PGE2 and other lipids in rheumatic diseases

Despite numerous options for treatment of rheumatic diseases, there is an unfulfilled clinical need for therapeutic strategies that can reduce inflammation and prevent tissue destruction. Lipid mediators (eicosanoids and fatty acids (FA)) are involved in the regulation of inflammatory processes and contribute to the pathogenesis of rheumatic diseases. Thus, selective targeting of the lipid mediators might enable improved antiinflammatory treatment. Microsomal prostaglandin synthase (mPGES) -1 produces prostaglandin E2 (PGE2) at sites of inflammation in rheumatic diseases. Inhibitors of mPGES-1 have been proposed as a more selective anti-inflammatory treatment retaining the therapeutic potential of non-steroidal anti-inflammatory drugs (NSAIDs) but with less severe side effects associated with NSAIDs. However, the impact of mPGES-1 inhibition on different pathological and physiological processes is not completely elucidated. Moreover, chronic inflammation might cause dysregulation of lipid and FA metabolism that may contribute to skeletal muscle weakness in patients with polymyositis (PM) and dermatomyositis (DM). The major aim of this thesis was to gain better understanding of the regulation of PGE2 and other lipid mediators in RA, PM and in DM to improve treatment of patients.

First, we have determined the catalytic mechanism of mPGES-1 activity by site-directed mutagenesis (Paper I). The amino acid residues arginine (Arg) 126 and aspartate (Asp) 49 were identified as essential for the catalytic activity of mPGES-1, as when exchanged, the enzyme variants lost their enzymatic activity. Previous high-resolution structural studies predicted a role for serine (Ser) 127 in the enzymatic activity of mPGES-1. In contrast, we have demonstrated that Ser127, as well as Arg73, do not seem to be significant to the catalytic mechanism because when exchanged, their variants retained considerable activity. These results are of relevance for the development of the new generation of mPGES-1 inhibitors.

Further, we studied whether mPGES-1 deletion might be beneficial for reducing inflammation via the suppression of platelet functions (Paper II). Platelet activation, the formation of platelet-leukocyte aggregates, and release of platelet-derived microparticles (PMP) were significantly reduced in mPGES-1 KO mice compared to WT after lipopolysaccharide (LPS) treatment. In addition, KO mice displayed a significant decrease in platelet aggregation ex vivo. The reduced activation of platelets may contribute to antiinflammatory effect and cardiovascular safety of mPGES-1 inhibitors.

In Paper III, we investigated effects of mPGES-1, PGIS, and cyclooxygenase (COX) -2 on vascular and renal pathways associated with asymmetric dimethylarginine (ADMA) and endothelial nitric oxide synthase (eNOS). WT mice treated with COX-2 inhibitor displayed no change in the plasma levels of cardioprotective prostacyclin (PGI2), while mPGES-1 KO mice showed significantly higher PGI2 levels in the plasma. In contrast to COX-2 inhibition, mPGES-1 deletion had no effect on genes responsible for the production or breakdown of ADMA in the kidney. Plasma creatinine and ADMA were elevated in mice treated with COX-2 inhibitor or PGIS KO mice but unaltered in mPGES-1 KO mice. Furthermore, the deletion of mPGES-1 significantly improved the eNOS-driven dilator response to acetylcholine in the aorta. These data further confirmed the cardioprotective effects of mPGES-1 deletion suggesting selective inhibitors of mPGES-1 as a safer alternative to NSAIDs.

To clarify mechanisms involved in muscle weakness, we examined effects of the conventional immunosuppressive treatment on global gene expression profiles in skeletal muscle from PM and DM patients (Paper IV). The genes related to immune response and inflammation including the interferon and the inflammasome pathways were downregulated by treatment. The genes involved in muscle tissue remodeling and growth were negatively affected by treatment. The immunosuppressive treatment caused an induction of gene markers of fast type II fibers. Furthermore, the fiber composition of the muscle tissue from patients was switched towards type II fibers after treatment. Importantly, the expression of genes involved in lipid metabolism was altered, signifying a probable lipotoxic effect on muscles, that at least partly might explain the persistent muscle weakness and fatigue observed in PM and DM patients despite treatment.

To confirm dysregulated lipid metabolism in myositis patients, we analyzed lipid and FA profiles in serum from patients with PM and DM in comparison to healthy individuals and response to immunosuppressive treatment (Paper V). FA composition of total serum lipids was changed in myositis patients compared to healthy individuals. In myositis patients, the levels of palmitic 16:0 acid was significantly higher while the levels of arachidonic 20:4(n-6) acid was significantly lower. The levels of serum lipid species within phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and triglycerides (TG) were also significantly changed in myositis patients compared to healthy individuals. Immunosuppressive treatment resulted in increased serum levels of C20:2(n-6) acid and C20:5(n-3) acids as well as in the changed serum PC, phosphatidylethanolamine (PE) and LPC profiles in myositis patients.

In conclusion, in this thesis, we have provided new knowledge on the catalytic mechanism and the impact of mPGES-1 on inflammation and cardiovascular safety. Furthermore, we have demonstrated that lipid metabolism is altered in PM and DM patients and might contribute to disease pathogenesis.

List of scientific papers

I. Arg126 and Asp49 are essential for the catalytic function of microsomal prostaglandin E2 synthase 1 and Ser127 is not. Joan Raouf, Nazmi Rafique, Michael Christopher Goodman, Helena Idborg, Filip Bergqvist, Richard N. Armstrong, Per-Johan Jakobsson, Ralf Morgenstern, Linda Spahiu. PLoS ONE. 2016.
https://doi.org/10.1371/journal.pone.0163600

II. mPGES-1 deletion affects platelet functions in mice. Joan Raouf, Fariborz Mobarrez, Karin Larsson, Per-Johan Jakobsson, Marina Korotkova. Clin Sci (Lond). 2016 Oct 7. pii: CS20160463.
https://doi.org/10.1042/CS20160463

III. mPGES-1 deletion increases prostacyclin and evades the elevated systemic ADMA associated with COX-2 inhibitors: relevance to cardiovascular safety of mPGES-1 inhibitors. Nicholas S. Kirkby, Joan Raouf, Blerina Ahmetaj-Shala, Bin Liu, Sarah I. Mazi MRes, Matthew L. Edin, Marina Korotkova, Darryl C. Zeldin, Yingbi Zhou, Per-Johan Jakobsson, Jane A. Mitchell. [Manuscript]

IV. Effects on muscle tissue remodeling and lipid metabolism in muscle tissue from adult patients with polymyositis or dermatomyositis treated with immunosuppressive agents. Ingela Loell, Joan Raouf, Yi-Wen Chen, Rongye Shi, Inger Nennesmo, Helene Alexanderson, Maryam Dastmalchi, Kanneboyina Nagaraju, Marina Korotkova, Ingrid E. Lundberg. Arthritis Research & Therapy. 2016 Jun 10; 18: 136.
https://doi.org/10.1186/s13075-016-1033-y

V. Serum lipid and fatty acid profiles are altered in patients with polymyositis or dermatomyositis. Joan Raouf, Helena Idborg, Petter Olsson, Helene Alexanderson, Maryam Dastmalchi, Per-Johan Jakobsson, Ingrid E Lundberg, Marina Korotkova. [Submitted]