Resolvins Decrease Oxidative Stress Mediated Macrophage and Epithelial Cell Interaction through Decreased Cytokine Secretion

Abstract

Background: Inflammation is a key hallmark of ALI and is mediated through ungoverned cytokine signaling. One such cytokine, interleukin-1beta (IL-1β) has been demonstrated to be the most bioactive cytokine in ALI patients. Macrophages are the key players responsible for IL-1β secretion into the alveolar space. Following the binding of IL-1β to its receptor, "activated" alveolar epithelial cells show enhanced barrier dysfunction, adhesion molecule expression, cytokine secretion, and leukocyte attachment. More importantly, it is an important communication molecule between the macrophage and alveolar epithelium. While the molecular determinants of this inflammatory event have been well documented, endogenous resolution processes that decrease IL-1β secretion and resolve alveolar epithelial cell activation and tissue inflammation have not been well characterized. Lipid mediator Aspirin-Triggered Resolvin D1 (AT-RvD1) has demonstrated potent pro-resolutionary effects in vivo models of lung injury; however, the contribution of the alveoli to the protective benefits of this molecule has not been well documented. In this study, we demonstrate that AT-RvD1 treatment lead to a significant decrease in oxidant induced macrophage IL-1β secretion and production, IL-1β-mediated cytokine secretion, adhesion molecule expression, leukocyte adhesion and inflammatory signaling.

Methods: THP-1 macrophages were treated with hydrogen peroxide and extracellular ATP in the presence or absence of AT-RvD1 (1000-0.1 nM). A549 alveolar-like epithelial cells were treated with IL-1β (10 ng/mL) in the presence or absence of AT-RvD1 (0.1 μM). Following treatment, cell lysate and cell culture supernatants were collected for Western blot, qPCR and ELISA analysis of pro-inflammatory molecules. Functional consequences of IL-1β induced alveolar epithelial cell and macrophage activation were also measured following treatment with IL-1β ± AT-RvD1.

Results: Results demonstrate that macrophages exposed to H2O2 and ATP in the presence of resolvins show decreased IL-1β production and activity. A549 cells treated with IL-1β in the presence of AT-RvD1 show a reduced level of proinflammatory cytokines IL-6 and IL-8. Further, IL-1β-mediated adhesion molecule expression was also reduced with AT-RvD1 treatment, which was correlated with decreased leukocyte adhesion. AT-RvD1 treatment demonstrated reduced MAP-Kinase signaling. Taken together, our results demonstrate AT-RvD1 treatment reduced IL-1β-mediated alveolar epithelial cell activation. This is a key step in unraveling the protective effects of resolvins, especially AT-RvD1, during injury.

Fig 1. H₂O₂ and ATP Treatment Results in Enhanced IL-1β Secretion in THP-1 Macrophages.

In order to reconstruct a physiologically relevant model of H₂O₂ induced macrophage secretion, primed THP-1 macrophages were treated with ATP (5 μM), H₂O₂ (10 mM) or combination of both ATP and THP1 for 1 hr. IL-1β released into the cell culture supernatant was analyzed. One way ANOVA was used with a tukey post-hoc test, where a p-value < 0.05 was deemed statistically significant (n = 4). Significance: ** = p < 0.01, *** = p < 0.001, vs no treatment control.

Fig 2. Omega-3 Treatment results in decreased H₂O₂-induced IL-1β secretion in THP1 macrophages.

In order to assess the therapeutic effects of omega-3 fatty acids DHA and EPA as well as resolvin metabolites on IL-1β secretion, primed THP-1 macrophages were treated with ATP (5 μM), H₂O₂ (10 mM) or combination of both ATP in the presence or absence of EPA, DHA, RvD1 and AT-RvD1 at indicated doses for 1 hr. IL-1β release into the cell culture supernatant was analyzed. One way ANOVA was used with a tukey post-hoc test, where a p-value < 0.05 was deemed statistically significant (n = 4). Significance: ** = p < 0.01, *** = p < 0.001, vs no treatment control.

Fig 3. Resolvin Treatment results in decreased H₂O₂-induced caspase-1 activation in THP1 macrophages.

In order to assess the role of RvD1 and AT-RvD1 on IL-1β processing enzyme caspase-1, primed THP-1 macrophages were treated with ATP (5 μM), H₂O₂ (10 mM) or combination of both ATP in the presence or absence of RvD1 and AT-RvD1 at indicated doses for 1 hr. Activated caspase-1 into the cell culture supernatant was analyzed by ELISA. One way ANOVA was used with a tukey post-hoc test, where a p-value < 0.05 was deemed statistically significant (n = 3).

Fig 4. Resolvin Treatment leads to reduced macrophage induced alveolar epithelial cytokine secretion.

