus (Adeno-GFP, Fig 2A). To further determine if Adeno-Ly-GDI could mediate Ly-GDI expression in mouse lungs, we intratracheally administrated different doses of Adeno-Ly-GDI (2.5×10 8 and 5 × 10 8 PFU) into mouse lung. 72 h later, whole lung homogenates were used to perform Western blot to examine Ly-GDI expression. As shown in Fig 2B, adenovirus-mediated Ly-GDI expression was evident at a dose of 5×10 8 PFU, and no strong Ly-GDI expression could not be observed in Adeno-GFP-and low dose Adeno-Ly-GDI-infected mouse lungs.
Next, we sought to determine the role of Ly-GDI in IgG IC-induced acute lung inflammation. 4 h after alveolar deposition of IgG IC, cell-free BAL fluids were harvested. Mouse albumin level in the BAL fluids was measured by ELISA, and permeability index was expressed as a ratio of total albumin in BAL fluid obtained from the inflamed lung to an average albumin content of the corresponding control group. We found that IgG IC treatment resulted in a marked increase in permeability index ( Fig 2C). Notably, permeability index was greatly reduced in mice receiving airway administration of Adeno-Ly-GDI when compared with mice that received Adeno-GFP (69%, p<0.001, Fig 2C). Next, we measured lung MPO activity, which is a marker for neutrophil accumulation. As shown in Fig 2D, MPO activity in IgG IC-injured lung was dramatically elevated compared with that of control mice receiving anti-BSA only. Consistent with permeability index, IgG IC-induced lung MPO activity was significantly lower in mice receiving Adeno-Ly-GDI when compared with control counterparts receiving Adeno-GFP (32%, p<0.01, Fig 2D). These data together indicate that Ly-GDI suppressed pulmonary capillary leakage and neutrophil recruitment during IgG IC-induced acute lung injury.
We further examined if mice that received intratracheal administration of Adeno-Ly-GDI exhibited attenuated lung injury by histological assay. We observed that airway injection of Adeno-GFP or Adeno-Ly-GDI had no influence on pulmonary inflammation and normal lung architecture (Fig 3A and 3C). Mice received Adeno-Ly-GDI together with IgG IC exhibited lung edema, hemorrhage, and neutrophil accumulation (Fig 3B). In contrast, all of these features were greatly attenuated in mice receiving Adeno-Ly-GDI during IgG IC-induced acute lung inflammation (Fig 3D).

IgG IC-Induced Leukocyte Accumulation in the Alveoli Is Decreased by Ly-GDI
We determined the total leukocyte and neutrophil contents in BAL fluids. As shown in Fig 4A  and 4h after IgG IC deposition in the lung, the number of leukocytes present in BAL fluids was significantly increased as compared with control mice challenged with anti-BSA alone. In injured lungs, the major cells recovered from BAL fluids were neutrophils (Fig 4). Importantly, we found that in the presence of IgG IC, mice receiving Adeno-Ly-GDI showed a significant decrease of total leukocytes and neutrophil accumulation in airspaces when compared with control mice receiving Adeno-GFP (40%, p<0.001 and 39%, p<0.001, respectively, Fig 4). Thus, Ly-GDI inhibited migration of leukocytes, especially neutrophils into the alveoli in IgG IC-injured mice.

