Protective Actions of Aspirin-Triggered (17R) Resolvin D1 and Its Analogue, 17R-Hydroxy-19-Para-Fluorophenoxy-Resolvin D1 Methyl Ester, in C5a-Dependent IgG Immune Complex–Induced Inflammation and Lung Injury

The IgG immune complex–induced lung injury model in rodents has been employed to determine the molecular mechanisms of acute lung inflammatory injury. In this model, intra-alveolar deposition of IgG immune complexes stimulates alveolar macrophages via cross-linking of FcγRs, which results in robust formation of the early response cytokines such as TNF-α and IL-6 (1, 2). These cytokines then activate transcription factors such as NF-κB and C/EBPs to induce expression of adhesion molecules and other inflammatory mediators such as CXC and CC chemokines on leukocytes and on endothelial cells and epithelial cells, all of which induce a strong proinflammatory cascade (1, 2). The formation of IgG immune complexes in lung also results in in situ generation of the complement activation product, C5a, a strong chemoattractant that is involved in the recruitment of inflammatory cells such as neutrophils (1, 2). These inflammatory events together led to the acute lung injury; however, the anti-inflammatory cascade, such as the molecular events that contribute to the resolution of immune complex-induced lung inflammation, is poorly understood.

Resolvin D1 (RvD1; 7S, 8R, 17S-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) belongs to a new class of specialized proresolving lipid mediators (SPMs), which is produced endogenously from essential ω-3-polyunsaturated fatty acids and docosahexaenoic acid (3, 4). Aspirin-triggered RvD1 (AT-RvD1) is the 17R epimer of RvD1 (7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid), which is more resistant to catalysis than RvD1 (5). Both RvD1 and AT-RvD1 have proven to be very potent in treating a number of inflammation-associated models of human diseases, including obesity-induced steatohepatitis (6), adjuvant-induced arthritis (7), inflammatory and postoperative pain (8, 9), peritonitis (10, 11), suture-induced or IL-1β–induced hemangiogenesis (12), ischemia/reperfusion kidney and lung injury (13, 14), dextran sulfate sodium–induced colitis (15), and sepsis (16). Of interest, recent studies indicate that RvD1 or AT-RvD1 plays a critical role in mitigating lung inflammation and injury (17, 18). Little is known about whether resolvins and other SPM could affect FcγR-mediated inflammatory responses. We hypothesize that the new classes of SPMs can regulate immune complex–induced inflammation and tissue injury. In the current studies, we sought to determine the role of AT-RvD1 and RvD1 metabolically stable analog, 17R-hydroxy-19-para-fluorophenoxy-RvD1 methyl ester (p-RvD1), during acute lung inflammation induced by IgG immune complexes. Our data indicate that administration of either AT-RvD1 or p-RvD1 reduces IgG immune complex–induced neutrophil accumulation and lung injury. AT-RvD1 or p-RvD1 also suppresses lung NF-κB and C/EBP activation in association with decreased bronchoalveolar lavage (BAL) fluid (BALF) levels of TNF-α, IL-6, and keratinocyte cell–derived chemokine (KC). Of interest, C5a levels in the BALF are significantly reduced by p-RvD1 and AT-RvD1. Furthermore, we provide evidence that AT-RvD1 has the ability to regulate the FcγR-mediated induction of inflammatory cytokines and chemokines in both macrophages and neutrophils. These findings suggest that AT-RvD1 is an important regulator of lung inflammatory injury after deposition of IgG immune complexes.

AT-RvD1 and RvD1 analog, p-RvD1, was prepared by total organic synthesis (14, 19). The 19-p-phenoxy-RvD1 methyl ester and AT-RvD1 methyl ester were used in the in vivo experiments. In some experiments, 17R-RvD1 with the same chemical structure as AT-RvD1 was purchased from Cayman Chemical (Ann Arbor, MI). Both AT-RvD1 and p-RvD1 are dissolved in ethanol. Vesicle control is the same amount of ethanol diluted in PBS.

Specific pathogen-free male C57BL/6 mice at the age of 8–12 wk (weighing 20–30 g) were obtained from The Jackson Laboratory (Bar Harbor, ME). All procedures involving mice were approved by the Animal Care and Use Committee of Harvard Medical School.

