New pro-resolving n-3 mediators bridge resolution of infectious inflammation to tissue regeneration

Abstract

While protective, the acute inflammatory response when uncontrolled can lead to further tissue damage and chronic inflammation that is now widely recognized to play important roles in many commonly occurring diseases, such as cardiovascular disease, neurodegenerative diseases, metabolic syndrome, and many other diseases of significant public health concern. The ideal response to initial challenges of the host is complete resolution of the acute inflammatory response, which is now recognized to be a biosynthetically active process governed by specialized pro-resolving mediators (SPM). These chemically distinct families include lipoxins, resolvins, protectins and maresins that are biosynthesized from essential fatty acids. The biosynthesis and complete stereochemical assignments of the major SPM are established, and new profiling procedures have recently been introduced to document the activation of these pathways in vivo with isolated cells and in human tissues. The active resolution phase leads to tissue regeneration, where we've recently identified new molecules that communicate during resolution of inflammation to activate tissue regeneration in model organisms. This review presents an update on the documentation of the roles of SPMs and the biosynthesis and structural elucidation of novel mediators that stimulate tissue regeneration, coined conjugates in tissue regeneration. The identification and actions of the three families, maresin conjugates in tissue regeneration (MCTR), protectin conjugates in tissue regeneration (PCTR), and resolvin conjugates in tissue regeneration (RCTR), are reviewed here. The identification, structural elucidation and the pathways and biosynthesis of these new mediators in tissue regeneration demonstrate the host capacity to protect from collateral tissue damage, stimulate clearance of bacteria and debris, and promote tissue regeneration via endogenous pathways and molecules in the resolution metabolome.

Figure 1. SPM biosynthetic pathways and substrate mobilization

(A) Human SPM biosynthesis. 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. Biosynthesis of D-series resolvins and Protectins are initiated by molecular oxygen insertion at carbon-17 position of DHA. Maresins are produced via initial lipoxygenation at carbon-14 position. The stereochemistry of each bioactive SPM is established, and SPM biosynthesis in human cells and tissues confirmed. (B) Substrate mobilization. In murine inflammation, edema carries substrate DHA and EPA from circulating blood to local inflamed site for SPM production. In human neural tissues, phospholipids are the source of DHA, which is released by cPLA2 for PD1 formation. In lymph nodes, sPLA₂G2D releases substrate to produce RvD1 and PD1 that reduce inflammation in the skin.

Figure 1. SPM biosynthetic pathways and substrate mobilization

(A) Human SPM biosynthesis. 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. Biosynthesis of D-series resolvins and Protectins are initiated by molecular oxygen insertion at carbon-17 position of DHA. Maresins are produced via initial lipoxygenation at carbon-14 position. The stereochemistry of each bioactive SPM is established, and SPM biosynthesis in human cells and tissues confirmed. (B) Substrate mobilization. In murine inflammation, edema carries substrate DHA and EPA from circulating blood to local inflamed site for SPM production. In human neural tissues, phospholipids are the source of DHA, which is released by cPLA2 for PD1 formation. In lymph nodes, sPLA₂G2D releases substrate to produce RvD1 and PD1 that reduce inflammation in the skin.

Figure 2. Main Actions of SPM in the Innate Immune System: Pro-Resolution and Anti-Inflammation Are Not Equivalent Mechanisms

The actions of SPM on innate immune system have been demonstrated with phagocytes including limiting PMN and stimulating macrophage functions. SPM also directly act on the adaptive immune cells, including T cells and B cells (see text for detail).

Figure 3. Demonstrations of SPM Production in Humans

(A) Illustration of functional metabololipidomics: human SPM production and assessment of their function. At time zero, individuals all ingested 1 gram of omega-3, containing 50% EPA and 20% DHA. At 2h, they took low-dose aspirin (81 mg). At 4h, blood was collected to carry out LM-metabololipidomics together with PCA analysis. In parallel, whole blood from the same subjects were used for functional assessment with phagocytosis. Whole blood was incubated with fluorescent-labeled E. coli ex vivo, and phagocyte ingestion of E. coli measured by flow cytometry. A cluster of SPM was elevated with acute n-3 and ASA intake, and correlated with increased phagocyte function in whole blood. This approach provides a tool for functional metabololipidomics. (B) Principal Component Analysis: Mastitis human milk gives altered LM-SPM profiles with higher levels of PG and LT, while healthy human milk contains higher amounts of SPM, including LX, Rv, PD and MaR1.