Ly-GDI Regulates Cytokine and Chemokine Expression in the Lung Challenged by IgG IC
We evaluated the role of Ly-GDI expression in inflammatory cytokine and chemokine generation in BAL fluids 4 h after pulmonary deposition of IgG IC. As shown in Fig 5, intratracheal administration of adenovirus alone without IgG IC challenge could not induce an increase in the levels of the indicated proinflammatory mediators. BAL fluids from IgG IC-injured mice receiving Adeno-GFP or Adeno-Ly-GDI exhibited dramatically augmented expressions of TNF-α, IL-6, and CC chemokines-MCP-1 and MIP-1α (Fig 5). Moreover, in IgG IC-injured When cytopathic effect became evident, the infected cells were lysed by using RIPA buffer. (B) Mice received intratracheal injection of Adeno-GFP or Adeno-Ly-GDI at the indicated doses. 3 days later, whole lung proteins were extracted by using RIPA buffer. Total proteins obtained from adenovirus-infected HEK 293 cells or mouse lungs were subjected to Western blot by using rabbit anti-Flag antibodies. The level of GAPDH was shown at the bottom as a loading control. (C and D) Adeno-GFP or Adeno-Ly-GDI was injected to mice via an airway instillation at a dose of 5 × 10 8 PFU. 3 days later, the mice were subjected to IgG immune complex-induced lung injury or control treatment (αBSA only). Mouse albumin levels (C) in BAL fluids were measured 4 h later, and the permeability index was expressed as the ratio of the albumin in the IgG immune complex-injured lungs versus that in the control-treated lungs of same type of mice. Lung MPO activity (D) was evaluated, and used as a marker for pulmonary neutrophil accumulation. Data were expressed as means ± S. E. M. N 3 mice for each group. ** and *** indicated statistically significant difference-p < 0.01 and p < 0.001, respectively. lungs, ectopic expression of Ly-GDI led to a significant reduced production of TNF-α, IL-6, MCP-1, and MIP-1α (by 51%, 68%, 71%, and 44%, respectively) when compared with mice receiving airway administration of Adeno-GFP (Fig 5). However, there was no statistical difference in MIP-1β, KC, and MIP-2 production between Adeno-GFP-treated mice and Adeno-Ly-   GDI-treaed mice in the presence of IgG IC (data not shown). These data suggest that Ly-GDI selectively inhibited certain pro-inflammatory mediators' production during IgG IC-induced acute lung injury.
To further determine the effect of Ly-GDI on IgG IC-stimulated inflammation, we assessed whether Ly-GDI affected luciferase expressions controlled by IL-6 promoter and TNF-α promoter, respectively, in alveolar macrophage, MH-S cells, incubated in the presence or absence of IgG IC. As shown in Fig 5E and 5F, both IL-6-and TNF-luciferase expression were significantly augmented by IgG IC stimulation. Notably, over-expression of Ly-GDI dramatically suppressed the IgG IC-induced luciferase activities in MH-S cells.

Ly-GDI Plays a Negative Role in C5a Level in BAL Fluids Recovered from IgG IC-Injured Lungs
Previous study has shown that C5a is required for the full production of TNF-α, pulmonary neutrophil accumulation, and lung injury in IgG IC-induced acute lung inflammation model [19]. Therefore, we sought to elucidate whether C5a level in BAL fluids could be regulated by Ly-GDI. We found that Adeno-Ly-GDI alone without IgG IC treatment resulted in a moderate increase in the BAL fluid C5a level as compared with Adeno-GFP treatment, but there was no statistical difference (Fig 6). Moreover, there was a significant increase in C5a content in BAL fluids obtained from IgG IC-inflamed lungs (Fig 6). Notably, in the presence of IgG IC deposition, Adeno-Ly-GDI mice showed a significantly reduced C5a level in BAL fluids when compared with Adeno-GFP mice (Fig 6).

Ly-GDI Inhibits IgG IC-Induced NF-κB Activity in the Lung and Alveolar Macrophages
We sought to determine the potential mechanism by which Ly-GDI mitigated IgG-IC-induced inflammation in the lung. We infected mice with Adeno-GFP or Adeno-Ly-GDI by airway injection. 3 d later, the mice were challenged with or without IgG IC for 4 h, and the whole lung nuclear extracts were subjected to EMSA. As shown in Fig 7A, IgG IC significantly induced NF-κB DNA binding activity when compared with anti-BSA challenge alone. Notably, the amount of lung NF-κB associated with its consensus sequence was markedly reduced in IgG IC-inflamed mice receiving airway administration of Adeno-Ly-GDI when compared with their littermates intratracheally injected with Adeno-GFP (Fig 7A). Alveolar macrophages contributed to full activation of NF-κB in IgG IC-induced acute lung inflammation model [23]. To elucidate the role of Ly-GDI in IgG IC-induced NF-κB activation in alveolar macrophages, we performed in vitro luciferase experiments by using MH-S cells. As shown in Fig 7B, IgG IC treatment led to an over 5-fold increase of luciferase production driven by NF-κB, and overexpression of Ly-GDI caused a significant decrease of luciferase expression in MH-S cells.

