Haha! Thanks, Lindsey!

Thanks, Niv!

I’m so happy to see this out! ☺️ I’ve been working on this >6 years - many setbacks along the way, but they've made getting this published all the more rewarding - perseverance is worth it! 💪

Very thankful for the support of @psalmotoxin.bsky.social, @mayadannawi.bsky.social & others! 🧵[10/10]

Taken together, we postulate that GPR65 on FLS orchestrates inflammatory joint pain through the release of mediators and subsequent crosstalk between neurons and immune cells. Thus inhibiting GPR65 might represent a means of relieving inflammatory pain. 🧵[9/10]

Increased expression of GPR65 has previously been reported in human osteoarthritis (OA). BTB also stimulated increases in pro-inflammatory cytokine production by FLS isolated from human OA patients. Further, synovial fluid samples from OA patients could activate GPR65. 🧵[8/10]

The role of GPR65 was confirmed by the inability of BTB to increase inflammatory mediator secretion by GPR65 knockout (KO) FLS, and lack of inflammation and pain-like behaviours when GPR65 KO mice were injected with BTB. 🧵[7/10]

…suggesting another cell type might be involved. Fibroblast-like synoviocytes (FLS), which line the knee joint, express GPR65. Stimulating FLS with BTB increased secretion of inflammatory cytokines, which could increase the excitability of naïve neurons. 🧵[6/10]

To investigate the cellular basis of the pain-like behaviours seen in mice, we explored whether BTB increased neuronal excitability. While neurons isolated from BTB-injected mice were hyperexcitable, we could not recreate this by incubating naïve neurons with BTB alone… 🧵[5/10]

Next, we injected BTB to the knee joint of mice. This caused both inflammation and an increase in pain-like behaviours, suggesting that activation of GPR65 in the knee joint is sufficient to drive inflammatory pain. 🧵[4/10]

…So, we first compared the signalling signatures of protons, BTB and psychosine (all reported to activate GPR65). We found that BTB recapitulated most features of proton-mediated signalling. Importantly, BTB was also shown to only activate GPR65 over other PS-GPCRs. 🧵[3/10]

Inflammatory conditions, like arthritis, are both painful and associated with acidosis. Thus we investigated if acidosis drives pain through GPR65 (a proton-sensing GPCR) - this isn’t an easy task though, as there are lots of receptors that can be activated by protons… 🧵 [2/10]