Vitamin D Deficiency in a Multiethnic Healthy Control Cohort and Altered Immune Response in Vitamin D Deficient European-American Healthy Controls

<div><p>Objective</p><p>In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.</p><p>Methods</p><p>Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African–Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.</p><p>Results</p><p>Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4⁺ (p = 0.04) and CD8⁺ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.</p><p>Conclusion</p><p>A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.</p></div

Analysis for proliferation of CD4+ T cells after delivery of the TT-derived T-cell epitope to slanDCs via the novel immuno targeting system.

<p>(A) CFSE labeled PBMCs were prepared and incubated at 37°C for eight days with either full length TT (b) or the linker module containing (d) or lacking (c) the TT-derived T-cell epitope TT_p and analysed by FACS. The data indicate that PBMCs of the selected donor contain anti-TT memory T cells that can be recalled by full length TT and to a less extent by the TT_p peptide linker but not by the linker peptide lacking the TT_p epitope. Untreated PBMCs (a). (B) CFSE labelled PBMCs were incubated with either the antigen-containing scaffold (c) or the single components (a,b) at 4°C. Unbound material was removed by washing.</p

Analysis of binding of the SL ScBsDb/linker complex to DD2 positive Jurkat cells.

<p>(A), (a, black graph) IgM isotype negative control. (a, red graph) Binding of the anti-slan IgM mab DD2 to slan(+) positive Jurkat cells. (b, black graph) Anti-penta-HIS, isotype negative control. (b, red graph) Lack of binding of the monovalent SL scBsDb (SL) in the absence of a linker peptide. (c,d black graph) Isotype negative control. (c,d, red graph) Bivalent or trivalent anti-slan scaffolds consisting of the SL scBsDb and the linker peptide l-GFP-l (c, red graph) or l-l-GFP-l (d, red graph). (B), Detection of anti-slan scaffolds on the surface of Jurkat cells by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Construction and characterisation of the SL scBsDb.

<p>(A) A schematic view of the structure and the complete AA sequence of the SL scBsDb. (B) The SL scBsDb was expressed and purified as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016315#s4" target="_blank">Materials and Methods</a>. The flow through fractions, wash fractions (wash1, wash2) and the eluted fractions (elution 1, elution 2) containing the SL scBsDb were analyzed by SDS-PAGE (C) and immunoblotting (B). After SDS-PAGE separated protein(s) were stained with Coomassie brilliant blue (C, CBB), and after transfer the membrane was developed with an anti-penta-HIS antibody (B). M, marker proteins.</p

Immuno targeting of slan(+) Jurkat cells with a multivalent anti-slan scaffold containing a TT-derived T-cell epitope.

<p>(a, black graph) Binding of the anti-slan mab DD2 to slan(+) Jurkat cells. (c,d, red graph) Binding of anti-slan scaffolds consisting of the SL scBsDb and trivalent linker peptides either containing (c) or lacking (d) theTT-derived peptide epitope TT_p. (b,c,d, black graph) Control binding of the single components of the anti-slan scaffolds including the SL scBsDb (b), the trivalent linker l-l-GFP-l (c), and the trivalent linker l- TT_p -l-GFP-l (d).</p

Characterisation of purified linker peptides.

<p>For the formation of bi- or trivalent anti-slan scaffolds peptide linker molecules were constructed containing the l-Tag twice (l-GFP-l) or three times (l-l-GFP-l; l-TT_p-l-GFP-l). The purified peptide linkers were analysed by SDS-PAGE/immunoblotting using the anti-La mab 7B6 directed to the l-Tag. (M) Marker proteins.</p

Binding of the SL scBsDb/linker complex to native slanDCs in PBMCs.

<p>(A) (a) Estimation of the amount of slan-DCs in a PBMC sample using the anti-slan IgM mab DD2. (d) Estimation of the amount of slanDCs in the same PBMC sample using the anti-slan scaffold consisting of the SL scBsDb and the l-l-GFP-l linker peptide. Control staining of the single components of the anti-slan scaffold including the SL scBsDb (b, SL) or the peptide linker module (c, l-l-GFP-l). (B) GFP-labelled anti-slan scaffolds identified by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Recombinant antibodies derived from IgM antibodies: Restoring avidity by oligomerisation on a modular peptide scaffold.

<p>(A) The IgM anti-slan mab DD2 is a pentameric molecule. Preparation of a scFv (B) resulted in a monovalent molecule which failed to bind to slanDCs (C, data not shown). For recovery of binding avidity a scBsDb (D, SL scBsDb) and suitable linking peptide molecules (F,G) were constructed (H,I). In the scBsDb the variable heavy and light chain domains of the two mabs (A, DD2, S; E, 7B6, L) are recombinantly fused via glycine serine linkers (D; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016315#pone-0016315-g003" target="_blank">Fig. 3</a>). This SL scBsDb is on the one hand directed to the slan epitope and on the other hand to a continuous peptide epitope (l-Tag). The linker peptide modules contain the l-Tag either two times (l-GFP-l, F) or three times (l-l-GFP-l, G(i); l-TT_p-l-GFP-l, G(ii)). Binding of the SL scBsDb to the respective linker peptides results in the formation of a divalent (H) or trivalent (I) anti-slan scaffold with increased avidity.</p

Male gender in European-Americans, increased BMI in females, and UV index in Native Americans are associated with vitamin D status.

<p>(A) Median 25(OH)D levels in males vs. females stratified by ancestral background. ****p<0.0001, Mann Whitney test; p<0.05, Shapiro-Wilk and D'Agostino normality test. (B) Median 25(OH)D levels in normal weight (BMI<25), overweight (BMI 25–30), obese (BMI 30–40), and morbidly obese (BMI>40) individuals stratified by gender. **p<0.01, ***p<0.001, Kruskal-Wallis test with Dunn's multiple comparison; p<0.05, Shapiro-Wilk and D'Agostino normality test. Error bars indicate interquartile range. (C) Mean 25(OH)D levels of individuals based upon the average UV index during the month in which their biological sample was obtained stratified by ethnicity. Error bars indicate SD; *r² = 0.04, p<0.05. (D) Mean 25(OH)D levels of individuals based upon the average UVB 305 nm during the month in which their biological sample was obtained stratified by ethnicity. Error bars indicate SD.</p

GM-CSF concentration was reduced in the vitamin D sufficient group.

<p>(A) Cytokine concentration ratio of vitamin D sufficient group to vitamin D severely deficient group. **p<0.01, Mann Whitney U Test. (B) Median concentration of GM-CSF in vitamin D severely deficient (n = 20) and sufficient groups (n = 20). *p<0.05, Mann-Whitney U Test corrected for BMI; p<0.05, Shapiro-Wilk and D'Agostino normality test. Error bars indicate interquartile range.</p