Structural elucidation and physiologic functions of specialized pro-resolving mediators and their receptors

Abstract

The acute inflammatory response is host-protective to contain foreign invaders. Many of today's pharmacopeia that block pro-inflammatory chemical mediators can cause serious unwanted side effects such as immune suppression. Uncontrolled inflammation is now considered a pathophysiologic basis associated with many widely occurring diseases such as cardiovascular disease, neurodegenerative diseases, diabetes, obesity and asthma, as well as the classic inflammatory diseases, e.g. arthritis, periodontal diseases. The inflammatory response is designated to be a self-limited process that produces a superfamily of chemical mediators that stimulate resolution of inflammatory responses. Specialized proresolving mediators (SPM) uncovered in recent years are endogenous mediators that include omega-3-derived families resolvins, protectins and maresins, as well as arachidonic acid-derived (n-6) lipoxins that stimulate and promote resolution of inflammation, clearance of microbes, reduce pain and promote tissue regeneration via novel mechanisms. Here, we review recent evidence from human and preclinical animal studies, together with the structural and functional elucidation of SPM indicating the SPM as physiologic mediators and pharmacologic agonists that stimulate resolution of inflammation and infection. These results suggest that it is time to develop immunoresolvents as agonists for testing resolution pharmacology in nutrition and health as well as in human diseases and during surgery.

Keywords: Inflammation; Leukocytes; Omega-3 PUFA; Resolvins; maresins; protectins.

Figure 1. The ideal outcome of inflammation: complete systems approach to mapping resolution

Injury, infection or surgery initiate acute inflammation that is normally a host-protective mechanism. First event in acute inflammation is edema formation, followed by infiltration of PMN, and then monocyte and macrophages that clear PMN leading to resolution. Using the systems approach to map resolution, we demonstrated temporal biosynthesis of SPM in the resolution phase of self-limited inflammation. These SPM are a super-family of endogenous mediators, first identified in resolving exudates. They promote resolution of inflammation, wound healing and reduce organ fibrosis, leading to homeostasis. Based on these findings, we proposed the sign of resolution as listed. Identification and structure elucidation of these SPM provided the first evidence that resolution of inflammation is an active process.

Figure 2. SPM Biosynthetic Routes

(A) Biosynthesis of E-series resolvins is initiated with molecular oxygen insertion at carbon-18 position of EPA, which is converted to bioactive E-series members resolvin E1, resolvin E2 and resolvin E3. (B) Resolution metabolome also activates 17-lipoxygenation of DHA; 17S-HpDHA is converted to resolvin-epoxide intermediates by the leukocyte 5-lipoxygenase that are transformed to resolvins D1-D6, which each carry potent actions. (C) 17-HpDHA is also precursor to 16,17-epoxide-protectin intermediate that is converted to protectin D1/neuroprotectin D1 and related protectins. Maresins are produced by macrophages via initial lipoxygenation at carbon-14 position by lipoxygenation and insertion of molecular oxygen, producing a 13S,14S-epoxide-maresin intermediate that is enzymatically converted to maresin family members. The stereochemistry of each bioactive SPM is established, and SPM biosynthesis in murine exudates and human tissues confirmed. See refs. (Serhan and Petasis, 2011; Winkler et al., 2016; Aursnes et al., 2015; Tungen et al., 2014; Dalli et al., 2016b) for original reports, total organic synthesis and stereochemical assignments and the text for further details.

Figure 2. SPM Biosynthetic Routes

(A) Biosynthesis of E-series resolvins is initiated with molecular oxygen insertion at carbon-18 position of EPA, which is converted to bioactive E-series members resolvin E1, resolvin E2 and resolvin E3. (B) Resolution metabolome also activates 17-lipoxygenation of DHA; 17S-HpDHA is converted to resolvin-epoxide intermediates by the leukocyte 5-lipoxygenase that are transformed to resolvins D1-D6, which each carry potent actions. (C) 17-HpDHA is also precursor to 16,17-epoxide-protectin intermediate that is converted to protectin D1/neuroprotectin D1 and related protectins. Maresins are produced by macrophages via initial lipoxygenation at carbon-14 position by lipoxygenation and insertion of molecular oxygen, producing a 13S,14S-epoxide-maresin intermediate that is enzymatically converted to maresin family members. The stereochemistry of each bioactive SPM is established, and SPM biosynthesis in murine exudates and human tissues confirmed. See refs. (Serhan and Petasis, 2011; Winkler et al., 2016; Aursnes et al., 2015; Tungen et al., 2014; Dalli et al., 2016b) for original reports, total organic synthesis and stereochemical assignments and the text for further details.

