Vitamin D Regulates MerTK-Dependent Phagocytosis in Human Myeloid Cells

Abstract

Vitamin D deficiency is a major environmental risk factor for the development of multiple sclerosis. The major circulating metabolite of vitamin D (25-hydroxyvitamin D) is converted to the active form (calcitriol) by the hydroxylase enzyme CYP27B1. In multiple sclerosis lesions, the tyrosine kinase MerTK expressed by myeloid cells regulates phagocytosis of myelin debris and apoptotic cells that can accumulate and inhibit tissue repair and remyelination. In this study, we explored the effect of calcitriol on homeostatic (M-CSF, TGF-β–treated) and proinflammatory (GM-CSF–treated) human monocyte-derived macrophages and microglia using RNA sequencing. Transcriptomic analysis revealed significant calcitriol-mediated effects on both Ag presentation and phagocytosis pathways. Calcitriol downregulated MerTK mRNA and protein expression in both myeloid populations, resulting in reduced capacity of these cells to phagocytose myelin and apoptotic T cells. Proinflammatory myeloid cells expressed high levels of CYP27B1 compared with homeostatic myeloid cells. Only proinflammatory cells in the presence of TNF-α generated calcitriol from 25-hydroxyvitamin D, resulting in repression of MerTK expression and function. This selective production of calcitriol in proinflammatory myeloid cells has the potential to reduce the risk for autoantigen presentation while retaining the phagocytic ability of homeostatic myeloid cells.

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Vitamin D deficiency is a major environmental risk factor for the development of multiple sclerosis (MS) (1). Although widely prescribed for patients with MS, the impact of vitamin D on disease course and severity, as well as its mechanisms of action, are poorly understood. Active vitamin D (calcitriol) is obtained from the cutaneous production of vitamin D3 (cholecalciferol) in the presence of sufficient UV B irradiation, as well as limited dietary sources. Cholecalciferol is converted to 25-hydroxyvitamin D (25OHD; calcifediol), the major circulating metabolite, and then to hormonally active 1,25-dihyroxyvitamin D (1,25(OH)₂D; calcitriol) through sequential hydroxylation, catalyzed by 25-hydroxylases (CYP2R1, CYP27A1) and 25-hydroxyvitamin D₃ 1-α-hydroxylase (CYP27B1), respectively (2). Levels of 25OHD are used clinically to assess vitamin D status (3). Calcitriol functions as a ligand for the vitamin D receptor, a member of the nuclear receptor family of hormone-regulated transcription factors (3). Catabolism of 25OHD and calcitriol is initiated by the CYP24A1 enzyme, whose expression is tightly regulated by calcitriol in a negative feedback loop. CYP27B1 is abundantly expressed in most biological systems, allowing for local calcitriol production in several tissues, including the CNS. Importantly, CYP27B1 expression is regulated by a complex cytokine network in immune cells, including cells of myeloid origin (4).

Cells of myeloid lineage, including endogenous microglia and infiltrating monocyte-derived macrophages (MDMs), are the dominant cell population within active MS lesions (5). We have previously shown that the myeloid cell–mediated phagocytic clearance of myelin debris, a process required for efficient remyelination, is regulated by MerTK, a member of the TAM family of receptor tyrosine kinases (6). MerTK deficiency results in delayed remyelination in the cuprizone model of demyelination (7). MDMs derived from MS patients show impaired ability to phagocytose myelin, a defect linked to a reduction in MerTK expression (8). In addition to clearing myelin debris, MerTK mediates the process of efferocytosis, the removal of dead/dying cells, which is important for autoreactive T cell fate determination in MS (9). The functions of myeloid cells are dependent on their state of activation. TGF-β, a key cytokine involved in CNS homeostasis, has been shown to maintain cells in a homeostatic state characterized by high expression of MerTK, TREM2, CSF1R, and MAFB (10). In contrast, MerTK expression is comparatively lower in proinflammatory myeloid cells, a population shown to contribute to MS pathogenesis (6). Genome-wide association studies (GWAS) have explained much of MS heritability. Single-nucleotide polymorphisms (SNPs) in CYP24A1 and CYP27B1, which tightly regulate the intracellular levels of calcitriol, have been associated with an increased risk of MS (11–13).