THP-1 macrophages were treated with ATP + H₂O₂ in the presence or absence of RvD1 (100 nM) and AT-RvD1 (100 nM). Following treatment, supernatant from each macrophage group was collected and used to treat A549 cells for 6 hours. After treatment, supernatant from alveolar epithelial cells was collected and analyzed for interleukin-8 via ELISA. One way ANOVA was used with a tukey post-hoc test, where a p-value < 0.05 was deemed statistically significant (n = 4). Significance: ** = p < 0.01, *** = p < 0.001, vs no treatment control.

Fig 5. Aspirin-Triggered Resolvin D1 Attenuated IL-1β-induced Cytokine/Chemokine Secretion.

A549 cells were seeded at a density of 0.5 x 10⁶ million cells per well in 12 well plates. When cells reached confluence, they were then serum starved and treated with IL-1β (10 ng/mL) in the presence or absence of aspirin-triggered resolvin D1 (AT-RD1, 100 nM) for 6 hours. Following treatment, cell culture supernatants were collected and the presence of (A) IL-8 and (B) IL-6 levels were analyzed by ELISA. A student t-test was used to determine statistical significance with p < 0.05 being statistically significant. ND = Not detected, limit of detection 2 pg/mL. n = 4 for both experiments.

Fig 6. Aspirin-Triggered Resolvin D1 Attenuate IL-1β-induced Intercellular Adhesion Molecule-1 Expression.

A549 cells were seeded at a density of 0.5 x 10⁶ million cells per well in 12 well plates. When cells reached confluence, they were then serum starved and treated with IL-1β (10 ng/mL) in the presence or absence of aspirin-triggered resolvin D1 (AT-RD1, 100 nM) for 6 hours. Following treatment, cells were lysed in Trizol and mRNA was extracted. Complimentary cDNA was produced and qPCR analysis was performed utilizing primers specific for ICAM-1. Statistical significance was determined using a standard student t-test with p < 0.05 being statistically significant (n = 5).

Fig 7. Aspirin-Triggered Resolvin D1 Attenuated IL-1β-mediated leukocyte-epithelial cell adhesion.

A549 cells were seeded in A) 12 well at a density of 0.75 x 10⁶ or B) 96 well plates at a density of 0.1 x 10⁶ cells per well. Following serum starvation the cells were treated with IL-1β (10 ng/mL) in the presence or absence of aspirin-triggered Resolvin D1 (100 nM) for 6 hours. Following IL-1β treatment, calcein AM loaded THP1 cells were co-incubated with the alveolar epithelial cells for 30 minutes. Unbound attached monocytes were washed away with media and leukocyte attachment was observed via (A) fluorescent microscopy and (B) quantified with a reading of mean fluorescence intensity at Ex: 495 Em: 515. Statistical significance was measured with a one way ANOVA with a tukey post-hoc test, where p < 0.05 was designated as statistically significant (n = 3).

Fig 8. Aspirin-Triggered Resolvin D1 does not rescue barrier function in IL-1β-treated A549 cells.

A549 cells were seeded in 8 well 8W1E ECIS arrays at a density of 5 x 10⁴ cells/mL. When cells reached confluence, they were treated with IL-1β (10 ng/mL) in the presence or absence of AT-RD1 (100 nM). Barrier resistance was recorded for 48 hours using ECIS. To eliminate variability; samples were treated, duplicated, and recorded 4 times at each time point. Statistical significance was measured with a one way ANOVA with a tukey post-hoc test, where p < 0.05 was designated as statistically significant (n = 4).

Fig 9. Resolvins regulate IL-1β-induced p38 and ERK 1/2 activity in alveolar epithelial cells.

A549 cells were seeded at a density of 0.5 x 10⁶ million cells per well in 12 well plates. When cells reached confluence, they were then serum starved and treated with IL-1β (10 ng/mL) in the presence or absence of aspirin-triggered resolvin D1 (AT-RvD1, 100 nM) for 6 hours. Following treatment, cells were harvested and lysed with RIPA buffer. Western blot analysis was performed and the samples were probed for the presence of total and phosphorylated (A) p38 and (B) ERK 1/2 MAPKs using enhanced chemiluminescence. n = 3 for both set of experiments.

Fig 10. AT-RvD1 blunts macrophage and epithelial communication through reduced cytokine signaling.

Exposure to oxidative stress leads to an enhanced secretion of proinflammatory cytokines, with IL-1β being the most bioactive for ALI patients. IL-1β secretion results in alveolar epithelial cell activation which is hallmarked by enhanced barrier function, cytokine secretion, and adhesion molecule expression. We found that AT-RvD1 was able to interrupt the macrophage to alveolar communication through the blockage of IL-1β signaling. Upon treatment of alveolar epithelial cells with IL-1β, there was an increase in inflammatory activation, which was significantly attenuated with AT-RvD1 treatment.