Vitamin D deficiency (VitD-) is commonly encountered in patients with rheumatoid arthritis (RA) (and other autoimmune diseases). Some studies show VitD levels impact expression of RA, but the mechanism(s) have not been well explored and have received scant attention in rodent models of RA. Interestingly, like VitD levels, in studies looking at only plasma gelsolin (pGSN), it was observed that pGSN levels also inversely correlate with C-reactive protein (CRP) in patients with RA. Gelsolin (GSN) has been shown to be protective in the TNFα transgenic (tg) mouse model of RA. It is known that sphingosine 1-phosphate (S1P) (acting through G protein- coupled receptors on T cells, B cells, monocytes/macrophages, and dendritic cells) affect immune and inflammatory responses and has been found to be elevated in RA synovial fluid but not osteoarthritis synovial fluid. Furthermore, pGSN binds to S1P, thereby regulating its ability to engage S1P receptors. We found humans, in addition to converting vitamin D3 (VitD3) to 25(OH)D3, also generate a noncalcemic analog, 20(OH)D3, with a serum concentration 1/20th of that of 25(OH)D3. In the type II collagen-induced arthritis (CIA), we found surprisingly that 20(OH)D3 treatment elevates pGSN levels and increases S1P levels in splenocyte cultures from these 20(OH)D3-treated mice. We discovered that supplementing VitD in humans elevates pGSN in sera and that 25(OH)D levels in normals and in RA and OA correlate fairly well with pGSN. Also, we found both 20(OH)D3 and 1,25(OH)2D3 increased sphingosine kinase (Sphk)2 mRNA in mouse splenocytes and increased GSN mRNA in cultured mouse myoblasts and pGSN production by explant cultures of mouse skeletal muscle. Knocking down GSN or Sphk2 worsens arthritis in mouse models of RA. Both GSN and Sphk2 regulate S1P levels in plasma or lymphoid tissue, which is important in immune function, including lymphocyte trafficking, Th1/Th2/Th17 cytokine expression, and control of FoxP3 regulatory T cells (Tregs). No studies have evaluated levels of 25(OH)D, S1P, and pGSN in the same RA population. We find 25(OH)D serum levels correlate fairly well with pGSN levels in RA patients, although concordance is not 100%. We further hypothesize that RA patients who have both high 25(OH)D and pGSN will have less RA disease activity score, while those with low pGSN and low 25(OH)D will have higher RA disease activity scores. Our overarching hypothesis is that there are interactions amongst VitD, GSN, and CD4+ T cell Sphk2, CD4+ T cell S1P receptor subtypes, and/or S1P that may be important in the modulation of autoimmunity and inflammatory arthritis. Whether administering VitD to humans with VitD deficiency/insufficiency will raise pGSN levels and modulate S1P, is unknown, and is the topic of Specific Aim 3. The three highly translational Specific Aims of this proposal are the following: Specific Aim 1A: Assess the association of serum levels of 25(OH)D, pGSN, and S1P separately, in patients with RA ...