Mice were anesthetized with i.p. ketamine (100 mg/kg body weight; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (12.5 mg/kg body weight; Ben Venue Laboratories, Benford, OH) for sedation. The trachea was surgically exposed by a midline incision, and 120 μg rabbit anti-BSA IgG (MP Biomedicals, LLC, Solon, OH) in 40 μl PBS was administered intratracheally by tracheal puncture with a 30-gauge needle. The incision was closed by two surgical clips, and 2 mg BSA (Sigma-Aldrich, St. Louis, MO) in a volume of 200 μl was injected i.v. immediately thereafter. When employed, AT-RvD1 (500 ng/mouse) or p-RvD1 (500 ng/mouse) was injected i.v. 5 min before the administration of anti-BSA. Control mice received anti-BSA intratracheally in the absence of an i.v. infusion of BSA.

BALFs were harvested for total leukocyte count, differential cell counts, and quantification of chemokine/cytokine production by ELISAs. Four hours after IgG immune complex deposition, the thorax was opened and 1 ml sterile PBS was instilled into the lung via a tracheal incision. The recovered BALFs were first used to determine the total leukocyte count via a hemocytometer. BALFs were centrifuged at 450 × g for 10 min, the cell-free supernatants were used for cytokine/chemokine measurements by sandwich ELISA, and the cell pellets were stained by HEMA3 stain set (Fisher Scientific, Kalamazoo, MI) for differential cell counts. The slides were quantified for macrophages, neutrophils, and lymphocytes by counting a total of 200 cells per slide in randomly selected high-powered fields (×400) as differential cell count. BAL levels of TNF-α, IL-6, KC, MIP-1α, and C5a were determined using ELISA kits (R&D Systems, Minneapolis, MN), according to the instructions of the manufacturer.

Mouse albumin levels in BALFs were measured using a mouse albumin ELISA kit purchased from Bethyl Laboratories (Montgomery, TX). The detection limit for this ELISA was 7 ng/ml. All procedures followed the protocol of the company.

Four hours after IgG immune complex deposition, lungs were fixed by intratracheal instillation of 1 ml buffered formalin (10%; Fisher Scientific, Fair Lawn, NJ), followed by further fixing in the 10% buffered formalin solution for histological examination to evaluate the lung injury by tissue sectioning and staining with H&E.

Nuclear extracts of whole lung tissues were prepared, as described previously (20). Briefly, fresh lungs were homogenized in Solution A containing 0.6% (v/v) Nonidet P-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, 0.5 mM PMSF, 2.5 μg/ml leupeptin, 5 μg/ml antipain, and 5 μg/ml aprotinin. The homogenate was incubated on ice for 5 min, and the nuclei were pelleted by centrifugation at 5,000 × g for 5 min at 4°C. Proteins were extracted from the nuclei by incubation at 4°C with Solution B containing 420 mM NaCl, 20 mM HEPES (pH 7.9), 1.2 mM MgCl₂, 0.2 mM EDTA, 25% (v/v) glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2.5 μg/ml leupeptin, 5 μg/ml antipain, and 5 μg/ml aprotinin. Nuclei debris was pelleted by centrifugation at 13,000 × g for 30 min at 4°C, and the supernatant extract was stored at −80°C. Protein concentrations were determined by Bio-Rad (Hercules, CA) protein assay kit. The EMSA probes were double-stranded oligonucleotides containing a murine IL-6 C/EBP binding site (5′-CTAAACGACGTCACATTGTGCAATCTTAATAAGGTT-3′ annealed with 5′-TGGAAACCTTATTAAGATTGCACAATGTGACGTCGT-3′, provided by R. Schwartz, Michigan State University) or a NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′; Promega, Madison, WI). C/EBP probes were labeled with α-[³²P]ATP (3,000 Ci/mmol at 10 mCi/ml; GE Healthcare, Piscataway, NJ). NF-κB probes were labeled with γ-[³²P]ATP (3,000 Ci/mmol at 10 mCi/ml; GE Healthcare). DNA-binding reactions were performed at room temperature, as described previously (20). Samples were electrophoresed through 5.5% polyacrylamide gels in 1× Tris borate EDTA, dried under vacuum, and exposed to x-ray film.

