Resolvin E1 and protectin D1 activate inflammation-resolution programmes

Abstract

Resolution of acute inflammation is an active process essential for appropriate host responses, tissue protection and the return to homeostasis. During resolution, specific omega-3 polyunsaturated fatty-acid-derived mediators are generated within resolving exudates, including resolvin E1 (RvE1) and protectin D1 (PD1). It is thus important to pinpoint specific actions of RvE1 and PD1 in regulating tissue resolution. Here we report that RvE1 and PD1 in nanogram quantities promote phagocyte removal during acute inflammation by regulating leukocyte infiltration, increasing macrophage ingestion of apoptotic polymorphonuclear neutrophils in vivo and in vitro, and enhancing the appearance of phagocytes carrying engulfed zymosan in lymph nodes and spleen. In this tissue terrain, inhibition of either cyclooxygenase or lipoxygenases--pivotal enzymes in the temporal generation of both pro-inflammatory and pro-resolving mediators--caused a 'resolution deficit' that was rescued by RvE1, PD1 or aspirin-triggered lipoxin A4 analogue. Also, new resolution routes were identified that involve phagocytes traversing perinodal adipose tissues and non-apoptotic polymorphonuclear neutrophils carrying engulfed zymosan to lymph nodes. Together, these results identify new active components for postexudate resolution traffic, and demonstrate that RvE1 and PD1 are potent agonists for resolution of inflamed tissues.

Figure 1. Pro-resolving lipid mediators RvE1, PD1 and ATLa direct local phagocyte tissue flux

(A) Peritonitis. ATLa, RvE1, or PD1 (300 ng, ip) was given either with zymosan (T₀, left) or after (12h, right), and peritoneal exudates collected (24h). Results are the mean ± s.e.m (n=4-8). *p<0.05, **p<0.01, ***p<0.001, compared to zymosan alone; ⁺p<0.05, ATLa vs. RvE1; ^#p<0.05, T₀ vs. 12h (B) Resolution Indices were defined in ref. 12 ( Supplemental Fig. 1), including ψ_max (maximal PMN), T_max (time point when PMNs reach ψ_max), T₅₀ (time point corresponding to ∼50% PMN reduction) and R_i (resolution interval, the interval between T_max and T₅₀). These indices were calculated when compounds were given at the initiation (T₀, solid lines and arrows) or peak (12h, dashed lines and arrows) of inflammation. Results are the mean ± s.e.m. (n=4-8). *p<0.05, ***p<0.001, compared to zymosan alone; ⁺p<0.05, ATLa vs. RvE1; ⁺⁺p<0.01, ATLa vs. PD1.

Figure 2. RvE1 and PD1 increase macrophage phagocytic activity in vivo and in vitro

(A) In vivo phagocytosis with representative dot plots of FACS analysis. (B) (Top) Time course of eicosanoid generation and phagocytosis activity in vitro. Results are the mean ± s.e.m. (n=3). LXA₄ amounts are expressed as pg/coincubation (0.4x10⁶ PMN+0.2x10⁶ macrophages in 0.2 ml). *p=0.01, **p=0.02 (vs. time 0 or 120 min). Phagocytosis activities are expressed as [F4/80⁺Gr-1⁺/F4/80⁺]x100%. ^#p=0.03, ^##p=0.01 (vs. time 120 min). (Inset) LXA₄ and LTB₄ generation at 60 min. (Bottom left) MS/MS spectrum of LXA₄. (Bottom right) RvE1 generation. Macrophages were incubated with ASA (500 μM, 30 min) followed by EPA (20 μM, 45 min) prior to incubation with apoptotic PMN. RvE1 was determined by LC/MS/MS and expressed as pg/coincubation (2x10⁶ PMN+1x10⁶ macrophages in 1 ml). (C) Phagocytosis of apoptotic PMN in vitro. Results are the mean ± s.e.m. (n=3-4) and expressed as percent increase of F4/80⁺Gr-1⁺ macrophages compared to vehicle alone. Cytokine/chemokine levels are the mean ± s.e.m. (n=3). *p<0.05, **p<0.01, ***p<0.001, compared to vehicle alone. (D) Phagocytosis of zymosan and latex particles in vitro. Results are the mean ± s.e.m. (n=3) and expressed as percent increase of F4/80⁺zymosan⁺ or F4/80⁺latex⁺ macrophages compared to vehicle alone. Results are the mean ± s.e.m. (n=3). *p<0.05, **p<0.01 compared to vehicle alone.

Figure 3. RvE1 and PD1 enhance leukocytes carrying phagocytozed zymosan in lymph nodes and spleen

(A) Leukocytes with engulfed-zymosan particles (zymosan^e) in the cortex of the LN and marginal zone of the spleen. The brown particles are positive staining of zymosan. Scale bars: 50 μm. (B) Quantification of zymosan. ATLa, RvE1 or PD1 was given at initiation (T₀) or peak (12 h) of inflammation. Results are mean ± s.e.m. (n=4-8). *p=0.05, **p<0.01, ***p<0.001. (C, D) COX or LOX inhibitor was given 30 min before zymosan challenge with or without mediators. Results are mean ± s.e.m. (n=3-4). *p=0.05, **p<0.01, ***p<0.001, compared with zymosan alone; ⁺p=0.05, ⁺⁺p<0.01, ⁺⁺⁺p<0.001, compared with (C) zymosan + COX-inhibitor or (D) zymosan + LOX-inhibitor.

Figure 4. Active removal of leukocytes from the inflammatory exudate

(A) Quantitation of FITC-zymosan^e leukocytes. Results are mean ± s.e.m. (n=3). *p=0.05 (4h vs. 24h). (B) Tracking of FITC-zymosan^e leukocytes 24h after FITC-zymosan injection in the (i) absence or (ii) presence of the primary anti-FITC Ab. The brown particles are positive staining of FITC-zymosan. (iii) Lipopassage (arrows). (iv) Zymosan^e leukocytes in perinodal adipose tissue. (v) In perinodal adipose tissue, lymph vessels (stars) conduct zymosan^e leukocytes in proximity to blood vessels. (vi) Zymosan^e leukocytes in the afferent lymph vessel (stars) and subcapsular sinus. (vii) In the lymph nodes, zymosan^e leukocytes were detected markedly in cortex areas. (viii, ix) Zymosan^e leukocytes consisted of monocytes/macrophages (arrowheads), and non-apoptotic PMNs with engulfed-zymosan (brown arrows) or without (blue arrows). (x) Dendritic-like (fine cell protrusions), zymosan^e leukocytes in the afferent lymph vessels (arrows). (xi, xii) In the spleen, some zymosan^e leukocytes also displaced morphological characteristics of DCs (arrows).