Figure 3. Demonstrations of SPM Production in Humans

(A) Illustration of functional metabololipidomics: human SPM production and assessment of their function. At time zero, individuals all ingested 1 gram of omega-3, containing 50% EPA and 20% DHA. At 2h, they took low-dose aspirin (81 mg). At 4h, blood was collected to carry out LM-metabololipidomics together with PCA analysis. In parallel, whole blood from the same subjects were used for functional assessment with phagocytosis. Whole blood was incubated with fluorescent-labeled E. coli ex vivo, and phagocyte ingestion of E. coli measured by flow cytometry. A cluster of SPM was elevated with acute n-3 and ASA intake, and correlated with increased phagocyte function in whole blood. This approach provides a tool for functional metabololipidomics. (B) Principal Component Analysis: Mastitis human milk gives altered LM-SPM profiles with higher levels of PG and LT, while healthy human milk contains higher amounts of SPM, including LX, Rv, PD and MaR1.

Figure 4

Resolvin E. coli Infectious Exudate Isolates Contain New Molecules that Promote Tissue Regeneration. To determine whether new signals were produced during self-limited infections chemical isolates were tested for their regeneration capacity (Left panel). Addition of these molecules to surgically injured planaria accelerated tissue regeneration by approximately 1 day, an action that was comparable to that displayed by the proresolving mediators MaR1.

Figure 5

Structure Elucidation of the Maresin Conjugates in Tissue Regeneration: Evidence for MCTR1 and MCTR2. Using several lines of evidence to interrogate the physical properties of the bioactive structures isolated from self-resolving infectious exudates, that included the incorporation of radiolabeled DHA, deuterium incorporation and methyl-ester derivatives as well as Rainey Nickel desulfurization and UV chromophores we established that the molecules carried a DHA backbone with conjugated triene double bond systems that was allylic to a peptide containing an auxochrome such as sulfur. Using these lines of evidence the structures were assigned as 13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid for MCTR1, and 13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid for MCTR2. These mediators are evolutionary conserved and their production was established in organisms as diverse as planaria, mice and humans.

Figure 6

MCTR Biosynthesis and Structures. The MCTR biosynthetic pathway is initiated by lipoxygenation of DHA at carbon position 14 leading to 14S-hydro(peroxy)-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid, this is converted by lipoxygenase activity to 13S,14S-epoxy −4Z,7Z,9E,11E,16Z,19Z- docosahexaenoic acid. Conversion of this allylic epoxide is to MCTR1 is catalyzed by either glutathione s-transferase mu4 (GSTM4) and by leukotriene C4 synthase (LTC4S). MCTR1 is converted by gammaglutamyl transferase (GGT) to MCTR2 that in turn is substrate for conversion by dipeptidase (DPEP) to MCTR3.

Figure 7. RCTR Biosynthesis and Structures

15-LOX type 1 is the initiating enzyme in the RCTR pathway which catalyzes the formation of 17S-hydro(peroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid via subsequent lipoxygenase activity this substrate is converted to 7, 8-epoxy, 17S-hydroxy-4Z,9,11,13,15,19Z-docosahexaenoic acid that is precursor to RCTR1. This mediator is converted to RCTR2 that in turn produces RCTR3.

Figure 8. PCTR Biosynthesis and Structures

Formation of 17S-hydro(peroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid from DHA by 15-LOX type 1 initiates the PCTR biosynthetic pathway. This hydroperoxide is converted to 16S,17S-epoxy-7Z,10Z,13E,14E,19Z- docosahexaenoic acid, this allylic epoxide is precursor to PCTR1, that is transformed to PCTR2 that is in turn precursor to PCTR3.