MH-S cells, obtained from American Type Culture Collection (Manassas, VA), were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 2 mM l-glutamine, 100 U/ml streptomycin, 100 U/ml penicillin, and 10% (v/v) FBS. Cells were stimulated by IgG immune complexes (100 μg/ml) with or without AT-RvD1 (100 nM) treatment (18). Supernatants were collected at 0, 2, 4, 8, and 24 h for determination of cytokines and chemokines via ELISA kits, as described above.

Mouse NF-κB–dependent promoter luciferase construct was obtained from Promega (Madison, WI). C/EBP-dependent promoter luciferase, the DEI-4 [DEI4-(-35alb) LUC], mouse TNF-α promoter luciferase, and mouse IL-6 promoter luciferase were provided by R. Schwartz (Michigan State University). The thymidine kinase promoter Renilla luciferase reporter plasmid is used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. Transient transfections were performed with 3 × 10⁵ cells plated in 12-well plates by using 0.5 μg DNA and 1.5 μl Fugene 6 Transfection Reagent (Roche, Indianapolis, IN) in 50 μl Opti-MEM I medium (Invitrogen, Carlsbad, CA). Under these conditions, the transfection efficiency is ∼20%. Unless otherwise indicated, 24 h after transfection, the cells were incubated with or without IgG immune complexes (100 μg/ml) and AT-RvD1 (100 nM) for 4 h. Cell lysates were subjected to luciferase activity analysis by using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).

Mouse peritoneal neutrophils were harvested 5 h after i.p. injection of 1.5 ml thioglycolate (BD Biosciences, Sparks, MD; 2.4 g/100 ml) by peritoneal lavaging peritoneum three times with 10 ml PBS. The cells were collected by centrifugation at 300 × g for 8 min at room temperature and washed twice with PBS. The cell pellets were stained by HEMA3 stain set (Fisher Scientific, Kalamazoo, MI) for differential cell counts. The slides were quantified for macrophages, neutrophils, and lymphocytes by counting a total of 200 cells per slide in randomly selected high-powered fields (×400) as differential cell count. The purity of cell suspension was at least 95% neutrophils. Neutrophils (5 × 10⁶ cells per experimental condition) were stimulated by IgG immune complexes (100 μg/ml) with or without AT-RvD1 (100 nM) treatment. Supernatants were collected at 0, 2, 4, 8, and 24 h for determination of cytokines and chemokines via ELISA kits, as described above.

All values were represented as the mean ± SEM. Significance was assigned in which p < 0.05. Data sets were analyzed using Student t test or one-way ANOVA, with individual group means being compared with the Student–Newman–Keuls multiple comparison test.

Our previous work in mice has shown that the pulmonary vascular permeability was increased after IgG immune complex deposition by measuring albumin level in the BALFs (21). Because AT-RvD1 partially resists metabolic inactivation compared with RvD1 (5), we chose to use AT-RvD1 for the study. IgG immune complex–induced lung injury was induced in the manner as described above, and the parameters of lung injury were determined at 4 h. As shown in Fig. 1A, the mean permeability index (albumin leakage) in the negative and positive controls is 1 ± 0.17 and 9.73 ± 0.93, respectively. However, the i.v. administration of AT-RvD1 (500 ng/mouse) resulted in a 59% decrease in lung permeability index (3.93 ± 0.44; p < 0.01). The major cells in BALFs from control lungs were macrophages and lymphocytes, whereas, in IgG immune complex–injured lungs, the majority of cells turned to neutrophils (data not shown). The neutrophil content in BALFs of animals undergoing IgG immune complex–induced lung injury reflects the degree of lung injury and correspondingly the protective effects of interventions (22, 23). As shown in Fig. 1B, AT-RvD1–treated mice exhibited significant attenuation of the neutrophils (by 81%; p < 0.05). To further examine whether AT-RvD1 treatment reduced lung injury, histological analyses were performed. As shown in Fig. 2A and 2C, mice receiving PBS (Fig. 2A) or AT-RvD1 (Fig. 2C) alone exhibited normal lung architecture with no evidence of inflammation. In the IgG immune complex–injured lung, significant hemorrhage, edema, and accumulation of neutrophils were observed (Fig. 2B). In AT-RvD1–treated mice, all of these features were attenuated 4 h after IgG immune complex deposition in the lung (Fig. 2D).