In the current study, we investigated calcitriol-mediated transcriptomic regulation of human MDMs and microglia. RNA sequencing (RNAseq) revealed significant calcitriol-mediated negative regulation of both phagocytic and Ag-presenting pathways in these cell types. We demonstrate that calcitriol represses MerTK expression and phagocytic capacity of primary myeloid cells and significantly downregulates components of the Ag presentation pathway. Notably, proinflammatory myeloid cells expressing the lowest levels of MerTK have the most active vitamin D metabolic processing pathway and are, therefore, able to respond to the precursor 25OHD. In contrast, lack of endogenous processing of 25OHD in homeostatic myeloid cells maintains high MerTK expression and, therefore, participation in the immunologically silent clearance of myelin debris and apoptotic cells.

Materials and Methods

MDMs

Human PBMCs were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation (GE Healthcare). Monocytes were isolated from PBMCs using magnetic CD14⁺ isolation beads (Miltenyi Biotec). Proinflammatory macrophages differentiated using GM-CSF (MØ_GMcsf) and alternative (M2) macrophages were generated by differentiating monocytes for 6 d in the presence of 25 ng/ml GM-CSF and M-CSF, respectively. To generate CNS homeostatic macrophages differentiated using M-CSF+TGFβ (MØ₀), TGF-β (50 ng/ml) was added to the M2 M-CSF culture conditions on days 3 and 6. A concentration of 10⁻⁷ M calcitriol (Selleck Chemicals) was added to designated macrophages on day 1 of culture and maintained throughout differentiation. Culture media was replenished every 2–3 d.

Microglia and astrocytes

Human adult microglia were isolated from brain tissue of patients undergoing brain surgery for intractable epilepsy. Cells were cultured in DMEM, 5% FBS, penicillin/streptomycin, and glutamine. Cell differentiation and calcitriol treatment was performed over 6 d as described above. Human fetal astrocytes were isolated, as previously described (14), from human CNS tissue from fetuses at 17–23 wk of gestation that were obtained from the University of Washington Birth Defects Research Laboratory (project no. 5R24HD000836-51) following Canadian Institutes of Health Research–approved guidelines.

Autologous T cells

Human T cells were isolated from the same PBMC fraction as described for macrophages, using magnetic CD3⁺ isolation beads (Miltenyi Biotec).

Proinflammatory cytokine assay

Following differentiation, macrophage cultures were supplemented with 10 ng/ml TNF-α or IL-1β for 24 h. Cells were then treated with 10⁻⁷ M 25OHD (Selleck Chemistry) for 48 h.

Phagocytosis assay

Human myelin was isolated as previously described (15). Myelin was found to be endotoxin-free using the Limulus amebocyte lysate test (Sigma-Aldrich). To evaluate myelin uptake, myelin was incubated with a pH-sensitive dye (pHRodamine; Invitrogen) for 1 h in PBS (pH 8). Dyed myelin was added to myeloid cells to a final concentration of 20 μg/ml and incubated for 1 h. Flow cytometry was performed using the FACS Fortessa (BD Biosciences). Live cells were gated based on live-dead staining, and doublets were excluded.

Flow cytometry

Human myeloid cells were detached gently using 2 mmol EDTA/PBS and blocked-in FACS buffer supplemented with 10% normal human serum and normal mouse IgG (3 mg/ml). Cells were incubated at 4°C for 15 min with Aqua viability dye (Life Technologies) and then subsequently incubated at 4°C for 30 min with either control isotype Ab or appropriate surface marker (MerTK, CD80, CD86, HLA-DR/DP/DQ, HLA-ABC, CD40, CD274) test Abs. Cells were washed, and flow cytometry was performed using the Attune NxT (Thermo Fisher Scientific). Myeloid cells were gated based on side scatter area and forward light scatter (FSC) area. Doublets were excluded using FSC area and FSC height. Live cells were gated based on live-dead staining (Aqua; Life Technologies).

