Lipoxygenase-derived oxylipins are enriched in anti-citrullinated protein antibody (ACPA)-positive individuals at risk for developing rheumatoid arthritis

Abstract

Background: Rheumatoid arthritis (RA) is typically preceded by an extended preclinical period where circulating autoantibodies, particularly anti-citrullinated protein antibodies (ACPA), are detectable in the absence of clinical arthritis. Increased dietary intake of anti-inflammatory omega-3 (ω3) polyunsaturated fatty acids (PUFA) has been shown to be associated with a lower the risk of developing incident RA in large epidemiological studies. It is currently not known how changes in fatty acid (FA) metabolism may impact on the progression towards RA in at-risk individuals. To begin to address this question, we profiled serum FAs and oxylipins in an established cohort of at-risk ACPA-positive first-degree relatives (FDR) of RA patients (N = 31), some of whom developed RA (N = 4), and compared their profile to ACPA-negative FDR from the same population (N = 10).

Methods: Gas chromatography (GC) was used for FA quantitation. Oxylipins were extracted and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS).

Results: Although we did not detect any meaningful differences in overall FA content between ACPA + and ACPA - FDR, the levels of oxylipins derived from FA metabolism demonstrated significant differences between the two groups, with the ACPA + group demonstrating enrichment in circulating arachidonic acid- and eicosapentaenoic acid-derived molecules. Compared with the ACPA - FDR group, the ACPA + FDR, including those who progressed into inflammatory arthritis, displayed higher levels of LOX-derived oxylipins.

Conclusion: ACPA seropositivity in otherwise unaffected individuals at-risk for developing future RA based on family history (FDR) is associated with alterations in the serum oxylipin profile that suggests dysregulated LOX activity.

Keywords: Fatty acids; Oxylipins; PUFA; Polyunsaturated fatty acids; Preclinical RA; Rheumatoid arthritis; ω-3 fatty acids; ω-6 fatty acids.

Fig. 1

Distribution of fatty acids and differences in ACPA + and ACPA − FDR. The samples used for this analysis were collected in/after 2013. A Bar plots showing relative proportion of unsaturated (UFA) and saturated (SFA) fatty acids in all samples. B Bar plot showing relative proportions of mono-unsaturated (MUFA), polyunsaturated (PUFA), and omega-3, omega-6, omega-9, and omega-5 fatty acids. C Volcano plot of differentially expressed FAs between ACPA + and ACPA − FDR. Analyzed by pairwise Wilcoxon rank sum test and adjusted for multiple comparisons. Significant FAs are annotated on the graph (erucic acid = C22_1, eicosatetraenoic acid = C20_3n3). D The ratio between omega-6 (ω6) and omega-3 (ω3) FA in ACPA − and ACPA + FDR. Differences determined with Wilcoxon rank-sum test

Fig. 2

Differences in oxylipin profiles in various pre-clinical RA stages. (A) Oxylipins derived from arachidonic Acid (AA), linoleic Acid (LA), dihomo-y-linolenic acid (DGLA), alpha-linolenic acid (ALA), eicosapentaenoic Acid (EPA) and Docosahexaenoic acid (DHA) in ACPA- FDR and ACPA+ FDR. Differences determined with Wilcoxon rank-sum test. (B) Ordered oxylipins by log2 fold change (vs ACPA- FDR) colored by derived fatty acid (legend). (C) Log2 fold change in Progressors (vs ACPA- FDR) or ACPA+ FDR (vs ACPA- FDR) categorized by log2 fold change > 1.5 (dotted lines). Pie charts are representative of the proportion of oxylipins based on the FA from which they are derived. Differences in proportion measured with chi-square test. The ratio between AA-derived oxylipins and EPA-derived oxylipins in ACPA+ FDR and ACPA+ Progressors. Differences determined with Wilcoxon rank-sum test

Fig. 3

LOX-derived oxylipins are increased in ACPA+ FDR and ACPA+ Progressors. (A) Omega-6 (ω6) and omega-3 (ω3) oxylipins derived from LOX (lipoxygenase), COX (cyclooxygenase), or CYP (cytochrome P450) pathways in ACPA- FDR, ACPA+ FDR and ACPA+ Progressor. Differences determined with Wilcoxon rank-sum test. Trend lines are drawn through the median values for each group (B) Arachidonic acid lipoxygenase pathway annotated by known steps of synthesis to derive oxylipins. Undetected oxylipins are grey, while detected are colored in black. Within each cell, there is a color gradient from left to right, based on the expression relative in ACPA+ FDR (left) and ACPA+ Progressors (right) relative to controls (log2 fold change)