Levels of TNF-α, IL-6, and KC that are involved in IgG immune complex–induced lung injury (1) were determined. Negative control mice had low levels of TNF-α (121 ± 85 pg/ml), IL-6 (165 ± 2 pg/ml), and KC (346 ± 16 pg/ml) (Fig. 3A–C). As expected, IgG immune complex deposition in the lung resulted in a substantial increase in BAL TNF-α (7637 ± 637 pg/ml), IL-6 (3725 ± 745 pg/ml), and KC (4020 ± 742 pg/ml) contents (Fig. 3A–C). The levels of all these inflammatory cytokines and chemokines were significantly decreased in AT-RvD1–treated mice (TNF-α by 61%, IL-6 by 76%, and KC by 62%, respectively). These results correlate with decreased albumin leakage, neutrophil, and histology changes, as described above.

Similar studies were conducted with RvD1 metabolically stable analog, p-RvD1, in the IgG immune complex model of lung injury. As shown in Fig. 1C, p-RvD1 treatment (i.v., 500 ng/mouse) significantly decreased the permeability values by 49.5% (p < 0.01). Next, BALFs were harvested from IgG immune complex injured to evaluate the effect of p-RvD1 on infiltration of the inflammatory cells. As shown in Fig. 1D, p-RvD1 treatment results in a 46% reduction in the number of neutrophils presented in the BALFs (3.88 ± 0.65 × 10⁶ cells/ml vs. 8.95 ± 1.39 × 10⁶ cells/ml; p < 0.01) when compared with IgG immune complex–injured mice with control treatment, whereas the numbers of mononuclear cells (chiefly lymphocytes and macrophages) show an increased tendency without significant difference (data not shown). To further examine whether p-RvD1 treatment reduces lung injury, histological analyses were performed. Similar to AT-RvD1 treatment, in the presence of p-RvD1, significantly reduced alveolar injury (hemorrhage) or inflammation (neutrophils) was found (Fig. 2E–H).

We examined TNF-α, IL-6, and KC in the BALF 4 h after deposition of IgG immune complexes in mice treated with either p-RvD1 or PBS. As shown in Fig. 3D–F, in the IgG immune complex–injured lungs, p-RvD1 reduced the BAL contents of TNF-α by 51% (p < 0.05), IL-6 by 64% (p < 0.05), and KC by 76% (p < 0.01), respectively. These results suggested that reduction of BAL TNF-α, IL-6, and KC by p-RvD1 in the IgG immune complex model is probably directly linked to the protective effects of this RvD1 metabolically stable analog, the results of which are associated with reduced lung content of neutrophils (Figs. 1D, 2H).

C5a is an inflammatory peptide with a broad spectrum of biological functions (24). Previous studies have demonstrated that C5a plays an essential role for the full production of TNF-α, albumin leakage, and neutrophil accumulation during IgG immune complex–induced lung injury (25, 26). To investigate whether p-RvD1 and AT-RvD1 can regulate the IgG immune complex–induced C5a activation in the lung, C5a levels in BALFs were assessed. As shown in Fig. 4A, negative control animals (anti-BSA only) had low levels of BAL C5a (89.96 ± 5.5). The level of C5a significantly increased in the BALFs from IgG immune complex–injured lungs when compared with that from control mice (326.2 ± 15.4; p < 0.0001) (Fig. 4A). However, the mice receiving p-RvD1 at the initiation of IgG immune complex deposition showed a marked decrease of the C5a content by 47.8% (190.1 ± 10.5; p < 0.0001) (Fig. 4A). Similarly, AT-RvD1 can also significantly decrease the C5a level in BALFs from IgG immune complex–injured lungs (p < 0.05, Fig. 4B). These findings indicate that p-RvD1 and AT-RvD1 may exert their protective roles in IgG immune complex–induced injury by inhibiting C5a production.

