Human lungs are exposed daily to thousands of liters of air containing numerous environmental insults and pro-inflammatory stimuli including dust, pollen, mold, ozone and hydrocarbon pollutants. Given this, regulating the balance between pro-inflammatory and pro-resolving signaling pathways is crucial to maintain normal lung homeostasis 
. Cigarette smoke is a powerful pro-inflammatory stimulus and a leading cause of lung, cardiovascular and other diseases 
. Resolvin D1 is a member of a novel class of lipid mediators with anti-inflammatory and pro-resolving functions 
. Here, we report that RvD1 is a potent inhibitor of cigarette smoke-induced pro-inflammatory signaling in human lung cells in vitro
and has potent anti-inflammatory and pro-resolving properties in a mouse model of acute cigarette smoke-induced lung inflammation.
We first investigated the response of primary cells to RvD1 in vitro
. RvD1 significantly reduced inflammatory responses in primary human lung fibroblasts and blood-derived monocytes using both IL-1β and CSE as inflammatory stimuli, and from human small airway epithelial cells stimulated with IL-1β ( and ). We used IL-1β as a model stimulus because we and others have shown that CS exposure upregulates IL-1β levels in both mice and humans exposed to cigarette smoke () 
. Therefore, lung cells are likely exposed to IL-1β following smoke exposure in vivo
. Cigarette smoke extract is widely used as an in vitro
analog to inhalation of smoke but an exact equivalence between in vitro
and in vivo
exposures can not be determined 
. It is interesting to note that RvD1 abolished IL-1β-induced PGE2
production in lung fibroblasts and inhibited PGE2
production in vivo
. The role of PGE2
in regulating inflammation is complex, as PGE2
has both pro-inflammatory and pro-resolving properties. PGE2
is a potent neutrophil chemokine, and is upregulated by cigarette smoke () 
. However, PGE2
also has anti-inflammatory properties in neutrophils, and is involved in activating lipid mediator class switching between pro-inflammatory and pro-resolving mediators 
. Here, RvD1 has a net anti-inflammatory effect, perhaps because its pro-inflammatory effects are suppressed by RvD1, while its pro-resolving effects are replaced by exogenous RvD1.
Cigarette smoking causes both acute and chronic lung inflammation with prominent involvement of neutrophils. Neutrophilic airway inflammation is one of the key underlying pathophysiological processes in the development of COPD 
. Recently, it was shown that cigarette smoke disrupts the normal resolution of neutrophilic lung inflammation by reducing neutrophil apoptosis and efferocytosis 
. Impairment of neutrophil clearance can lead to irreversible local tissue damage via production of mediators including neutrophil elastase and reactive oxygen species 
. Here we report that RvD1 dampens neutrophil recruitment and enhances neutrophil clearance in acute CS-induced inflammation. These results suggest that RvD1 or 17R-RvD1 might be useful as therapies to reduce chronic inflammation and lung injury in diseases of chronic smoke exposure such as COPD.
Alternatively activated or type II macrophages (M2) are immunoregulatory cells that possess anti-inflammatory properties and eliminate tissue debris and apoptotic bodies via phagocytosis. Previous reports have shown that RvE1 and RvD2 enhanced macrophage phagocytosis of bacterial ligands and apoptotic neutrophils in sepsis and asthma models 
. Here we show that RvD1 promotes differentiation of pro-resolving macrophages that have enhanced phagocytic activity in vitro
and in vivo
( and ). It was recently reported that RvD1 regulates pro- and anti-inflammatory micro RNAs via signaling through its G-protein receptors ALX and GPR32, resulting in increased production of IL-10 and decreased NF-κB activity 
. Additionally, it is known that IL-10 promotes the M2 phenotype 
. These results, combined with our findings that IL-10 is elevated in RvD1-treated lungs () and alveolar macrophages (), demonstrate that RvD1 and IL-10 enhance M2 polarization by a positive feedback mechanism initiated by RvD1-mediated G-protein signaling.
Although neutrophil chemokines (MIP-2, KC) and broad-spectrum pro-inflammatory signals (IL-1β, IL-6) were strongly inhibited by RvD1, the macrophage chemoattractant MCP-1, which was also induced by CS, was not reduced by RvD1 in mouse lung. Interestingly, it has been reported that MCP-1 enhances efferocytosis by alveolar macrophages, and that MCP-1 is important for the resolution of inflammation 
. This is consistent with our observations that macrophage numbers were not reduced by RvD1 and that RvD1 enhances efferocytosis (). RvD1 also up-regulated production of IL-10, a potent immunoregulatory cytokine that is reported to be essential for the resolution of lung inflammation 
and which is significantly decreased in sputum samples from COPD patients 
. Taken together, this suggests that RvD1 is not an indiscriminate anti-inflammatory agent, but rather selectively activates specific anti-inflammatory and pro-resolving pathways that include inhibition of neutrophilic inflammation and the activation of a subset of anti-inflammatory, pro-resolving macrophages.
Smoking-related inflammation of the lungs, cardiovascular system and other organs persists for months or even years after smoking cessation, and COPD not only persists but also may continue to worsen 
. This suggests that development of novel therapeutics should be focused on promoting resolution, rather than preventing inflammation. Here, a single dose of 17R-RvD1 given after the final smoke exposure resulted in accelerated reduction in BAL neutrophils and IL-6 (). The pro-resolving activity was maintained for at least 3 days, with continued reductions in neutrophils and key pro-inflammatory mediators. These results are similar to results recently reported from Dr. Serhan’s laboratory that RvD1 and 17R-RvD1 accelerated the resolution of allergic airway inflammation in the mouse ovalbumin model 
. 17R-RvD1 is an alternate epimer of RvD1 produced endogenously in the presence of aspirin, which acetylates the COX-2 resulting in a shift from the 17S to the 17R epimer 
. The 17S epimer is subject to rapid inactivation by the endogenous enzyme 15-prostaglandin dehydrogenase/eicosanoid oxidoreductase (15-PGDH) whereas the 17R epimer is not. While RvD1 was effective at inhibiting inflammation when given immediately prior to smoke exposure, we were concerned that it may not have a long enough half-life to be effective at promoting resolution when given after smoke exposure. 17R-RvD1 has similar biological activity and uses the same receptors 
but is resistant to 15-PGDH and has a longer in vivo
. It is also possible that 17R-RvD1 would have been more efficacious than RvD1 in the pre-treatment model (, , ), although this has not been tested.
Cigarette smoke is a direct cause of several devastating diseases including COPD for which there are few effective therapeutic agents. Long-term use of glucocorticoids carries risks of infection and other adverse effects potentially outweighing their benefits 
, and many long-term smokers and emphysema patients exhibit steroid resistance 
. By blocking utilization of arachidonic acid, COX-2 inhibitors not only block pro-inflammatory signals, but also inhibit resolvin biosynthesis 
. Resolvins offer significant clinical potential because they promote resolution and repair without being immunosuppressive. Here, we have demonstrated that RvD1 possesses potent anti-inflammatory and pro-resolving activity in vivo
and in vitro
. Inhalation therapy with RvD1 may offer an alternative approach for treating human inflammatory lung diseases including chronic lung injury and COPD resulting from cigarette smoking and other chronic injuries.