20 S-Hydroxyvitamin D3, a Secosteroid Produced in Humans, Is Anti-Inflammatory and Inhibits Murine Autoimmune Arthritis

Abstract

The ability to use large doses of vitamin D3 (D3) to chronically treat autoimmune diseases such as rheumatoid arthritis (RA) is prohibitive due to its calcemic effect which can damage vital organs. Cytochrome P450scc (CYP11A1) is able to convert D3 into the noncalcemic analog 20S-hydroxyvitamin D3 [20S(OH)D3]. We demonstrate that 20S(OH)D3 markedly suppresses clinical signs of arthritis and joint damage in a mouse model of RA. Furthermore, treatment with 20S(OH)D3 reduces lymphocyte subsets such as CD4⁺ T cells and CD19⁺ B cells leading to a significant reduction in inflammatory cytokines. The ratio of T reg cells (CD4+CD25+Foxp3+ T cells) to CD3+CD4+ T cells is increased while there is a decrease in critical complement-fixing anti-CII antibodies. Since pro-inflammatory cytokines and antibodies against type II collagen ordinarily lead to destruction of cartilage and bone, their decline explains why arthritis is attenuated by 20(OH) D3. These results provide a basis for further consideration of 20S(OH)D3 as a potential treatment for RA and other autoimmune disorders.

Keywords: 20S(OH)D3; RA; arthritis; collagen; mouse; vitamin D.

Figure 1

20S(OH)D3 suppresses CIA: 24 DBA/1 Lac J female mice at 6 wks of age were immunized with bovine CII and 14 days later 12 mice were treated daily with intraperitoneal (i.p.) 50 µl S.O. and 12 mice with 50 µl S.O. containing 2.4 µg/kg 20S(OH)D3. Arthritis severity (A) and incidence (B) were assessed by a blinded observer and each paw was scored on a scale of 0 to 4 with 16 being the maximal score per mouse. All mice survived to day 40. On Day 40, mice were euthanized, sera obtained, and paws processed for histology and scored histologically as described in Methods. An H&E stained section of the decalcified hind paw of a mouse treated with 20S(OH)D₃ shows preservation of joint structures with minimal inflammation (10X magnification, b, bone, * identifies joint space) (C) while S.O.-treated mouse hind paws showed marked joint destruction with extensive loss of cartilage and bone with invasion of pannus (20X magnification, b, bone; p, pannus) (D). 20S(OH)D3- treated CIA mice had a lower histologic score than S.O. treated CIA mice (E). Photographs of typical paws for the scoring system we used are available online at the Hooke Laboratories Web Site (https://hookelabs.com/services/cro/cia/MouseCIAscoring.html).

Figure 2

20(OH)D3 reduces serum levels of anti-CII antibodies in CIA mice: Aliquots of sera harvested at day 40 post-immunization with CII from mice described in Figure 1A were analyzed for levels of CII specific murine IgG1, IgG2a and IgG2b antibodies as described in Methods. 20S(OH)D3 treatment significantly reduced levels of anti CII IgG1 and IgG2a (A, B) but not of anti-CII IgG2b antibodies (data not shown).

Figure 3

Suppression of CIA by 20S(OH)D3 given by gavage: 36 DBA/Lac J female mice 6 weeks of age were immunized with bovine CII (Day 0) and groups of 12 were gavaged orally beginning on day 13 post CII immunization with daily 15 µg/kg 20S(OH)D_{3 in} 50 µl 1:5 diluted PG and 50 µl normal saline by oral gavage every 7 days; oral gavage daily with 50 µl 1:5 diluted PG and every 7 days by oral gavage with MTX 2.5 mg/kg dissolved in 50 µl normal saline; or oral gavage daily with 50 µl 1:5 diluted PG and 50 µl normal saline by oral gavage every 7 days. Treatment began at Day 13 after CII immunization and continued through 48 days post CII immunization. Arthritis severity (A) and arthritis incidence (B) were assessed every 2 days. All mice survived until termination of the experiment.

Figure 4

20S(OH)D3 given by oral gavage Suppresses established arthritis: 24 6 wk old DBA/1 Lac J female mice were immunized on day 0 with CII and scored for arthritis by a blinded observer every other day for 64 days post CII immunization. Treatment was started day 20 post CII immunization by daily oral gavage with (N=12) 100 µl/day PPG 1:5 dilution in sterile saline (N=12) or 100µl of the PPG 1:5 dilution containing 30 µg/kg 20S(OH)D3.