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. Author manuscript; available in PMC: 2020 May 3.
Published in final edited form as: Tuberculosis (Edinb). 2019 May 3;116 Suppl:S131–S137. doi: 10.1016/j.tube.2019.04.021

Vitamin D modulates human macrophage response to Mycobacterium tuberculosis DNA

Jorge L Cervantes 1,*, Esther Oak 2, John Garcia 3, Hongfei Liu 4, Paolo A Lorenzini 4,5, Deepika Batra 6, Arvind Chhabra 6, Juan C Salazar 7,8, Xavier Roca 4
PMCID: PMC6626683  NIHMSID: NIHMS1528528  PMID: 31085128

Abstract

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen that infects macrophages where it avoids elimination by interfering with host defense mechanisms, including phago-lysosome fusion. Endosomal Toll-like receptors (TLRs) generate Type I Interferons (IFNs), which are associated with active tuberculosis (TB).

We aimed to explore if DNA from different Mtb lineages lead to differences in the inflammatory response of human monocytic/macrophage cells. THP-1 cells which express two inducible reporter constructs for interferons (IFNs) as well as for NF-κB, were stimulated via endosomal delivery of Mtb DNA as a nanocomplex with PEI. DNA from different Mtb phylogenetic lineages elicited differential inflammatory responses in human macrophages. An initial relatively weak IRF-mediated response to DNA from HN878 and H37Rv increased if the cells were pre-treated with Vitamin D (Vit D) for 72 hrs. RNAseq of THP-1 under different transformation conditions showed that pre-treatment with Vit D upregulated several TLR9 variants, as well as genes involved in inflammatory immune response to infection, immune cell activation, Type I IFN regulation, and regulation of inflammation.

Vit D appears to be important in increasing low IRF responses to DNA from certain lineages of Mtb. Variations in the IRF-mediated response to DNA derived from different Mtb genotypes are potentially important in the pathogenesis of tuberculosis since Type I IFN responses are associated with active disease. The role of Vit D in these responses could also translate into future therapeutic approaches.

Keywords: Mycobacterium tuberculosis, Type I IFNs, Vitamin D

Graphical Abstract

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1. Introduction

Tuberculosis (TB) is one of the oldest illnesses in history and still remains a major health problem worldwide, with more than 8 million new cases and more than 1.8 million deaths occurring every year [1]. Mycobacterium tuberculosis (Mtb) mainly infects professional phagocytes in the lungs where it uses strategies such as prevention of phagolysosome maturation and subversion of host cell death pathways, to survive and replicate [2].

In order to mount an appropriate antimicrobial response, the host’s innate immune response first detects pathogens through pattern recognition receptors (PRRs), which recognize conserved pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) expressed on macrophages can recognize PAMPs on Mtb [3, 4] and mediate the production of immune-regulatory cytokines such as tumor necrosis factor (TNF) and type I Interferons (IFNs). A type I IFN gene signature is associated with active TB [5, 6]. Type I IFNs (IFN-α and IFN-β) appear to suppress type II Interferon (IFN-γ)-triggered responses [7, 8], including Mtb killing by macrophages [9]. Although different isolates from Mtb have shown differences in invasiveness [10] and proinflammatory cytokine induction [11, 12], no study has evaluated differences in Type I IFN induction in humans with different Mtb strains. A differential induction of Type I IFNs might explain how TB manifests in some individuals, as type I IFNs have been demonstrated to strongly inhibit monocytes’ and macrophages’ responsiveness to IFN-γ, and are associated with active TB disease [7-9].

The role of Vitamin D (Vit D) in the treatment of tuberculosis has been portrayed in the early 20th century with the use of heliotherapy, and then continued in the form of Vit D supplementation in the pre-antibiotic era [13]. Vit D appears to be crucial for macrophage activation and early Mtb clearance [14-16]. Vit D enhances innate immunity through TLR and IFN-γ, reversal of phagosome maturation arrest, increased expression of various antimicrobial peptides, and induction of autophagy in infected cells, thus restricting Mtb intracellular growth in macrophages [17].