Figure 2. SPM Biosynthetic Routes

(A) Biosynthesis of E-series resolvins is initiated with molecular oxygen insertion at carbon-18 position of EPA, which is converted to bioactive E-series members resolvin E1, resolvin E2 and resolvin E3. (B) Resolution metabolome also activates 17-lipoxygenation of DHA; 17S-HpDHA is converted to resolvin-epoxide intermediates by the leukocyte 5-lipoxygenase that are transformed to resolvins D1-D6, which each carry potent actions. (C) 17-HpDHA is also precursor to 16,17-epoxide-protectin intermediate that is converted to protectin D1/neuroprotectin D1 and related protectins. Maresins are produced by macrophages via initial lipoxygenation at carbon-14 position by lipoxygenation and insertion of molecular oxygen, producing a 13S,14S-epoxide-maresin intermediate that is enzymatically converted to maresin family members. The stereochemistry of each bioactive SPM is established, and SPM biosynthesis in murine exudates and human tissues confirmed. See refs. (Serhan and Petasis, 2011; Winkler et al., 2016; Aursnes et al., 2015; Tungen et al., 2014; Dalli et al., 2016b) for original reports, total organic synthesis and stereochemical assignments and the text for further details.

Figure 3. SPM receptors: Pro-resolving GPCRs

(A) Specific SPM receptors in human and mouse. LXA₄ and RvD1 both activate ALX receptor. RvD1 also activates DRV1, which was an orphan receptor GPR32 with human cells. RvE1 functions as an agonist for ChemR23/ERV and an antagonist for LTB₄ receptor BLT1 on PMN. RvD2 activates DRV2/GPR18 to control phagocyte functions in human and mouse. These receptors contribute to the SPM’s pro-resolving actions on select cell types and also in vivo as demonstrated using transgenic and KO mice for ALX, ERV, BLT1 and DRV2. (B) RvD2-GPR18/DRV2 resolution axis. A novel RvD2 receptor, namely DRV2/GPR18, was identified by GPCR screening. RvD2 directly binds to and activates recombinant DRV2. RvD2 activates endogenous human and mouse GPR18 to stimulate macrophage and PMN functions. In addition, RvD2 accelerates resolution of bacterial infections, improves survival in sepsis, and gives organ protection in sterile injury. These actions were diminished in DRV2 KO mice.

Figure 3. SPM receptors: Pro-resolving GPCRs

(A) Specific SPM receptors in human and mouse. LXA₄ and RvD1 both activate ALX receptor. RvD1 also activates DRV1, which was an orphan receptor GPR32 with human cells. RvE1 functions as an agonist for ChemR23/ERV and an antagonist for LTB₄ receptor BLT1 on PMN. RvD2 activates DRV2/GPR18 to control phagocyte functions in human and mouse. These receptors contribute to the SPM’s pro-resolving actions on select cell types and also in vivo as demonstrated using transgenic and KO mice for ALX, ERV, BLT1 and DRV2. (B) RvD2-GPR18/DRV2 resolution axis. A novel RvD2 receptor, namely DRV2/GPR18, was identified by GPCR screening. RvD2 directly binds to and activates recombinant DRV2. RvD2 activates endogenous human and mouse GPR18 to stimulate macrophage and PMN functions. In addition, RvD2 accelerates resolution of bacterial infections, improves survival in sepsis, and gives organ protection in sterile injury. These actions were diminished in DRV2 KO mice.

Figure 4. Steps to Human Translation: Structure and functional elucidation of SPM

Illustration of the key steps on structures and function of SPM and steps towards human translation. SPM were first isolated from resolving exudates, and reduced PMN infiltration and transmigration; their structures were elucidated and the biosynthesis was reconstructed with human cells. Their structures were then confirmed using synthetic material for matching and stereochemistry assignments. SPM structures proved to be highly conserved and are produced not only in mouse and human tissues but also in fish and planaria. Their actions on the single-cell level were demonstrated using microfluidic chamber with only one drop of human blood. In addition, we established bioactions in vivo animal models and define resolution indices. SPM production was documented in human cells, tissues and organs using mass spectrometry-based profiling. Criteria for pro-resolving mediators are listed in the box. These steps focusing on structure and function elucidation of SPM provided molecular basis for resolution pharmacology towards human translation.