Discussion
Ly-GDI suppresses the activation of small GTPases of the Rho family by inhibiting dissociation of GDP from Rho proteins. Although the Rho family plays critical roles in inflammatory signal transduction cascades, however, the function of Ly-GDI in immune system remains largely unknown. Ly-GDI was originally identified as a metastasis suppressor in bladder cancer during expression from a co-transfected thymidine kinase reporter gene. These values were also normalized to a relative value of 1 for the control plasmidtransfected cells that did not receive IgG immune complex stimulation. Data were expressed as means ± S. E. M. N 3 mice for each group. *, ** and *** indicated statistically significant difference-p<0.05, p < 0.01 and p < 0.001, respectively. doi:10.1371/journal.pone.0140804.g005 studies of the differential invasive and metastatic properties of isogenic human bladder carcinoma cell lines [24]. Using Ly-GDI knock-out mice, Yin et al show that Ly-GDI is not essential for development of the immune system or many immunological functions, but significantly influences lymphocyte growth and survival during in vitro cultivation [25]. In addition, Ly-GDI associates with CARD9, a caspase-recruitment domain (CARD)-containing protein, in phagosomes after bacterial and fungal infection, and binding of CARD9 suppressed LyGDImediated inhibition of the GTPase Rac1, thereby leading to bacterial killing in macrophages [26]. Ectopic expression of Ly-GDI greatly inhibited FcγR-mediated phagocytosis in human monocytes, but its role in FcγR activation-triggered inflammatory reactions was still unknown [1]. In the current report, we show that Ly-GDI expression was significantly reduced in lungs during IgG IC-induced ALI. Importantly, Ly-GDI plays a negative role in IgG IC-induced inflammatory responses in the lung and alveolar macrophages.
During IgG IC-induced ALI, alveolar macrophages are activated, and play a critical role in IgG IC-stimulated generation of pro-inflammatory mediators including a variety of chemokines and cytokines [27]. For example, production of TNF-α that is considered as an early response cytokine was dramatically increased in IgG IC-injured lungs, which contributed to the elevated synthesis of chemokines such as MIP-1α, and final pulmonary injury including increased lung microvascular permeability, neutrophil accumulation, and pulmonary hemorrhage [17,19,20,28,29]. MIP-1α belongs to CC chemokine family, and is required for formation of chemoattractant gradients and subsequent transmigration of polymorphonuclear neutrophils (PMNs) from vascular vessels into lungs [30]. MCP-1 is also a member of CC chemokine family, and plays an essential role in regulating macrophage infiltration. Our finding that over-expression of Ly-GDI led to a decreased expression of various pro-inflammatory mediators including TNF-α, IL-6, MCP-1, and MIP-1α suggests that Ly-GDI regulates IgG IC- induced ALI by cytokine and chemokine pathways. In addition, the reduced MCP-1 production may contribute to decreased accumulation of macrophages in lungs, which further leads to inhibition of IgG IC-induced lung inflammation and injury. Interestingly, a recent study implicates that Ly-GDI suppresses cancer metastasis by reducing MCP-1 expression in tumor microenvironment, which was required for macrophage infiltration [31].