Apoptosis assay

Isolated T cells were collected and resuspended to 1 × 10⁶ cells per milliliter in PBS. Cells were exposed to UV for 1 h. Following exposure, cells were collected, pelleted, and processed for phagocytosis, as previously described, for myelin. pHRodamine-dyed cells were inoculated into macrophage cultures at a density of 5:1 T/M and left to incubate for 1 h. Assessment of apoptosis was done by flow cytometry using Alexa 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific).

RNAseq

Control and calcitriol-treated MDMs and microglia were collected in TRIzol reagent (Invitrogen), and RNA was extracted according to the manufacturer’s protocol (Qiagen). Smart-Seq2 libraries were prepared by the Broad Technology Labs and sequenced by the Broad Genomics Platform. cDNA libraries were generated from the Smart-seq2 protocol (16). RNAseq was performed using Illumina NextSeq500 and a High Output v2 Kit to generate 2 × 25 bp reads. Reads were aligned to the hg19 genome with STAR aligner and quantified by the Broad Technology Labs' computational pipeline using Cuffquant version 2.2.1 (17, 18). Raw counts were normalized using trimmed mean of maximum values normalization and then log₂-transformed. The read counts for each sample were used for differential expression analysis with the edgeR package (19, 20). The differentially expressed genes were identified using p value <0.05 and log₂ fold change >1. Principle component analysis (PCA) was carried out using built-in R function, prcomp, and visualized using gplot package. Heatmaps were created using ggplot2 package in R. The full list of identified genes was used to generate volcano plots in R. For PCA and heatmap graphs, variance of genes across all macrophage phenotypes was calculated and the top-500 highly variable genes were used for further analysis.

Quantitative PCR

Cells were lysed in TRIzol (Invitrogen). Total RNA extraction was performed using standard protocols followed by DNAse treatment according to the manufacturer’s instructions (Qiagen). For gene expression analysis, random hexaprimers and Moloney murine leukemia virus reverse transcriptase were used to perform standard reverse transcription. Analysis of individual gene expression was conducted using TaqMan probes to assess expression relative to Gapdh.

Study approval

All studies that were performed have been conducted according to Declaration of Helsinki principles and with approval of the Research Ethics Office at McGill University.

Statistics

Paired Student t test and one-way ANOVA were used to determine significance of results.

Results

Calcitriol mediates significant transcriptional changes in human MDMs

We have previously identified MerTK as an important phagocytic receptor for the immunologically silent clearance of myelin debris (6). To identify compounds that are known to alter MERTK gene expression, we used a data integration approach known as Integrated Complex Traits Network (iCTNet) (21). iCTNet retrieves information from multiple databases and creates a single network with user-defined parameters for visualization. Calcitriol was revealed as a regulatory factor upon visualization of a subset of Food and Drug Administration–approved compounds (gray) and diseases (pink) related to MERTK (Fig. 1A).

To examine the effect of calcitriol on MDMs in different states of polarization (Supplemental Fig. 1A, 1B), we analyzed the transcriptomic profile of homeostatic (MØ₀) and MØ_GMcsf MDMs generated in vitro and subjected to bulk RNAseq. MØ₀ show high expression of CNS homeostatic myeloid markers such as TREM2, CSF1R, IL-10, and MAFB (Supplemental Fig. 1C). Proinflammatory MØ_GMcsf cells expression signatures show typical inflammatory markers such as IL-6, NLRP1, CCL22, MMP9, and ITGAX, as well as induction of inflammatory programs involving the transcription factor BHLHE40, identified as part of the disease-associated transcriptomic signature (22). PCA (Fig. 1B) and heatmap (Fig. 1C) analyses showed that MØ₀ and MØ_GMcsf cells cluster separately based on their phenotypes with calcitriol-treated cells clustering together regardless of their starting phenotype (Fig. 1B, 1C). Volcano plot analysis confirms this calcitriol-mediated shift in the transcriptomic signature and highlights that both phenotypes responded to calcitriol by upregulating known calcitriol target genes CYP24A1 and cathelicidin (CAMP) (Fig. 1D). Finally, overrepresentation analysis (ORA) was carried out using significantly differentially expressed genes in both MØ₀ and MØ_GMcsf cells exposed to calcitriol (Fig. 1E). Set nodes represent biological processes colored based on p value (red to light yellow, most significant to least significant). The size of the node corresponds to the number of genes associated with the biological process that correlates with the function of these genes. Smaller unlabeled nodes represent individual genes (red, upregulated; green, downregulated). Downregulated genes of interest (MERTK and HLA-DRB1) with their link to relevant biological processes (regulation of endocytosis and adaptive immune response) are highlighted.