In the model of IgG immune complex–induced lung injury, activation of NF-κB is known to be required for production of relevant inflammatory mediators (27, 28). In addition, our recent studies show that C/EBP transcription factors play a critical role in FcγR signaling in macrophages and IgG immune complex–induced lung injury (29, 30). To determine the potential mechanisms whereby p-RvD1 and AT-RvD1 suppress IgG immune complex–induced inflammation, we performed EMSA of nuclear proteins from control and IgG immune complex–injured lungs in the presence or absence of p-RvD1 or AT-RvD1 to evaluate NF-κB and C/EBP activation. As shown in Fig. 5A and 5B, very little NF-κB and C/EBP were found in lung nuclear proteins obtained from mice receiving PBS, AT-RvD1, or p-RvD1 in the presence of anti-BSA alone. In mice undergoing IgG immune complex deposition treated i.v. with PBS, there were clear evidences of increased DNA-binding activities for both NF-κB and C/EBP (Fig. 5A, 5B). Importantly, in mice undergoing IgG immune complex deposition and treated with AT-RvD1 or p-RvD1, there was reduced activation of NF-κB and C/EBP (Fig. 5A, 5B, right four lanes).

We next determined whether AT-RvD1 could affect NF-κB and C/EBP promoter luciferase activity in alveolar macrophage cells (MH-S). As shown in Fig. 5C and 5D, IgG immune complex stimulation led to a significant increase of NF-κB and C/EBP promoter luciferase activity (∼2-fold; p < 0.05). Although AT-RvD1 treatment had no effect on the basal activity of luciferase, it caused a significant decrease of the NF-κB and C/EBP promoter luciferase expression induced by IgG immune complexes (p < 0.05; Fig. 5C, 5D). Together, these data suggest that the reduction of NF-κB and C/EBPs activity is a potential mechanism whereby AT-RvD1 and p-RvD1 suppress IgG immune complex–induced cytokine and chemokine production in the lung.

We evaluated the effects of AT-RvD1 treatment on the cytokine production in the MH-S cells. We showed the secretions of TNF-α and IL-6 were significantly induced from IgG immune complex–stimulated MH-S cells over a 24-h period (Fig. 6A, 6B). Interestingly, there were rapid increases in the production of TNF-α, peaking at 2 h after IgG immune complex stimulation, followed by a gradual decline, whereas the secretion of IL-6 shows a progressive increase, peaking at 24 h (Fig. 6A, 6B). Moreover, on IgG immune complex stimulation, AT-RvD1 led to a decreased production of both TNF-α and IL-6 in all time points when compared with control-treated MH-S cells (Fig. 6A, 6B).

To further examine the mechanisms by which AT-RvD1 suppresses the production of TNF-α and IL-6 induced by IgG immune complexes, we performed transient transfection assay with TNF-α and IL-6 promoter luciferase constructs. As with the endogenous promoter, IgG immune complex stimulation induced luciferase expression by >3-fold and 4-fold, for TNF-α and IL-6 promoter luciferase, respectively. AT-RvD1 treatment led to a significant decrease in TNF-α (∼30%; p < 0.05) and IL-6 (∼40%; p < 0.05) promoter luciferase expression induced by IgG immune complexes (Fig. 6C, 6D). These results suggested that, in alveolar macrophages, AT-RvD1 inhibits IgG immune complex–induced TNF-α and IL-6 production at transcription level.

In the IgG immune complex–induced lung injury model, recruitment of neutrophils and their subsequent activation by immune complexes led to the generation of oxidants and release of proteinases, eventually causing lung injury characterized by increased vascular permeability and alveolar hemorrhage (1, 2). We evaluated AT-RvD1 treatment on the expression of cytokines and chemokines in primary peritoneal neutrophils. As shown in Fig. 7, the secretions of TNF-α, IL-6, KC, and MIP-1α were all significantly induced from IgG immune complex–stimulated neutrophils. Moreover, AT-RvD1 treatment led to a significant decrease in IgG immune complex–induced secretion of these cytokines and chemokines from neutrophils (TNF-α and KC at all time points, Fig. 7A, 7C; IL-6 and MIP-1α at 4–8 h and after, Fig. 7B, 7D) when compared with control-treated cells. These results suggest one potential mechanism whereby AT-RvD1 disrupts IgG immune complex–induced lung injury is via its effects on neutrophil inflammatory responses (Fig. 8).