We herein show that Mtb nucleic acid of different lineages elicit differential inflammatory responses in human macrophages. We also investigated the effect of Vit D in these responses and observed that cells which initially showed a weak Interferon regulatory factor (IRF)-mediated response to DNA from certain Mtb strains, could increase it if they were pre-treated with Vit D. Our findings show that both, NF-κB and IRF-activation in macrophages, are differentially affected by the lineage of Mtb, and that Vit D appears to be important in increasing the IRF response when this is low.

2. Material and Methods

2.1. Human cell lines

Human monocytic cell line THP-1 (ATCC TIB-202), and dual THP-1 (Invivogen) cell lines were used for our experiments. The latter expresses two inducible reporter constructs for interferon regulatory factors (IRFs) as well as for NF-κB. The activities of these reporters were measured through a colorimetric assay for the secretory embryonic alkaline phosphatase (SEAP) reporter gene that is linked to NF-κB activation using the Quanti-blue substrate (InvivoGen), and a Luciferase reporter gene linked to IRFs using the QuantiLuc substrate (Invivogen), according to manufacturer’s instructions. Measurement of NF-kB and IRF activation were expressed as a response ratio for each stimulus relative to the reporter activity in unstimulated cells.

THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/ml Penicillin and 100 μg/ml Streptomycin at 37 °C and 5% CO2. Differentiation and pre-treatment of THP-1 cells were done with 100 ng/mL phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA, Sigma-Aldrich) or 0.5μM 1,25-dihydroxyvitamin D3 (Vitamin D3, Sigma-Aldrich) for 72 hours. For some experiments, cells were also activated with 100 ng/mL LPS plus 20 ng/mL IFN-γ for 8 hrs. Reagents were used in working dilutions (1/100) in PBS from stock solutions in DMSO.

2.2. Bacterial nucleic acid stimulation

Genomic DNA from different Mtb lineages: Indo-Oceanic T17X strain (NR-44096) (Lineage 1), HN878 strain (bei NR-14867) (Lineage 2), East-African-Indian strain (NR-44095) (Lineage 3), and Mtb strains H37Rv (NR-14865) and H37Ra (ATCC 25177D-5) (Lineage 4), as well as DNA from Mycobacterium bovis (ATCC BAA-935D-2) were acquired from ATCC / bei resources. Strains and correspondent lineages [18] are summarized in Table 1. CpG 2395 (Invivogen) was used as a control ligand.

Table 1.

Mtb Genomic DNA used in this study

Strain ATCC Lineage
Indo-Oceanic T17X NR-44096 Lineage 1
HN878 NR-14867 Lineage 2
East-African-Indian NR-44095 Lineage 3
H37Rv NR-14865 Lineage 4
H37Ra 25177D-5
M. bovis BAA-935D-2
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Cells, at a density of 2.4×104 cells/well, were stimulated for 24 hours with 100ng of DNA delivered with the use of the cationic polymer polyethilenimine (PEI) according to manufacturer instructions (Polyplus, France). PEI is a transfection reagent that wraps negatively charged RNA and DNA with subsequent transport of the PEI/nucleic acid nanoparticle complex to early and late endosomes [19]. We have previously utilized this endo-lysosomal delivery system for bacterial nucleic acid to evaluate endosomal TLR activation and cytokine induction, including type I IFN in human phagocytic cells [20].

2.3. RNA-Sequencing library preparation

After removing the medium, THP-1 cells were washed twice with cold PBS and centrifugation to get rid of floating cells. Afterwards, total RNA was isolated using RNesay Kit together with QIAshredder (Qiagen). Residual DNA was removed by 30-min DNase treatment (TURBO DNA-free Kit, Ambion) at 37 °C. Subsequently, RNA was purified with RNA Clean and Concentrator (Zymo Research), and its concentration and integrity was checked using ribogreen assay and bioanalyzer (Agilent Technologies 2100 Bioanalyzer). Only RNA samples with RIN number above 8 were further processed. RNA library preparation was carried out using the TruSeq Stranded mRNA Library Prep Kit (Illumina). HiSeq 2500 System Rapid mode (Illumina) was used for RNA-sequencing, giving rise to paired-end reads of 101 bp. A total number of reads per sample was obtained in the range of 123-189×106, with 85.8-91.0% mapped reads.

