In our mouse model of LPS-induced ALI, pretreatment with RvD1 reduced levels of TNF-α, IL-6 and neutrophils in BALF, and it attenuated inflammation in lung tissues. RvD1 inhibited degradation of IκBα and it decreased the levels of NF-κB p65 subunit in the nucleus as well as the DNA-binding activity of nuclear NF-κB. Administering the PPARγ antagonist GW9662 before injecting RvD1 partially reversed these anti-inflammatory effects of RvD1. These results suggest that RvD1 may attenuate lung inflammation of LPS-induced ALI by suppressing NF-κB activation in a process that depends partly on PPARγ activation.
Proinflammatory cytokines play a critical role in ALI and ARDS: persistently elevated levels of proinflammatory cytokines such as TNF-α and IL-6 are associated with worse outcome in patients with ALI or sepsis . Since TNF-α and IL-6 are considered markers of inflammation, the fact that their levels increased after LPS administration in our mouse model of ALI suggests that the model is valid.
Using this model, we found that RvD1 down-regulated levels of TNF-α and IL-6 in BALF of mice with LPS-induced ALI, and that it did so in a dose-dependent manner. These results are consistent with previous work in patients and animal models of disease. Feeding patients with rheumatoid arthritis or multiple sclerosis fish oils rich in ω-PUFA, from which RvD1 is derived, can reduce TNF-α and IL-6 levels . RvD1 has been shown to reduce levels of TNF-α and IL-6 in mouse models of colitis, d-galactosamine-sensitized endotoxin shock and ALI [21–23]. In addition, the aspirin-triggered epimer of RvD1 (AT-RvD1; 7S,8R,17R-trihydroxy- 4Z,9E,11E,13Z,15E,19Z-docosahexzenoic acid) has been shown to decrease levels of TNF-α and IL-6 in a mouse model of hydrochloric acid-induced ALI .
Our findings, together with those of previous studies, suggest that RvD1 can down-regulate proinflammatory cytokines during the early stages of several inflammatory diseases, including ALI.
In many inflammatory diseases, neutrophils are rapidly recruited to sites of inflammation; there they kill invading bacteria through phagocytosis involving the release of preformed granular enzymes and proteins, as well as the production of a range of oxygen species. However, the highly destructive activity of neutrophils can also pose a risk to healthy tissues. This helps to explain why one of the first steps in the resolution of inflammation, which occurs when the initial injury or microbial invasion has been limited and injurious stimuli or microbes have been neutralized , is the loss of neutrophils from the inflamed area. In fact, research over the last decade has shown that resolution of inflammation is a programmed process that is actively regulated by various pro-resolving lipid mediators derived from ω-PUFA, including LXA4, RvE1, and PD1 . In our mouse model of LPS-induced ALI, RvD1 at a dose of 600 ng/mouse significantly and rapidly reduced the numbers of neutrophils in BALF and attenuated inflammation and ALI scores in lung tissues.
RvD1 may decrease the numbers of neutrophils at inflammatory sites through the same mechanisms as other pro-resolving lipid mediators derived from ω-PUFA such as LXA4, RvE1, and PD1. Mechanisms proposed for these other mediators include: (i) limiting polymorphonuclear leukocyte (PMN) infiltration to inflamed sites, (ii) up-regulating CCR5 expression on apoptotic neutrophils, and (iii) enhancing apoptotic PMN engulfment by macrophages . We speculate that RvD1 down-regulates neutrophils at inflammation sites through multiple mechanisms. One is that RvD1 exerts anti-inflammatory effects by down-regulating levels of TNF-α and IL-6, and perhaps also levels of neutrophil chemoattractants such as IL-8 and LTB4 . In this way, RvD1 would reduce the number of neutrophils entering the BALF. At the same time, RvD1 exerts pro-resolving effects, as do other lipid mediators, by accelerating the exit of neutrophils from sites of inflammation. It may do this by up-regulating CCR5 expression on apoptotic neutrophils, stimulating their uptake by macrophages and the subsequent exit of phagocytes from the exudate via the lymphatic system . This is of particular relevance to ALI, since ALI/ARDS involves extensive inflammation with PMN activation in lungs, accompanied by interstitial edema and an intense inflammatory response. Neutrophil apoptosis and clearance are delayed in sepsis and ARDS, which exacerbates inflammatory injury to the parenchyma ; as a result, enhancing neutrophil apoptosis in ALI can decrease mortality and ameliorate lung damage . In sum, we suggest that the ability of RvD1 to reduce neutrophil numbers at inflammatory sites and to attenuate inflammation in lung tissue in our mouse model of ALI involves a combination of anti-inflammatory and pro-resolving mechanisms.