Calcitriol regulation of MerTK expression and function in human MDMs

Use of the ingenuity pathway analysis (IPA) bioinformatic tool highlighted “phagosome formation” as one of the top canonical pathways affected by calcitriol in MDMs (Supplemental Fig. 2). Visualization of this pathway highlighted the downregulation of a number of phagocytic and immune-sensing receptors, including complement receptors, Fc receptors, and integrins, suggesting that calcitriol may influence the cells’ ability to phagocytose a range of substrates (Fig. 2A). We identified a list of 30 genes associated with phagocytosis by myeloid cells and assessed their expression in response to calcitriol in both MØ₀ and MØ_GMcsf cells. A total of seven genes were significantly downregulated in MØ₀ and four genes in MØ_GMcsf in response to calcitriol treatment. MERTK was the only gene significantly downregulated in both MØ₀ and MØ_GMcsf cells (Fig. 2B). We validated this RNAseq finding by quantitative RT-PCR. Regardless of phenotype, calcitriol significantly downregulated MERTK mRNA (Fig. 2C) and protein expression, as measured by flow cytometry (Fig. 2D).

To assess if reduced expression of MerTK would have a functional impact on the cells, we measured the ability of calcitriol-treated MDMs to phagocytose myelin debris, autologous apoptotic T cells, and opsonized RBCs (oRBCs). Calcitriol-treated MDMs displayed a reduced capacity to phagocytose pHRhodamine-labeled human myelin regardless of cellular phenotype (Fig. 2E).

In addition to myelin, MerTK has been extensively characterized as a mediator of apoptotic cell clearance (9). To investigate whether calcitriol also inhibited this process, pHRhodamine-labeled apoptotic T cells were incubated with autologous MDMs. We observed a significant inhibition of apoptotic T cell phagocytosis by calcitriol-exposed MØ₀ but not MØ_GMcsf cells. This is indicative of an MØ_GMcsf–specific efferocytotic receptor that can compensate for the calcitriol-mediated downregulation of MerTK (Fig. 2F). Finally, to validate the specificity of calcitriol in regulating MerTK-dependent phagocytosis, we assessed the uptake of oRBCs by both MDM phenotypes. Phagocytosis of oRBCs occurs through Fc receptor–mediated endocytosis, an MerTK-independent pathway. In all cases, calcitriol had no influence on the ability of MDMs to phagocytose oRBCs, suggesting a specificity to the calcitriol-mediated inhibition of phagocytosis by human MDMs (Fig. 2G).

