CYP450-derived oxylipins mediate inflammatory resolution

Abstract

Resolution of inflammation has emerged as an active process in immunobiology, with cells of the mononuclear phagocyte system being critical in mediating efferocytosis and wound debridement and bridging the gap between innate and adaptive immunity. Here we investigated the roles of cytochrome P450 (CYP)-derived epoxy-oxylipins in a well-characterized model of sterile resolving peritonitis in the mouse. Epoxy-oxylipins were produced in a biphasic manner during the peaks of acute (4 h) and resolution phases (24-48 h) of the response. The epoxygenase inhibitor SKF525A (epoxI) given at 24 h selectively inhibited arachidonic acid- and linoleic acid-derived CYP450-epoxy-oxlipins and resulted in a dramatic influx in monocytes. The epoxI-recruited monocytes were strongly GR1(+), Ly6c(hi), CCR2(hi), CCL2(hi), and CX3CR1(lo) In addition, expression of F4/80 and the recruitment of T cells, B cells, and dendritic cells were suppressed. sEH (Ephx2)(-/-) mice, which have elevated epoxy-oxylipins, demonstrated opposing effects to epoxI-treated mice: reduced Ly6c(hi) monocytes and elevated F4/80(hi) macrophages and B, T, and dendritic cells. Ly6c(hi) and Ly6c(lo) monocytes, resident macrophages, and recruited dendritic cells all showed a dramatic change in their resolution signature following in vivo epoxI treatment. Markers of macrophage differentiation CD11b, MerTK, and CD103 were reduced, and monocyte-derived macrophages and resident macrophages ex vivo showed greatly impaired phagocytosis of zymosan and efferocytosis of apoptotic thymocytes following epoxI treatment. These findings demonstrate that epoxy-oxylipins have a critical role in monocyte lineage recruitment and activity to promote inflammatory resolution and represent a previously unidentified internal regulatory system governing the establishment of adaptive immunity.

Keywords: epoxygenase; monocyte; oxylipins; phagocytosis; resolution.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

LC/MS/MS analysis of oxylipin-generating pathways in the mouse peritoneal cavity during acute inflammation and resolution initiated by zymosan A. (A) Absolute levels of oxylipins determined in naive peritoneal cavity lavage fluid of male C57BL/6 mice. (B) Sum of oxylipin-generated COX, CYP450, CYP450-lipoxygenase–like (CYP-L), or LO pathways using either arachidonic acid (AA), linoleic acid (LA), DHA, or EPA as a substrate in the naive peritoneal cavity. (C) Heatmap showing fold changes in oxylipin formation following zymosan A treatment (1 mg, i.p.) from 0 to 4 h (peak of acute inflammation), 24 h, and 48 h (resolution). (D and E) Fold-normalized square root of oxylipins produced by COX-AA (prostanoids), CYP-AA (DHETs), CYP-L-AA (19- and 20-HETE), CYP-DHA (19,20-EpDPE and 19,20-DiHDPA), CYP-EPA (17,18-DHEQ), and 5-LO-AA (5-HETE) pathways (D) and CYP-LA (EpOMEs and DiHOMEs), 8/12/15-LO (8-, 12-, and 15-HETE), and LO-LA (HODEs) pathways (E) in response to zymosan A over 48 h. Pathways in D and E represent two distinct responses to challenge with zymosan A. Data represent the mean ± SEM from n = 4–8 mice per group.

Fig. 2.

Epoxygenase products form in a biphasic manner mirroring acute inflammation and inflammatory resolution: selective inhibition of CYP-AA and CYP-LA products by epoxI. (A and B) Mean fold change in (A) CYP-AA products: DHETs and (B) CYP-LA (DiHOME), CYP-DHA (DiHDPA), and CYP-EPA (DHEQ) products, 0–48 h. Each mediator was normalized to its paired levels found in the naive cavity in each experiment; the levels found in the naive cavity were given an arbitrary value of 1. *P < 0.05 by one-sample t test from 0 h. †P < 0.05 by unpaired t test between the values observed at 24 and 48 h. (C) Mean fold-change in epoxygenase product formation in the presence or absence of epoxI (30 mg/kg). EpoxI was given at 24 and 36 h. Epoxy-oxylipins were measured at 48 h and are represented as fold change from the respective mean levels observed in the naive cavity at 0 h. *P < 0.05 by unpaired t test. Data represents mean ± SEM.

Fig. 3.

Epoxygenase inhibition during resolution causes Ly6chi monocyte recruitment and Ccl2 generation. (A) Change in lymphocytes, monocytes/macrophages, and PMNs in the peritoneal cavity of mice 48 h postzymosan treatment. At 24 and 36 h, mice were given either sterile PBS or epoxI (30 mg/kg, i.p.). Total cell numbers were counted on a hemocytometer and the proportion of each cell type was determined by FACS. Data represent the mean ± SEM from n = 6 mice per group; *P < 0.05 by unpaired t test between vehicle (Cont; PBS) and epoxI-treated mice. (B) Example of FACS analysis of total cell (Upper) and GR+ populations (Lower) of cells elicited by zymosan alone (control; Left) or in the presence of epoxI (Right) in terms of size (FSc) and granularity (SSc). (C–F) Relative expression of Ly6c (C), Ccr2 (D), CX3CR1 (E), and CCL2 (F) mRNA compared with β-actin in elicited cells from zymosan + control or epoxI-treated mice taken at 48 h. (G) CCL2 generation in cells from zymosan + control or epoxI-treated mice. Cells were elicited at 36 h and left for a further 8 h ex vivo and CCL2 was measured by ELISA. (H–K) CCL2 (H), iNOS (I), IL-12 (J), and TNFα (K) mRNA expression in zymosan-elicited cells at 36 h treated with 14,15-EET or 11,12-EET. Cells were elicited at 36 h and treated for a further 7 h with EETs. Data represent the mean ± SEM from n = 3–4 mice per group; *P < 0.05 by unpaired t test between vehicle (Cont; PBS) and epoxI-treated mice.