As a first step towards elucidating these mechanisms, we examined whether RvD1 down-regulates inflammatory mediators and neutrophils in our mouse model through a PPARγ/NF-κB pathway. PPARγ is a ligand-activated nuclear transcription factor involved in cellular differentiation, cancer, inflammation, insulin sensitization, atherosclerosis, and metabolic diseases, and it has been shown to inhibit NF-κB and to play important roles in several inflammatory processes . Various PUFA, especially ω-PUFA, are natural ligands of PPARγ . PPARγ is found in both the cytoplasm and nucleus under normal conditions; when activated, cytosolic PPARγ translocates to the nucleus, where it induces gene transcription [30, 31]. In our study, RvD1 increased the level of PPARγ in the nucleus in a dose-dependent manner. Pretreatment with GW9662 partially reversed the RvD1-induced increase in nuclear PPARγ. A simple explanation of our results is that RvD1 activates cytosolic PPARγ, causing it to translocate into the nucleus, and that this activation occurs via a PPARγ-dependent pathway.
Using our animal model, we also found that RvD1 decreased the level of nuclear NF-κB p65 subunit and the DNA-binding activity of nuclear NF-κB. Pretreatment with GW9662 partially reversed the RvD1-induced inhibition of NF-κB activation. These results suggest that RvD1 inhibits NF-κB activation through a pathway at least partially dependent on PPARγ activation. These findings are consistent with several studies in animals and tissue culture. Oxidized eicosapentaenoic acid (EPA) and DHA, from which RvD1 is derived, have been shown to act as potent endogenous PPARγ ligands and to inhibit NF-κB DNA-binding activity in vitro and in vivo . In fact, both EPA and DHA down-regulated LPS-induced activation of NF-κB via a PPARγ-dependent pathway in human kidney-2 cells . Therefore, RvD1 may exert both its anti-inflammatory and pro-resolving effects through a PPARγ/NF-κB pathway. This is particularly relevant to ARDS, because inhibition of NF-κB activation in patients with the disease reduced not only the levels of proinflammatory mediators but also the numbers of activated resident neutrophils, thereby accelerating the resolution of lung injury .
Taken together, our results strongly suggest that RvD1 interacts with a PPARγ/NF-κB pathway, but whether that interaction occurs by direct binding of RvD1 to PPARγ remains unclear. Krishnamoorthy and coworkers  transiently transfected HEK-293 cells with expression vectors encoding PPARγ receptors coupled to Gal4 and found that RvD1 did not activate PPARγ directly. They also showed that RvD1 specifically interacted in vitro with both lipoxin A4 receptor ALX and orphan receptor GPR32. These results do not necessarily have to apply in vivo, so future studies should examine this question in animals.
Indeed future studies should clarify several limitations of the present work. First, our results do not identify the mechanism(s) by which RvD1 can reduce neutrophils in BALF. Second, inhibition of PPARγ only partially reversed the effects of RvD1, suggesting that other signaling pathways may help mediate the effects of the drug. For example, RvD1 has been found to protect mice from LPS-induced ALI by interacting with MAP kinases as well as with the NF-κB pathway . Another study found that ω-PUFA exerted broad anti-inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages through stimulation of G protein-coupled receptor 120 (GPR 120) . Future studies should consider these and other pathways when examining downstream effectors of RvD1 anti-inflammatory and pro-resolving activity.