Calcitriol downregulates the expression of Ag presentation molecules

Engagement of the adaptive immune system through the reactivation of antimyelin T cell responses in the CNS acts as a key pathogenic step in the initiation and exacerbation of MS (23). Activation of CD8⁺ and CD4⁺ T cells requires recognition of cognate Ags loaded on the surface of APCs. The strongest MS risk loci maps to the HLA region, which is a gene complex encoding the major histocompatibility family of proteins (MHC). GWAS has identified the HLA-DRB1 as the strongest risk locus, conferring a 3-fold–increased MS risk (24). Activation of T cells requires expression of MHC class molecules by APCs (signal one) in addition to a “second” signal in the form of expression of costimulatory molecules such as CD40 and CD86, both also identified as MS risk loci (15, 25, 26). IPA analysis of our sequencing results highlights the “Ag presentation pathway” as a significantly affected pathway (Supplemental Fig. 2), with downregulation of both MHC class I and MHC class II molecules as indicated using the pathway visualization tool (Fig. 3A). We identified a list of 24 genes associated with Ag presentation in our dataset and assessed expression in response to calcitriol in MØ₀ and MØ_GMcsf cells (Fig. 3B). Expression of a large number of HLA/MHC genes were downregulated by calcitriol treatment in both cellular phenotypes, including the major MS risk gene HLA-DRB1. We validated these sequencing findings by measuring protein expression using flow cytometry. Protein expression of both MHC class I (HLA-ABC) and MHC class II (HLA-DR/DP/DQ) molecules were downregulated by calcitriol treatment in both MØ₀ and MØ_GMcsf cells (Fig. 3C). Expression of costimulatory molecules CD86 and CD40 were also significantly reduced in response to calcitriol (Fig. 3D). Interestingly, we observed increased expression of immune checkpoint molecule CD274(PD-L1) both at the mRNA (Fig. 3B) and protein (Fig. 3E) level following treatment with calcitriol. CD274(PD-L1) suppresses the adaptive immune response by inducing apoptosis in CD279-expressing T cells (27). Moreover, previous work has shown that the human CD274(PD-L1) gene is a direct target of the 1,25(OH)₂D–regulated vitamin D receptor (28). Finally, a “third” signal in the form of proinflammatory cytokine release from the APC is suggested to be necessary for the induction of T cell proliferation. IL-6 is a cytokine that, when released from APCs, can promote the differentiation of IL-17–producing Th-17 cells, known to be highly pathogenic in MS (29). We observed a significant decrease in IL-6 mRNA and protein release by ELISA in response to calcitriol (Fig. 3F) in MØ_GMcsf cells.

Endogenous production of calcitriol inhibits MerTK selectively in proinflammatory MDMs

The in vivo–circulating concentrations of calcitriol (40–100 pM) are much lower than those of 25OHD (20–150 nM). It is therefore important to determine whether there is sufficient intracellular metabolism of 25OHD to calcitriol within MDMs to affect MerTK expression. As shown in Fig. 4A, proinflammatory MØ_GMcsf cells exhibited the highest expression of the calcitriol-producing enzyme, CYP27B1 (Fig. 4E). This high level of CYP27B1 expression negatively correlated with MERTK expression. Cells that expressed the lowest levels of MERTK (MØ_GMcsf) expressed the highest levels of CYP27B1 and, conversely, cells (MØ₀) that expressed the highest levels of MERTK displayed the lowest expression of CYP27B1 (Fig. 4A, 4B). CYP27B1 expression is regulated by a complex network of cytokines (4); we therefore assessed the impact of proinflammatory cytokines known to play a role in MS pathology (TNF-α and IL-1β) on CYP27B1 expression, 25OHD metabolism, and MerTK expression (30). We observed that the addition of TNF-α (and, to a lesser degree, IL-1β) enhanced the expression of CYP27B1 in MØ_GMcsf cells but not MØ₀ (Fig. 4C). To assess the capacity of the vitamin D metabolic pathway to regulate MerTK expression, cells were treated with the major circulating metabolite 25OHD. Despite the increased basal expression of CYP27B1 in MØ_GMcsf cells, exposure to 25OHD did not significantly alter MerTK expression (Fig. 4D). However, combinatorial treatment of MDMs with 25OHD and TNF-α (and, to a lesser degree, IL-1β) selectively and significantly downregulated MerTK expression in MØ_GMcsf cells to a similar degree as calcitriol (Fig. 4D). TNF-α alone did not change MerTK expression. Altogether, we show that proinflammatory MØ_GMcsf cells are the only cells capable of converting 25OHD to active calcitriol, leading to the downregulation of the myelin-phagocytic receptor MerTK.

