Monocytic Cytokines in Autoimmune Polyglandular Syndrome Type 2 Are Modulated by Vitamin D and HLA-DQ

Abstract

Context: Autoimmune polyglandular syndrome (APS-2: autoimmune Addison's disease or type 1 diabetes) is conferred by predisposing HLA molecules, vitamin D deficiency, and heritable susceptibility. Organ destruction is accompanied by cytokine alterations. We addressed the monocytic cytokines of two distinct APS-2 cohorts, effects of vitamin D and HLA DQ risk.

Methods: APS-2 patients (n = 30) and healthy controls (n = 30) were genotyped for HLA DQA1/DQB1 and their CD14+ monocytes stimulated with IL1β and/or 1,25(OH)₂D₃ for 24 h. Immune regulatory molecules (IL-6, IL-10, IL-23A, IL-15, CCL-2, PD-L1), vitamin D pathway gene transcripts (CYP24A1, CYP27B1, VDR), and CD14 were analyzed by enzyme-linked immunosorbent assay and RTqPCR.

Results: Pro-inflammatory CCL-2 was higher in APS-2 patients than in controls (p = 0.001), whereas IL-6 showed a trend - (p = 0.1). In vitro treatment with 1,25(OH)₂D₃ reduced proinflammatory cytokines (IL-6, CCL-2, IL-23A, IL-15) whereas anti-inflammatory cytokines (IL-10 and PD-L1) rose both in APS-type 1 diabetes and APS-Addison´s disease. Patients with adrenal autoimmunity showed a stronger response to vitamin D. Expression of IL-23A and vitamin D pathway genes VDR and CYP27B1 varied by HLA genotype and was lower in healthy individuals with high-risk HLA (p = 0.0025; p = 0.04), while healthy controls with low-risk HLA showed a stronger IL-10 and CD14 expression (p = 0.01; p = 0.03).

Conclusion: 1,25(OH)₂D₃ regulates the monocytic response in APS-2 disorders type 1 diabetes or Addison´s disease. The monocytic cytokine profile of individuals carrying HLA high-risk alleles is proinflammatory, enhances polyglandular autoimmunity and can be targeted by vitamin D.

Keywords: Addison’s disease; HLA DQ haplotypes; autoimmune polyglandular syndrome type 2; autoimmune thyroiditis; cytokine gene expression; type 1 diabetes.

Figure 1

Organs affected in autoimmune polyglandular syndrome type 2. The autoimmune polyglandular syndrome type 2 can affect a wide variety of organs in the body, triggering various autoimmune diseases. For reasons that are still unclear, APS-2 destroys particularly endocrine organs, leading to the development of type 1 diabetes, Addison’s disease, Hashimoto’s thyroiditis, and Graves’ disease. However, non-endocrine organs such as the skin, the gastrointestinal tract, the brain, the liver, and also muscles and cartilage may be affected by the body’s own destruction, which is often driven by antibodies and autoreactive lymphocytes.

Figure 2

Effect of 1,25(OH)₂D₃ on vitamin D pathway genes and CD14 in human monocytes. Effect of 1,25(OH)₂D₃ on expression of vitamin D pathway genes (A–C) and CD14 (D) in IL1β stimulated monocytes of both APS-2 cohorts and HC. Data are presented as median obtained from 15 AD/AIT patients, 15 T1D/AIT patients and 30 healthy controls (HC). T1D, type 1 diabetes; AIT, autoimmune thyroiditis; AD, Addison’s disease; HC, healthy controls. Values ​​are considered to be statistically significant when p <0.05.

Figure 3

Vitamin D mediated reduction of pro-inflammatory cytokines. Gene expression (A, C, E, G) and protein secretion (B, D, F) of pro-inflammatory cytokines IL-6, CCL-2, IL-23, and IL-15 after in-vitro 1,25(OH)₂D₃ in both APS-2 cohorts and HC. Data are presented as median values obtained from 15 AD/AIT patients, 15 T1D/AIT patients and 30 healthy controls (HC). T1D, type 1 diabetes; AIT, autoimmune thyroiditis; AD, Addison’s disease; HC, healthy controls. Values ​​are considered to be statistically significant when p <0.05.

Figure 4

Vitamin D mediated enhancement of anti-inflammatory molecules IL-10 and PD-L1. Gene expression (A, C) and protein secretion (B, D) of anti-inflammatory cytokine IL-10 and immune regulator programmed death ligand 1 (PD-L1) after in-vitro 1,25(OH)₂D₃ administration in IL1β-activated monocytes of APS-2 patients and HC. Data are presented as median values obtained from 15 AD/AIT patients, 15 T1D/AIT patients and 30 healthy controls (HC). T1D, type 1 diabetes; AIT, autoimmune thyroiditis; AD, Addison’s disease; HC, healthy controls. Values ​​are considered to be statistically significant when p <0.05.

Figure 5

High-risk HLA affects VDR and CYP27B1 mRNA transcription levels. Gene expression levels of HC were stratified according to their HLA types. VDR (A) and CYP27B1 (B) gene expression of high-risk HLA and intermediate-risk HLA carriers are lowered compared to carriers with low-risk HLA. Low-risk HLA type is associated with increased CD14 (C) and IL-10 (D) expression in HC. HLA frequencies in HC: high-risk HLA (n = 3), intermediate-risk HLA (n = 13), low-risk HLA (n = 14).