Vitamin d deficiency reduces the immune response, phagocytosis rate, and intracellular killing rate of microglial cells

Abstract

Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality and neurological sequelae. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides (AMPs) and suppression of T-cell proliferation, but its influence on microglial cells is unknown. The purpose of the present study was to determine the effects of vitamin D deficiency on the phagocytosis rate, intracellular killing, and immune response of murine microglial cultures after stimulation with the Toll-like receptor (TLR) agonists tripalmitoyl-S-glyceryl-cysteine (TLR1/2), poly(I·C) (TLR3), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9). Upon stimulation with high concentrations of TLR agonists, the release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was decreased in vitamin D-deficient compared to that in vitamin D-sufficient microglial cultures. Phagocytosis of E. coli K1 after stimulation of microglial cells with high concentrations of TLR3, -4, and -9 agonists and intracellular killing of E. coli K1 after stimulation with high concentrations of all TLR agonists were lower in vitamin D-deficient microglial cells than in the respective control cells. Our observations suggest that vitamin D deficiency may impair the resistance of the brain against bacterial infections.

FIG 1

25-OH D₃ concentrations in mouse serum. Six weeks after mice were fed a diet containing a low vitamin D concentration (vitamin D concentration under detection level), the serum 25-OH D₃ concentrations were significantly lower than in mice fed a diet containing normal amounts of vitamin D (1,500 IU/kg) (P < 0.0001). Each symbol represents the serum concentration of an individual mouse. The values for both mice with the lowest 25-OH D₃ values in the vitamin D-deficient group were below the limit of quantification. For statistical analysis, unpaired t test was used. ***, P < 0.001.

FIG 2

Flow cytometric analysis of microglial cell surface marker proteins. Vitamin D-sufficient and vitamin D-deficient microglial cells were left undisturbed under medium condition or stimulation with Pam₃CSK₄ (10 μg/ml) or CpG (10 μg/ml) for 24 h. (A) Expression of the pan-populational surface marker protein CD11b. CD11b-positive vitamin D-sufficient and vitamin D-deficient microglial cells showed positivity for the pan-populational surface marker protein CD45, as exemplified for medium (B) and Pam₃CSK₄ stimulation (C). Levels of expression of the induction marker F4/80 on the surface of CD11b⁺ CD45⁺ microglia 24 h after stimulation were not significantly different between vitamin D-sufficient and vitamin D-deficient microglial cells (D). Only stimulated microglial cells expressed F4/80 (C and D). Data are provided as means ± SE (n = 4 per condition from two independent experiments) and were analyzed using one-way ANOVA followed by Bonferroni's correction for multiple comparisons.

FIG 3

Cell viability of vitamin D-sufficient and vitamin D-deficient microglial cells after treatment with Pam₃CSK₄ at 1 μg/ml, poly(I·C) at 10 μg/ml, LPS at 1 μg/ml, and CpG at 10 μg/ml (n = 4), shown as means ± SE. Treatment with all TLR agonists did not result in a loss of microglial viability in both groups compared to the viability of untreated cells. Values are as follows: medium, vitamin D sufficient, 100% ± 7.5%, and vitamin D deficient, 100% ± 1.5%; Pam₃CSK₄ at 1 μg/ml, vitamin D sufficient, 100.8% ± 1.9%, and vitamin D deficient, 99.5% ± 1.9%; poly(I·C) at 10 μg/ml, vitamin D sufficient, 101.7% ± 2.4%, and vitamin D deficient, 103.8% ± 3.2%; LPS at 1 μg/ml, vitamin D sufficient, 97.7% ± 5.4%, and vitamin D deficient, 102.1% ± 6.4%; and CpG at 10 μg/ml, vitamin D sufficient, 102.% ± 2.6%, and vitamin D deficient, 101.7% ± 3.1%. Data were analyzed using one-way ANOVA followed by Bonferroni's correction for multiple comparisons. No significant differences were noted.

FIG 4

Phagocytosis of E. coli K1 by microglial cells after 24 h of prestimulation with various TLR agonists [Pam₃CSK₄, poly(I·C), LPS, and CpG] at different concentrations. The number of ingested bacteria was determined by quantitative plating of the cell lysates on blood agar plates. Data are shown as recovered bacterial CFU per ml (median and 25% and 75% interquartile ranges). Prestimulation with TLR agonists increased the number of bacteria ingested by microglia in comparison to unstimulated cells in both groups. After stimulation with high concentrations of TLR3, -4, and -9 agonists, vitamin D-deficient microglial cells ingested significantly fewer E. coli than did vitamin D-sufficient microglial cells. Data were analyzed using two-way ANOVA followed by Bonferroni's correction for multiple comparisons. Filled black circles, vitamin D-sufficient microglial cells; open grey circles, vitamin D-deficient microglial cells. *, P < 0.05.

FIG 5

NO release in primary mouse microglial cells upon stimulation with high concentrations of TLR1/2, -3, -4, and -9 agonists. NO release after stimulation with high concentrations of the agonists did not differ between vitamin D-deficient and vitamin D-sufficient microglia. Nitric oxide concentrations in the supernatants are given as means plus SE. Data were analyzed using one-way ANOVA followed by Bonferroni's correction for multiple comparisons. No significant differences were noted.

FIG 6

Cytokine and chemokine release of unstimulated and stimulated vitamin D-sufficient and vitamin D-deficient microglial cells. IL-6 (circles), TNF-α (triangles), and CXCL1 (squares) concentrations in the supernatants of microglial cell cultures after 24 h of stimulation with Pam₃CSK₄ (P3C), poly(I·C), LPS, and CpG are shown. Stimulation of vitamin D-deficient microglia by TLR agonists induced decreased TNF-α and IL-6 release compared to that of vitamin D-sufficient microglia but no differences in CXCL1 release. Data are given as median and interquartile ranges (25% and 75%). Data were analyzed using two-way ANOVA followed by Bonferroni's correction for multiple comparisons. Filled black symbols, vitamin D-sufficient microglial cells; open grey symbols, vitamin D-deficient microglial cells. *, P < 0.05.