Calcitriol regulation of MerTK expression in primary human glia

In addition to recruited MDMs, both resident microglia and astrocyte populations take part in the neuroinflammatory process and the phagocytic clearance of myelin debris. We therefore examined the effect of calcitriol on human microglia isolated from resected brain tissue and astrocytes derived from the fetal human CNS. Microglia were polarized to CNS homeostatic (MG₀) and proinflammatory (MG_GMcsf) phenotypes. Similar to MDMs, cells were exposed to M-CSF (MG₀) or GM-CSF (MG_GMcsf) over a 6-d period, with homeostatic cells receiving additional TGF-β. Confirmation of these phenotypes is highlighted by expression of established CNS homeostatic markers, including microglia-specific markers TMEM119, SALL1, and OLFML3 (Supplemental Fig. 1C). Proinflammatory microglia are characterized by high expression of canonical inflammatory myeloid markers including genes that show relative specificity to microglia, CCL17 and IL-1α (Supplemental Fig. 1D). Bulk RNAseq was carried out on calcitriol-treated MG₀ and MG_GMcsf cells. PCA of these samples showed that, similar to MDMs, microglia cluster along the first principal component based on their cellular phenotype (MG₀ and MG_GMcsf) and along the second principal component based on treatment with calcitriol (Fig. 5A). ORA carried out on differentially expressed genes in both phenotypes exposed to calcitriol show a similar pattern of calcitriol-responsive biological processes, including “inflammatory response” and “cytokine production/secretion” (Fig. 5B). These transcriptomic results were validated in vitro whereby calcitriol downregulated MERTK mRNA and MerTK protein in human microglia (Fig. 5C). Finally, calcitriol had no influence on MerTK mRNA or protein expression in human fetal astrocytes (Fig. 5D), indicating that the regulation of MerTK expression by calcitriol is specific to cells of the myeloid lineage.

Discussion

The link between vitamin D and MS risk and the overrepresentation of genes involved in vitamin D metabolism as part of the genetic architecture of MS highlights the need for understanding the functional pathways under the control of vitamin D. In this study, we explored the response of human myeloid populations to calcitriol (1,25(OH)₂D) on the level of the whole transcriptome. Using network-based analysis, we observed significant modulation of both Ag presentation and phagocytosis pathways in MDMs and primary human microglia. We report that calcitriol controls expression of the phagocytic receptor MerTK and subsequent uptake of myelin debris and apoptotic cells. Calcitriol also establishes an immune-regulatory phenotype in these myeloid cells, significantly reducing expression of inflammatory mediators and Ag presentation machinery while increasing the expression of immune checkpoint molecules.

Mendelian randomization studies have shown that genetically determined variations in 25OHD serum levels play a causal role in MS (31, 32). Clinical studies are ongoing (vitamin D to ameliorate MS and efficacy of vitamin D supplementation in MS), yet a reproducible benefit of vitamin D supplementation has not been evident thus far. Standard of care preparations of vitamin D are comprised of precursors such as 25OHD, the major circulating form of vitamin D. 25OHD is processed locally to biologically-active calcitriol yet, it is 25OHD levels that define an individual’s vitamin D “status” (3). Both systemic and intracellular conversion of 25OHD to calcitriol is dependent on sufficient expression of the enzyme CYP27B1 (2). Our study demonstrates a significant effect of the activation state of the cell on CYP27B1 levels. Cells exposed to inflammatory cytokines expressed the highest levels of CYP27B1 and had an enhanced ability to respond to 25OHD. Based on our findings, we would predict that circulating 25OHD may not be as important as the CYP27B1-mediated production of intracellular calcitriol and subsequent transcriptional regulation of cellular function, particularly in cells of the innate immune system. Therefore, supplementation that increases serum 25OHD levels may not be targeting the cellular functions relevant to the pathogenesis of MS. Based on our results, we would propose that an individual’s ability to respond to vitamin D supplementation may fluctuate with time based on their inflammatory status and their cells’ abilities to produce active calcitriol from circulating 25OHD.