Fig. 4.

Endogenous oxylipins regulate Ly6chi monocyte, F4/80 macrophage, and CD19 and CD3 cell populations during resolution. Changes in (A) Ly6chi cell populations, (B) Ly6g+ cell populations, (C) F4/80 expression (mean fluorescence intensity; MFI) (Left) and F4/80++ cell populations (Right), and (D) CD19+, (E) CD3+, and (F) MHCII+ CD11c+ dendritic cell populations in control and epoxI-treated mice (solid black bars; Left) and in wild-type (wt) and sEH−/− mice (Right). The proportion of cells in each group was determined by FACS and related back to cell numbers. Data represent the mean ± SEM from n = 4–5 mice per experimental group; *P < 0.05 by two-way ANOVA or unpaired t test.

Fig. 5.

Epoxygenase inhibition alters the resolution phenotype of recruited cells of the monocyte lineage. Inflammation was initiated by zymosan (1 mg, i.p.), and mice were treated with vehicle control (PBS) or epoxI (30 mg/kg, i.p.) at 24 and 36 h. Cells were collected and pooled from n = 10 mice and Ly6chi, Ly6clo monocytes, resident macrophages, and recruited dendritic cells were sorted on a FACSAria as detailed in Materials and Methods. A qRT-PCR resolution monocyte panel (n = 3–6) was then used to examine the phenotype of each cell type. (A–C) In the presence of epoxI, (A) Plxcd2, Ccna2, Ccnb2, Aspa, F5, Tgfb2, and Timd4 were found to be down-regulated in the cell types; (B) Ccr2, CCL2, Ms4A7, CD86, and IL1F9 were up-regulated in the cell types; and (C) Stfa2l1 was up- and down-regulated in a cell type-specific manner. *P < 0.05 by unpaired t test. (D, Upper) Summary of the relative basal levels of each transcript in each cell type. (D, Lower) Summary of the effect of epoxI on the different transcripts. Data represents mean ± SEM.

Fig. 6.

Epoxygenase inhibition regulates monocyte and macrophage differentiation. Inflammation was initiated by zymosan (1 mg, i.p.), and mice were treated with vehicle control (PBS) or epoxI (30 mg/kg, i.p.) at 24 and 36 h. Cells were collected at 48 h and pooled from n = 9–18 mice. Ly6c+ monocytes, resident macrophages, and monocyte-derived macrophages were sorted on a LSR Fortessa as detailed in Materials and Methods. (A) Representative zebra plots of the cell populations from control and epoxI-treated mice expressing Ly6c and F4/80, with labeled populations representing A: Ly6c+ monocytes; B: resident macrophages (Resi-Mϕ); and C: monocyte-derived macrophages (Mono-Mϕ). (B) Representative dot plots of the Ly6c+ monocytes, resident macrophages, and monocyte-derived macrophages from control and epoxI-treated mice expressing PKH26 and CD11b (Upper) and CD64 and MerTK (Lower). (C) Changes in cell numbers, and expression (MFI in relative fluorescence units; RFUs) for F4/80, CD11b, MHCII, CD103, CD64, MerTK, and Timd4 in the Ly6c+, Resi-Mϕ, and Mono-Mϕ cell populations. Data are mean ± SEM from n = 5 mice per group; *P < 0.05 by unpaired t test.

Fig. 7.

Epoxygenase inhibition reduces the phagocytic activity of monocyte-derived and resident macrophages. Inflammation was initiated by zymosan (1 mg, i.p.), and mice were treated with vehicle control (PBS) or epoxI (30 mg/kg, i.p.) at 24 and 36 h. Cells were collected at 48 h and pooled from n = 9–18 mice. Ly6c+ monocytes, resident macrophages, and monocyte-derived macrophages were sorted on a FACSAria as detailed in Materials and Methods. Sorted cells were then tested for their ability to phagocytose CFSE-labeled apoptotic cells (thymocytes) or FITC-labeled zymosan BioParticles over 30 min. Cells were gated using F4/80 or Ly6c on an ImageStreamX Mark II. (A) ImageStream analysis differentiates cells that phagocytose apoptotic cells or zymosan (internalization score >0) from those where particles or bodies just stick to the cell (internalization score 0). (B) Ex vivo phagocytosis of apoptotic cells (Left) and zymosan (Right) by Ly6c+, Resi-Mϕ, and Mono-Mϕ cell populations from control and epoxI-treated mice. Data are mean ± SEM from n = 3–6 pooled samples; *P < 0.05 by unpaired t test.

Fig. 8.

Role of epoxy-oxylipins in the resolution phenotype. The resolution of zymosan-initiated inflammation involves monocytes, dendritic cells, and T- and B-cell recruitment and the differentiation of monocytes into resolution-type macrophages. In this process (as revealed by epoxI treatment and sEH−/− mice), epoxy-oxylipins, most likely EETs, limit Ccl2 and Ccr2 expression, Ly6chi monocyte accumulation, and T- and B-cell recruitment and encourage the formation of mature phagocytic resolution macrophages. Ly6chi monocytes, Ly6clo monocytes, dendritic cells, and monocyte-derived and resident macrophages are all activated in the presence of epoxI.