OH vitamin D down-regulates in vitro production of antierithrocyte antibodies in autoimmune haemolytic anaemia

Background: Vitamin D is a well known regulator of bone and calcium balance that has multiple immune-modulating activities. Reduced levels of 25-OH vitamin D have been found in various autoimmune diseases, such as systemic lupus systemic lupus erythematous and multiple sclerosis, and its levels and supplementation did correlate with disease clinical severity. Aims: We evaluated a) the effect of vitamin D on the in vitro production of antiRBC autoantibodies in patients with autoimmune haemolytic anaemia (AIHA); b) vitamin D levels, VDR expression and immunemodulatory cytokines. Methods: Clinical and haematological parameters, serum samples and informed consent were collected at the time of enrolment from January 2013. 25-OH vitamin D levels, VDR and IL-6, IL-10, IL-17, TNF-alfa and IFN-gamma were evaluated in 40 patients and in 40 age and sex matched healthy controls, using ELISA kits. Heparinized blood samples from 7 AIHA patients were tested for anti-RBC production in unstimulated and PWM-stimulated 48h cultures with or without vitamin D at increasing concentrations (10, 20 and 40 ng/mL). The number of immunosuppressive therapy lines (steroids, immunosuppressors, rituximab, splenectomy) were retrospectively collected. Results: Laboratory features of the patients (15 males and 25 females; mean age 58 years; 18 CAD, 18 WAIHA and 4 DAT negative AIHA) are shown in Figure 1a. Vitamin D levels were significantly reduced in patients versus controls, regardless sex, age nor season at sampling. VDR was increased in patients compared to controls; IL-6, IL-10, IL-17 and IFN-gamma serum levels were reduced. As shown in Figure 1b, vitamin D (at 10, 20, and 40 ng/mL) exerted a dose dependent inhibition on in vitro production of anti-RBC antibodies, with a delta% reduction of 6, 16 and 31 in unstimulated conditions, and 2, 1, 16 in PWM-stimulated conditions, respectively. As regards treatment at sampling, 24 patients were under low dose steroids (e.g. 0,2-0,5 mg/Kg/day prednisone); vitamin D status was comparable in treated and untreated cases. Retrospectively, 2 cases were therapy naïve, 13 cases had been treated with steroid only, 15 cases with both steroid and a second line treatment (9 rituximab and 4 cyclophosphamyde), and 10 with more than 2 lines of therapy (4 splenectomy, 10 rituximab and 6 cyclophosphamyde). Vitamin D levels were significantly lower in patients who had been treated with 2 or more lines of therapy (1,72±1 versus 2,78±2 ng/mL, p=0.04). Summary and Conclusions: Vitamin D deficiency/insufficiency was observed in patients with AIHA, both therapy naive or previously treated, with concomitant alteration of immuno-modulatory cytokine levels. Moreover, we found increased VDR expression, possibly reflecting an up-regulation due to ligand deficiency or increased shedding because of reduced receptor recruitment. Vitamin D deficiency was more evident in relapsed/refractory patients, suggesting a more pronounced immune disregulation in these cases. In vitro studies demonstrated a dose dependent inhibitory effect of vitamin D on the production of anti-RBC auto-antibodies even at very low concentrations and suggested that vitamin D supplementation may be useful in AIHA.