Although inflammation is usually a local, protective reaction to injury or invasive microbes, these immune responses may sometimes injure the host in both acute and chronic circumstances. For example, tissue injury and destruction may result from the vigorous responses with which leukocytes destroy pathogens and pathogen-infected cells, and dispose of dead cells and their products instead of the direct effects of the pathological agents themselves (1). Accordingly, the inflammatory responses must be precisely regulated. The recent discovery of SPM, derived from polyunsaturated fatty acids, such as lipoxins, D-series resolvins, E-series resolvins, neuoprotectins, and maresins, has uncovered molecular mechanisms that regulate the progression and resolution of inflammation (31). However, the detailed events in which SPM controls inflammation-triggered tissue injury remain of interest. Resolvins of the D-series (RvD1–RvD6) are derived from docosahexaenoic acid (C22:6) (31). The biosynthesis of both D-series and aspirin-triggered D-series resolvins has been described (19, 31, 32). Among them, RvD1/AT-RvD1 is proved to be a potent D-series resolvin that protects from excessive inflammation (31). In the current study, we determined the actions of AT-RvD1 and its analog, p-RvD1, on FcγR-mediated inflammatory responses.

Lung inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes has proven to be an important model for developing an understanding of the role of various mediators in events that lead to tissue injury (1). In this model, intra-alveolar deposition of IgG immune complexes results in an acutely damaging process that includes a vascular leak syndrome, significant recruitment and activation of leukocytes, and damage of vascular endothelial cells and alveolar epithelial cells (1). These types of events are observed in many diseases, including autoimmune diseases and specific types of immune-mediated diseases such as allergic aspergillosis (33). Using this neutrophil-dependent lung injury model, to our knowledge we have demonstrated for the first time that AT-RvD1– and p-RvD1–treated mice have significantly reduced lung inflammatory responses and reduced lung injury after IgG immune complex deposition. This was indicated by reduced lung vascular permeability (albumin leak), lung histology, BAL neutrophil influx, and cytokine/chemokine levels (Figs. 1–3). These results suggest that AT-RvD1and p-RvD1 play a critical role in IgG immune complex–induced inflammatory responses and injury in the lung.

Earlier studies, including ours, suggest that activation of transcription factors NF-κB and C/EBPβ plays a central role in the pulmonary inflammatory response to IgG immune complexes (28, 30). Both NF-κB and C/EBPβ are known regulators of various genes involved in the inflammation, such as those coding for cytokines, chemokines, their receptors, and acute-phase proteins. In the current study, we show that AT-RvD1 and p-RvD1 inhibited the activities of NF-κB and C/EBPs in both lung and alveolar macrophages, suggesting that the reduction of NF-κB and C/EBP activity is a potential mechanism whereby AT-RvD1 and p-RvD1 suppress IgG immune complex–induced cytokine/chemokine production and injury in the lung. Interestingly, recent studies show that RvD1 reduces NF-κB pathway in human monocytes and macrophages by regulating specific microRNAs (32, 34). Whether the similar mechanism is involved in the AT-RvD1 regulation of C/EBPβ remains an interesting question to determine.

Alveolar macrophage activation is a key initiation signal for acute lung injury (35–38). By airway instillation of liposome-encapsulated dichloromethylene diphosphonate, Lentsch et al. (39) shows that depleting alveolar macrophages significantly reduced NF-κB activation in the IgG immune complex–injured lungs. Moreover, our recent study demonstrates that lung C/EBP activation induced by IgG immune complexes is suppressed by depletion of alveolar macrophages (30). Furthermore, intrapulmonary instillation of phosphonate-containing liposomes or C/EBPβ gene knockout led to substantially reduced BAL levels of TNF-α, the CXC chemokines, neutrophil accumulation, and lung injury (30, 39, 40). Interestingly, lung instillation of rTNF-α in alveolar macrophage–depleted animals restores the NF-κB activation response in the whole lung (39). These data together suggest that initial activation of NF-κB and C/EBP in alveolar macrophages and the ensuing production of TNF-α and other inflammatory mediators play an important role in the initial pathogenesis of IgG immune complex–induced lung injury. Data in the current study show that AT-RvD1 suppresses IgG immune complex–induced TNF-α and IL-6 production from alveolar macrophages at both transcriptional and translational levels (Fig. 6). In addition, AT-RvD1 treatment also led to a significant decrease of the NF-κB and C/EBP promoter luciferase expression induced by IgG immune complexes (Fig. 5C, 5D). These data suggest that alveolar macrophage is an important target of RvD1 upon immune complex stimulation. Interestingly, we previously show that Stat3 plays an important regulatory role in the pathogenesis of IgG immune complex–induced acute lung injury (21). Furthermore, it has been demonstrated that Stat3 is involved in the IL-6–induced upregulation of C/EBPβ and δ gene promoters (41). Thus, it is reasonable to speculate that IgG immune complex–activated IL-6–Stat3–C/EBP signal is a critical circuit regulated by RvD1. However, Stat3 can also be activated in response to IL-10, which is an important regulator of lung inflammatory injury after deposition of IgG immune complexes and contains the extent of injury (42). Thus, in future studies, it is interesting to investigate how Stat3 activation through different receptors (IL-6 or IL-10 receptors) can be differentially regulated by RvD1 in immune effector cells, leading to controlled inflammatory responses.

