Background/Purpose: Gout is characterized by deposition of monosodium urate (MSU) crystals in articular joints, where they activate macrophages inducing NLRP3 inflammasome activation and bioactive IL-1β release. However, it is not fully understood how the initiation of gouty inflammation takes place. Intake of meat and seafood is associated with frequency and intensity of gout flares. Choline is a vitamin-like essential nutrient, mainly found in meat and seafood. Choline transporter CTL1, and Choline Kinase (ChoKα), enzyme that converts choline into phosphocholine, are highly expressed in macrophages and fibroblast like synoviocytes in inflamed joints. Here we study whether choline uptake and phosphorylation contribute to NLRP3 inflammasome activation and gouty inflammation.

Methods: Wild type or AMPKα1 knockout primary bone marrow derived macrophages (BMDM) were cultured in L929 media for 7 days. shCtrl, shCTL1 and shChoKα were generated by lentiviral infection. Choline and phosphocholine were measured by ¹HMRS. Choline deficiency was studied using media without or with 3.54μM choline. ChoKα inhibitor RSM932A was used at 5μM and colchicine at 10nM. BMDM were treated with 100ng/ml LPS for 4h and 400mg/ml MSU for 3h. IL-1β was measured by ELISA. Protein levels were examined by immunoblot, and mRNA expression by QPCR. Mitophagy was defined by p62, LC3 or DRP1 recruitment to mitochondria by confocal microscopy (CM). MSU crystals-induced peritonitis was induced by 3mg/ml MSU crystals i.p. injection (n=4 mice/group). For synovium-like air pouch model, 2.5mg/Kg ChoKα inhibitor MN58b or PBS was injected 24h before adding 3mg/ml MSU crystals into the air pouch (n=6 mice/group). Cells from pouch fluids were counted; pouch tissue stained for F4/80, CTL1 and ChoKα and visualized by CM.

Results:

LPS increased 6 fold CTL1 expression, and boosted intracellular choline by 238% (p<0.01) and phosphocholine by 559% (p<0.001). Choline deficiency reduced MSU-induced IL-1β release by 60% (p<0.001), shCTL1 by 57% (p<0.05), and shChoKα by 89% (p<0.05) and ChoKα inhibition via RSM932A by 71% (p<0.005). Impaired choline uptake or ChoKα activity promoted AMPK activation and DRP1, LC3 and p62 recruitment to mitochondria. AMPK deletion blocked choline deficiency effect on IL-1β release. Of note, treatment with colchicine, previously defined to inhibit NLRP3 inflammasome activity and activate AMPK, also reduced CTL1 (90%) and ChoKα levels (85%). In vivo, after MSU crystal injection, cells collected from peritoneal cavity strongly expressed both CTL1 (p<0.05) and ChoKα (p<0.01). F4/80 positive myeloid cells recruited into the air pouch also expressed ChoKα and CTL1. Last, treatment with ChoKα inhibitor MN58b reduced MSU crystal-induced leukocyte recruitment by 47% (p<0.05) and IL-1β release by 66% (p<0.01).

Conclusion: Our results show that Choline, which is mainly sourced from diet, contributes to gouty inflammation. MSU crystal-induced activation of myeloid cells causes overexpression of genes involved in choline uptake and metabolism, and this is prevented by Colchicine treatment. Targeting choline transporter CTL1 or ChoKα, and limiting dietary choline, could be novel therapeutic anti-inflammatory approaches for gouty arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-choline-in-gouty-inflammation/