The molecular mechanism by which Ly-GDI affects cytokine and chemokine gene expression is not clear. RhoA, Rac, and Cdc42 all participate in the signaling pathways required for the activity of NF-κB, leading to the induction of cytokines and chemokines [32,33]. Furthermore, the activation of NF-κB has been linked to Rho-dependent activation of the c-Jun NH2-terminal kinase/stress-activated protein kinase pathway [32,34]. Our current study shows that Ly-GDI reduces the activation of both lung NF-κB (Fig 7) and Stat3 (data not shown). These two transcription factors are important regulators of inflammatory mediators. Thus, it is possible that Ly-GDI mitigates IgG IC-induced cytokine and chemokine expression by regulating NF-κB and Stat3 activity in the lung. However, the detailed pathways for this regulation remain to be determined.
Another interesting finding in this study is that C5a generation in BAL fluids was significantly reduced in mice receiving Adeno-Ly-GDI when compared with control mice treated with Adeno-GFP during IgG IC-induced ALI. Our previous studies showed that pulmonary STAT3 was activated after airway deposition of IgG IC, which positively regulated IgG ICinduced acute pulmonary inflammation [15,18]. In addition, STAT3 activation was dependent Adeno-GFP or Adeno-Ly-GDI was injected to mice via an airway instillation at a dose of 5 × 10 8 PFU. 3 days later, the mice were subjected to IgG immune complex-induced lung injury or control treatment (αBSA only). Lungs were harvested 4 h later. Whole lung nuclear proteins were extracted, and subjected to EMSA to determine the NF-κB DNA binding activity. (B) MH-S cells were transiently transfected with indicated genes. 48 h later, the cells were treated with or without 100 μg/ml IgG immune complexes for 4 h. The cell lysates were used to evaluate luciferase expression. Luminometer values were normalized for expression from a co-transfected thymidine kinase reporter gene. These values were then normalized to a relative value of 1 for the control plasmid transfected cells that did not receive IgG IC challenge. The data were expressed as means ± S. E. M. N = 3. * and *** indicated statistically significant difference-p < 0.05, and p < 0.001, respectively. on C5a/C5aR signaling pathway in lungs challenged by IgG IC [15]. Thus, Ly-GDI-mediated C5a reduction may lead to STAT3 inhibition in IgG IC-injured lung.
Fcγ receptors (FcγRs), which bind to the Fc portion of IgG and induce inflammatory reactions, play essential roles in immune defense system. The roles of FcγRs in IgG IC-induced autoimmune diseases are extensively investigated, but growing evidences suggest that they also play an important role in the regulation of innate immunity. For example, it was recently shown that binding of IgG IC that is formed by interaction between peptidoglycan (PGN) and the corresponding IgG antibody to FcγRIIA was indispensible for PGN-stimulated inflammation [35]. Because PGN is a major structural component of Gram-positive bacteria, these data suggest that FcγRIIA may be involved in Gram-positive bacteria-mediated systemic inflammatory responses [35]. This is supported by the finding that high expression of FcγRIIA on human neutrophils is a sepsis biomarker [36,37]. Moreover, there may be a crosstalk between FcγRs and Toll-like receptors (TLRs) signaling pathways. For example, IgG IC could stimulate interaction between TLR4 and FcγRIII, and disruption of the association by knockout or mutation of TLR4 led to decreased inflammatory responses triggered by IgG IC [38]. Interestingly, TLR4-induced TRIF-dependent pathway was positively regulated by FcγIIA, whereas TLR4-triggered MyD88-dependent signaling pathway was suppressed by FcγIIA in human monocytes [39]. Here, we showed that over-expression of Ly-GDI resulted in inhibition of IgG IC-induced NF-κB transcriptional activity; however, whether Ly-GDI could regulate TRIF/ MyD88 pathways or suppress the interaction between TLR4 and FcγRs to affect NF-κB remains an open question. Taken together, these studies and our current finding that Ly-GDI is an important regulator of lung inflammatory injury after deposition of IgG IC may help to understand a new signaling pathway which is important for both inflammation/infection and autoimmune diseases.