Neutrophil activation and transmigration into the alveolar compartment play a key role in the development of IgG immune complex–induced lung injury. Our current study provides the evidence that AT-RvD1 and p-RvD1 appear to reduce leukocyte recruitment into the alveolar space (Fig. 1B, 1D). In addition, AT-RvD1 suppressed cytokine and chemokine secretion from primary neutrophils when incubated with IgG immune complexes. Interestingly, a recent study demonstrates that the RvD1 is able to limit the human neutrophil recruitment under shear conditions in a mechanism dependent on its receptors, ALX/FPR2 and GPR32 (43). Furthermore, both AT-RvD1 and RvD1 analogs effectively activated ALX/FPR2 and GPR32 in GPCR-overexpressing β-arrestin systems (44). Importantly, neutrophil infiltration in self-limited peritonitis was reduced in human ALX/FPR2-overexpressing transgenic mice (44). Together with our current results, these studies suggest that regulation of neutrophil activation and migration is another important mechanism in RvD1 mitigation of IgG immune complex–induced inflammatory responses. Both human neutrophils and macrophages express ALX/FPR2 and GPR32 (45); however, the detailed molecular mechanisms whereby RvD1 regulates FcγR-mediated signals in phagocytes remain to be determined.

Probably one of the most important findings in the current study is that p-RvD1 and AT-RvD1 treatment led to a significant reduction in the IgG immune complex–induced C5a production in BALFs (Fig. 4). C5a is a powerful proinflammatory anaphylatoxin. In the model of IgG immune complex acute lung injury, anti-C5a treatment significantly reduced the increase in vascular permeability and neutrophil recruitment (25). The protective effects of anti-C5a appeared to be related to its ability to suppress lung alveolar macrophage production of TNF-α (25). Similarly, mice deficient in C5 and C5aR were protected from IgG immune complex–induced alveolitis (26, 46). In addition, early IgG immune complex–induced C5a and its interaction with C5aR led to induction of activating FcγRIII and suppression of inhibitory FcγRII on alveolar macrophages, which appears crucial for cytokine production and neutrophil recruitment in the IgG immune complex–injured lung (26). The detailed mechanisms by which p-RvD1 and AT-RvD1 suppress C5a production in the lung remain to be determined. Interestingly, C/EBPβ plays a critical role in the transcriptional induction of C3 (47). Thus, a possible mechanism of RvD1 involvement in C5a production is its regulation on C/EBPβ transcriptional activities.

In summary, our studies provide evidence that AT-RvD1 and its metabolically stable analog, p-RvD1, play a critical role in blocking acute inflammatory responses induced by IgG immune complexes both in vitro and in vivo in the lungs. More detailed understanding of the cross-talk between resolvins and FcγR-mediated inflammatory responses and the underlying mechanisms may provide new therapeutic strategies for diseases with an inflammatory component, including acute hypersensitivity pneumonitis.

C.N.S. is an inventor on patents (resolvins) assigned to Brigham and Women’s Hospital and licensed to Resolvyx Pharmaceuticals. C.N.S. is a scientific founder of Resolvyx Pharmaceuticals and owns equity in the company. C.N.S.’s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. N.A.P. is an inventor on patents (resolvins) assigned to the University of Southern California and licensed for clinical development and retains stock in Resolvyx Pharmaceuticals.