A Role for Iodide and Thyroglobulin in Modulating the Function of Human Immune Cells

Abstract

Iodine is an essential element required for the function of all organ systems. Although the importance of iodine in thyroid hormone synthesis and reproduction is well known, its direct effects on the immune system are elusive. Human leukocytes expressed mRNA of iodide transporters (NIS and PENDRIN) and thyroid-related proteins [thyroglobulin (TG) and thyroid peroxidase (TPO)]. The mRNA levels of PENDRIN and TPO were increased whereas TG transcripts were decreased post leukocyte activation. Flow cytometric analysis revealed that both PENDRIN and NIS were expressed on the surface of leukocyte subsets with the highest expression occurring on monocytes and granulocytes. Treatment of leukocytes with sodium iodide (NaI) resulted in significant changes in immunity-related transcriptome with an emphasis on increased chemokine expression as probed with targeted RNASeq. Similarly, treatment of leukocytes with NaI or Lugol's iodine induced increased protein production of both pro- and anti-inflammatory cytokines. These alterations were not attributed to iodide-induced de novo thyroid hormone synthesis. However, upon incubation with thyroid-derived TG, primary human leukocytes but not Jurkat T cells released thyroxine and triiodothyronine indicating that immune cells could potentially influence thyroid hormone balance. Overall, our studies reveal the novel network between human immune cells and thyroid-related molecules and highlight the importance of iodine in regulating the function of human immune cells.

Keywords: NIS; RNAseq; iodine; iodine deficiency; nutritional immunology; pendrin; thyroglobulin; thyroid hormones.

Figure 1

Human immune cells express and regulate iodide transporters, thyroglobulin, and thyroid peroxidase. (A) Leukocytes were left unstimulated (US) (−) or activated (+) with PMA and ionomycin for 18 h, total RNA was extracted, and then the cDNA from samples was amplified with PCR. Shown is DNA gel electrophoresis image of two representative donors. (B) Leukocytes mRNA was amplified using real-time quantitative PCR, and then each gene was normalized to internal B2M control. Fold increase over US controls was graphed ± SEM of nine independent donors. (C) Leukocytes were stained with no antibody (control), NIS, or PENDRIN antibodies. The cells were then stained with secondary Alexa-647 and primary-conjugated CD45 Krome-orange antibodies. Leukocyte subsets (granulocytes, monocytes, and lymphocytes) were gated on based on CD45 and side-scatter characteristics. The gated subset histograms are shown as cell counts and antibody staining intensity representative of six donors. (D) Median fluorescent intensity (MFI) values from panel (C) were subtracted from background staining and then graphed ± SEM of six to seven independent donors.

Figure 2

Targeted RNASeq analysis of iodide-treated leukocytes via next-generation sequencing. (A) Viability analysis of iodide-treated leukocytes—5 × 10⁶ leukocytes were left untreated (control—PBS) or incubated with 1 mM NaI for up to 3 days. Cell counts and viability utilizing trypan blue exclusion were determined with TC20 automated cell counter. (B) Targeted RNASeq—leukocytes were left unstimulated with PBS or incubated with 1 mM NaI for 48 h, total RNA was extracted, and then targeted RNASeq libraries were created for a total of 475 genes. The libraries were indexed (multiplexed) and then loaded onto Illumina MiSeq sequencer. The data were de-multiplexed, and unique molecular tags were identified utilizing Qiagen’s RNASeq bioinformatics software. Total molecular tag counts were normalized to counts of 10 housekeeping genes and then quantified based on fold expression relative to each untreated control. Average fold increases were obtained from 5 to 10 independent donors. Genes that were significantly increased or decreased (p < 0.05) were selected and displayed in bar graph ± SEM of at least five independent donors. See Table S1 in Supplementary Material for quantifications and p values.

Figure 3

Increased release of cytokines and chemokines by iodide-treated leukocytes. (A) 5 × 10⁶ leukocytes were left unstimulated (US) with PBS or treated with 1 mM NaI for 72 h. The supernatants were collected, and protein levels of cytokines/chemokines were analyzed utilizing enzyme-linked immunosorbent assay. (B) Same as in panel (A), but instead leukocytes were incubated with 500 µM Lugol’s iodine for 72 h, and then the supernatant was analyzed for cytokine levels. Detected ranges for cytokine secretion are (IFNγ: 5–1,005 pg/mL, IL6: 10–2,500 pg/mL, IL10: 26–482 pg/mL, IL8-CXCL8: 0.3–230 ng/mL, and CCL2: 15–65 ng/mL). Cytokine concentrations were normalized based on fold changes over each US pair and was then averaged and graphed as fold increase ± SEM of at least seven donors.

Figure 4

Thyroglobulin (TG) is utilized by leukocytes to increase levels of thyroid hormones. (A) 5 × 10⁶ leukocytes were left unstimulated with PBS or treated with 1 mM NaI for 72 h, and then levels of total and free thyroid hormones in media were detected utilizing ECiQ diagnostic immunoassay instrument. Shown are averaged concentrations ± SEM of six donors. (B) 5 × 10⁶ leukocytes were incubated with TG derived from human thyroid tissue at a concentration of 20 µg/mL for 3 days in serum free media, and then levels thyroid hormones (T₄ and T₃) in media were detected utilizing ECiQ diagnostic immunoassay instrument. The data were averaged after background subtraction of TG signal. Shown are averaged concentrations ± SEM of six donors. (C) Same as in panel (B), but instead leukocytes were incubated with thyroxine (T₄) at 2.4 µg/mL for 2 days in serum free media. Levels of T₃ were analyzed utilizing diagnostic immunoassay instrument ECiQ. The data were averaged after background subtraction of T₄ signals. Data shown represent averaged quantifications ± SEM of six donors.

Figure 5

Current model—immune cells regulate their function via iodide and increase levels of thyroid hormones by processing thyroid-derived thyroglobulin (TG). (A) Immune cells express surface iodide transporters (NIS and PENDRIN) that are upregulated during cellular activation. The cells are able to accumulate iodide that could alter the transcription of multiple immune mediators and possibly other genes. The changes are functional since the higher mRNA levels correlate with increased cytokine release at the basal state. The effect is systemic and is not polarized to either pro- or anti-inflammatory genes. During an immune response, the presence of sufficient amounts of iodide allows for a “primed” state of cells that are ready to proliferate upon activation. The effects of iodide on immune cells could have an impact during early conception wherein immune cells could release more factors to support blood vessel and optimal pregnancy. (B) In an iodine deficiency state, TSH is secreted to command an increase of mostly T₄ and some T₃ by the thyroid. The thyroid responds by elevating NIS and PENDRIN surface expression and the production of more TG, some of which is released in the blood stream. Increased severity of iodine deficiency raises TG levels in the blood accordingly. Based on our findings, we propose that leukocytes could uptake TG from the blood or tissues and release T₄, which will eventually raise T₃ levels by deiodinase activity. This has a systemic effect since it increases levels of hormones to local tissues and/or in the blood for increased metabolism. Released thyroid hormones are also known to affect the immune system by enhancing cytokine expression and altering phenotypes of immune cells.