Reduced vitamin D-induced cathelicidin production and killing of Mycobacterium tuberculosis in macrophages from a patient with a non-functional vitamin D receptor: A case report

Abstract

Tuberculosis (TB) presents a serious health problem with approximately a quarter of the world's population infected with Mycobacterium tuberculosis (M. tuberculosis) in an asymptomatic latent state of which 5-10% develops active TB at some point in their lives. The antimicrobial protein cathelicidin has broad antimicrobial activity towards viruses and bacteria including M. tuberculosis. Vitamin D increases the expression of cathelicidin in many cell types including macrophages, and it has been suggested that the vitamin D-mediated antimicrobial activity against M. tuberculosis is dependent on the induction of cathelicidin. However, unraveling the immunoregulatory effects of vitamin D in humans is hampered by the lack of suitable experimental models. We have previously described a family in which members suffer from hereditary vitamin D-resistant rickets (HVDRR). The family carry a mutation in the DNA-binding domain of the vitamin D receptor (VDR). This mutation leads to a non-functional VDR, meaning that vitamin D cannot exert its effect in family members homozygous for the mutation. Studies of HVDRR patients open unique possibilities to gain insight in the immunoregulatory roles of vitamin D in humans. Here we describe the impaired ability of macrophages to produce cathelicidin in a HVDRR patient, who in her adolescence suffered from extrapulmonary TB. The present case is a rare experiment of nature, which illustrates the importance of vitamin D in the pathophysiology of combating M. tuberculosis.

Keywords: cathelicidin; hereditary vitamin D-resistant rickets (HVDRR); macrophage; tuberculosis; vitamin D.

Figure 1

Plasma cathelicidin levels are independent of 25(OH)D and VDR function (A) Plasma cathelicidin levels in control subjects (VDR^WT), heterozygous family members (VDR^WT/R80W) and the HVDRR patient (VDR^R80W). (B) Representative Western blot analysis of plasma cathelicidin from one control subject (VDR^WT) and the HVDRR patient (VDR^R80W) both run in triplicates. A titration of His-tagged recombinant cathelicidin used for the standard curve was included in each analysis. (C) Plasma cathelicidin versus 25(OH)D concentrations (C) or versus 1,25(OH)2D concentrations (D) in the control subjects (VDR^WT), heterozygous family members (VDR^WT/R80W) and the HVDRR patient (VDR^R80W).

Figure 2

Normal VDR expression levels and activation of macrophages from the HVDRR patient (A) Overview of the experimental setup; Mtb (M. tuberculosis). (B) VDR, (C) CYP24A1, (D) IL-6, (F) TNFα and (H) IL-1β mRNA in macrophages and (E) IL-6, (G) TNFα and (I) IL-1β protein in the supernatants harvested at 168 h from control subjects (VDR^WT) and the HVDRR patient (VDR^R80W). The macrophages were treated with 1,25(OH)₂D₃ and M. tuberculosis as indicated below the graphs. (B, D, F, H) The expression levels of the indicated targets were normalized to the levels in untreated macrophages from control subjects. (C) CYP24A1 levels were normalized to the mean CYP24A1 level in untreated macrophages from control subjects. (B–I) Data from three independent experiments each with macrophages from three control subjects and the HVDRR patient. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 3

Reduced vitamin D-induced cathelicidin expression and M. tuberculosis elimination in macrophages from the HVDRR patient (A) mRNA and (B) protein levels of cathelicidin in macrophages from control subjects (VDR^WT) and the HVDRR patient (VDR^R80W). The macrophages were treated with 1,25(OH)₂D₃ and M. tuberculosis as indicated below the graphs. The cathelicidin levels were normalized to the cathelicidin levels in (A) untreated and (B) 1,25(OH)₂D₃-treated control cells. (C, D) Estimation plots of the CFU levels in macrophages infected with M. tuberculosis in the absence and presence of vitamin D from control subjects (VDR^WT) and the HVDRR patient (VDR^R80W). (A–D) Data from three independent experiments each with macrophages from three control subjects and the HVDRR patient. ns: not significant, *p < 0.05; ****p < 0.0001.