Myelin clearance through myeloid cell–mediated phagocytosis is an essential process that allows for efficient remyelination and CNS repair (33). We and others have reported reduced MerTK expression and phagocytic capacity in myeloid cells of MS patients (8). Expression of both membrane-bound and -soluble forms of MerTK are elevated in MS lesional tissues (34). In animal models, MerTK and its cognate ligand Gas6 have shown to play protective roles, particularly in the cuprizone toxin model in which Gas6-knockout mice develop a more severe level of demyelination coupled with a delayed remyelination process (7). Experimental evidence strongly supports a functional role for MerTK in inflammation resolution, debris clearance, and repair (35). GWAS has identified several SNPs in the MERTK gene as independently associated with the risk of developing MS (12, 25). Fine-mapping of the MERTK locus identifies a risk variant that operates in trans with the HLA-DRB1 locus and is associated with higher expression of MerTK in MS patient monocytes (13). This particular SNP (rs7422195) displays discordant association depending on the individual’s HLA-DRB1*15:01 status, conferring increased risk, but converting to a protective effect on an HLA-DRB1*15:01 homozygous background. The stratification of risk based on DRB1 status is strongly suggestive of a functional interplay or crosstalk between phagocytosis and Ag presentation in cells capable of carrying out such functions. The beneficial role of high MerTK expression is dependent on the underlying pathology, the phase of the disease, and the activation status of the cell in which it is expressed. A recent study has shown polymorphisms in the MERTK gene that drive low expression of the protein in Kupffer cells to protect against the development of liver fibrosis in nonalcoholic steatohepatitis (36). In addition to the genomic determinants of MerTK expression and function, our study highlights how environmental factors can also influence expression of this key phagocytic and immunomodulatory receptor.

In addition to myelin debris, impaired clearance of cells undergoing apoptosis leads to sustained proinflammatory responses as cells progress to secondary necrosis (37). Digestion of phagocytosed substrate and presentation as Ags loaded on MHC molecules (signal 1) coupled with costimulation (signal 2) and secretion of inflammatory cytokines (signal 3) from APCs play a critical role in stimulating the adaptive immune response (38). GWAS has identified an extended HLA haplotype, HLA DRB1*15:01, DQA1*0102, DQB1*0602, within the MHC class II region that is strongly associated with MS risk. In accordance with previous reports, we observed that calcitriol downregulated the expression of both MHC class I and II molecules on the surface of myeloid cells including HLA DRB1/DQA1/DQB1. Calcitriol downregulated the expression of major costimulatory molecules and upregulated immune checkpoint molecule CD274 (PD-L1), as previously reported (28). Calcitriol also inhibited IL-6 expression and release. These combined data highlight the ability of calcitriol to modulate both the ingestion of material and the expression of molecular machinery involved in Ag presentation, potentially lowering the risk of autoantigen presentation to the adaptive immune system.

In summary our data demonstrates that calcitriol downregulates MerTK expression and MerTK-mediated phagocytosis in primary human myeloid cells. Intracellular production of active calcitriol from its precursor and resultant repression of MerTK is limited to proinflammatory myeloid cells (due to high expression of CYP27B1). This proinflammatory-specific effect may underlie a beneficial mechanism of vitamin D in MS. Proinflammatory myeloid cells are potent Ag presenters; selective inhibition of myelin uptake by these cells may lower the risk of myelin Ag presentation to infiltrating T cells. In contrast, maintenance of MerTK, and therefore phagocytic function in homeostatic myeloid populations (due to low expression of CYP27B1), would allow these cells to maintain clearance of myelin debris and contribute to the process of repair. Overall, we uncover a functional interaction between one of the strongest environmental modulators of MS risk (vitamin D) and the MerTK pathway that is selective to disease-relevant populations of primary human myeloid cells.

Disclosures

The authors have no financial conflicts of interest.

References

Footnotes