Modulation of arachidonic and linoleic acid metabolites in myeloperoxidase-deficient mice during acute inflammation

Abstract

Acute inflammation is a common feature of many life-threatening pathologies, including septic shock. One hallmark of acute inflammation is the peroxidation of polyunsaturated fatty acids forming bioactive products that regulate inflammation. Myeloperoxidase (MPO) is an abundant phagocyte-derived hemoprotein released during phagocyte activation. Here, we investigated the role of MPO in modulating biologically active arachidonic acid (AA) and linoleic acid (LA) metabolites during acute inflammation. Wild-type and MPO-knockout (KO) mice were exposed to intraperitoneally injected endotoxin for 24 h, and plasma LA and AA oxidation products were comprehensively analyzed using a liquid chromatography-mass spectrometry method. Compared to wild-type mice, MPO-KO mice had significantly lower plasma levels of LA epoxides and corresponding LA- and AA-derived fatty acid diols. AA and LA hydroxy intermediates (hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids) were also significantly lower in MPO-KO mice. Conversely, MPO-deficient mice had significantly higher plasma levels of cysteinyl-leukotrienes with well-known proinflammatory properties. In vitro experiments revealed significantly lower amounts of AA and LA epoxides, LA- and AA-derived fatty acid diols, and AA and LA hydroxy intermediates in stimulated polymorphonuclear neutrophils isolated from MPO-KO mice. Our results demonstrate that MPO modulates the balance of pro- and anti-inflammatory lipid mediators during acute inflammation and, in this way, may control acute inflammatory diseases.

Fig. 1. Increased production of EET and EpOME by PMNs isolated from wild-type compared to PMNs isolated from MPO-KO mice

Relative levels of EETs (AA epoxides) and EpOMEs (LA epoxides) in control and PMA/calcium ionophore activated PMNs incubated in the absence or the presence of AA and LA. Results are expressed as the percentage (mean ± SEM) of wild-type controls incubated in the absence of AA and LA and are derived from at least three samples (pooled from four or five animals). p < 0.05 MPO-KO vs. wild-type, #p < 0.05 control MPO-KO vs. LPS treated MPO-KO. Average values in control samples of WT mice 5(6)-EET - 4.72 nM; 8(9)-EET - 2.33 nM; 11(12)-EET - 2.02 nM; 9(10)-EpOME - 2.22 nM; and 12(13)-EpOME - 2.97 nM. Concentrations of 14(15)-EET were under detection limits in samples from both wild-type and MPO-KO mice. Limits of quantification are in Supplement Table 1.

Fig. 2. Increased production of DHET and DHOME by PMNs isolated from wild-type compared to PMNs isolated from MPO-KO mice

Relative levels of DHETs (AA dihydroxy metabolites) and DHOMEs (LA dihydroxy metabolites) in control and PMA/calcium ionophore activated PMNs incubated in the absence or the presence of AA and LA. Results are expressed as the percentage (mean ± SEM) of wild-type controls incubated in the absence of AA and LA. All results are derived from at least three samples (pooled from four or five animals). p < 0.05 MPO-KO vs. wild-type, +p < 0.05 control wild-type vs. LPS treated wild-type, #p < 0.05 control MPO-KO vs. LPS treated MPO-KO. Average values in control samples of WT mice 11,12-DHET - 0.10 nM;14,15-DHET - 0.09 nM; 9,10-DHOME - 0.10 nM; 12,13-DHOME - 0.22 nM. Concentrations of 5,6-DHET and 8,9-DHET were under detection limits in samples from both wild-type and MPO-KO mice. Limits of quantification are in Supplement Table 1.

Fig. 3. Increased production of HETE and HODE by PMNs isolated from wild-type compared to PMNs isolated from MPO-KO mice

Relative levels of AA- (HETEs) and LA-derived (HODEs) lipid metabolites in control and PMA/calcium ionophore activated PMNs incubated in the absence or the presence of AA and LA. Results are expressed as the percentage (mean ± SEM) of wild-type controls incubated in the absence of AA and LA and are derived from at least three samples (pooled from four or five animals). p < 0.05 MPO-KO vs. wild-type, +p < 0.05 control wild-type vs. LPS treated wild-type, #p < 0.05 control MPO-KO vs. LPS treated MPO-KO. Average values in control samples of WT mice 5-HETE - 17.72 nM, 8-HETE - 0.1 nM, 9-HETE - 0.1 nM, 9 HODE - 73.07 nM, and 13 HODE - 65.76 nM. Concentrations of 11-HETE, 12-HETE, 15-HETE, 19-HETE, and 20-HETE were under detection limits in samples from both wild-type and MPO-KO mice. Limits of quantification are in Supplement Table 1.

Fig. 4. Increased production of oxo ODEs and oxo EETs by PMNs isolated from wild-type compared to PMNs isolated from MPO-KO mice

Relative levels of oxo ODEs (LA degradation products) and oxo-EETs (AA degradation products) in control and PMA/calcium ionophore activated PMNs incubated in the absence or the presence of AA and LA. Results are expressed as the percentage (mean ± SEM) of wild-type controls incubated in the absence of AA and LA and are derived from at least three samples (pooled from four or five mice). p < 0.05 MPO-KO vs. wild-type, +p < 0.05 control wild-type vs. LPS treated wild-type, #p < 0.05 control MPO-KO vs. LPS treated MPO-KO. Average values in control samples of WT mice 9-oxo-ODE - 10.27 nM, 13-oxo-ODE - 20.62 nM, 5-oxo-EET - 2.11 nM, 15-oxo-EET - 5.75 nM. Limits of quantification are in Supplement Table 1.