2.4. RNA-Seq data analysis

The original read quality of our RNA-Seq data was first confirmed by FastQC (Simon Andrews, Babraham Bioinformatics). For gene expression analysis, the paired-end reads were aligned to human genome (UCSC-hg19) using Tophat2 [21] with Bowtie2 [22]. In RNA-Seq data analysis, the default parameters for each tool were used unless otherwise stated. For Tophat2, a maximum of 2 mismatches were allowed and two parameters as --library-type=fr-firststrand and --read-realign-edit-dist=0 were set. Cufflinks, Cuffmerge and Cuffdiff [23] were used for transcripts assembly and quantification of the Tophat2 output. Annotated transfer RNA, ribosomal RNA and mitochondrial transcripts were masked out in Cuffdiff analysis by using the parameter –mask-file with corresponding GTF file. The common FPKM (fragments per kilobase of transcript per million fragments mapped) were used as the expression-levels measure for each gene or transcript.

2.5. Polymerase chain reaction (PCR)

2.5.1. Semi-quantitative PCR

Characterization of expression of TLR-9 and inteferon-1beta was done by semi-quantitative PCR, as described before (Chhabra et al. 2017, JI). In brief, cDNAs were prepared from mRNAs derived from THP-1 cells stimulated with DNA from different MTB strains, using the SuperScript cDNA synthesis kit (Invitrogen Inc.), cDNAs were quantified and 2.5ng cDNA was used for each PCR reaction of 50 ul volume. GAPDH gene amplified as the loading control. PCR conditions included 1 cycle of denaturation step at 94C for 5 min, followed by 40 cycles of amplification step comprised of denaturation at 94C for 30sec, annealing at 56C for 30sec and extension at 72C for 30 sec., followed by one cycle at 72C for 10 min. (PCR Primers: TLR-9 F: 5‘-GAATGCCAGTTGGTTCCGTG-3‘, TLR-9 R: 5‘-TTCAGAGCTGGGAGTGTCCA-3‘, IFN-1b F: 5‘-GCCGCAGTGACCATCTATGA-3‘, IFN-1b R: 5‘-CAGTGACTGTCCTCCTTGGC-3‘, GAPDH F: 5‘-GAAGGTGAAGGTCGGAGTC-3, GAPDH R: 5‘-GAAGATGGTGATGGGATTTC-3‘).

2.5.2. Real-Time quantitative PCR

RT-qPCR analysis was also performed for TLR9 using the previously described cDNA as template. Complementary DNA samples were diluted 1:10 (vol:vol) in deionized water, samples were stored at −20°C. Amplification reactions were performed in triplicate for each tested gene. Each reaction contained 1 μl of cDNA, 5 μl of iQ™ SYBR® Green Supermix (Bio-Rad), 3.7 μl of water and 150 nM of each primer to a final volume of 10 μl. Primer sequences for TLR9, and GAPDH, have been published elsewhere[24, 25]. Non-template controls (NTC) were included in triplicate for each primer pair confirming for the absence of contamination. Amplification reactions were performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). PCR conditions were performed as follows, denaturation at 95°C for 3 min, 40 cycles of denaturation at 95°C for 10 sec and annealing/extension at 60°C for 30 sec. To ensure the presence of a single amplicon, a melting curve analysis was performed at the end of each PCR run. Cycle threshold (Ct) values for each gene were used to calculate the transcript levels of TLR9 in reference to the endogenous control GAPDH. The relative changes in gene expression generated were calculated using the 2−ΔΔCt method [26].

2.6. Endosomal TLR Blocking.

A specific immunoregulatory DNA sequence (IRS869: 5’-TCCTGGAGGGGTTGT-3’) [27] (Integrated DNA Technologies) was used to block TLR9 signaling. IRS869 was used at a concentration of 1.4 μM, as it has shown previously to result in optimal effectiveness and specificity of the inhibitory sequences [27, 28]. The ODN was added 1 hour prior to cell stimulation.

2.7. Statistical Analysis

General statistical analysis was performed using GraphPad Prism 7.01 (GraphPad Software, San Diego, CA). For comparison or response between Untreated vs. Vitamin D-treated for each stimulus a t-test or a non-parametric test (Mann-Whitney) was used, depending on if the data followed a normal distribution or not. p values of <0.05 were considered significant. A two-way ANOVA was performed to assess the interaction of Vit D with the responses upon stimulation with the different Mtb DNAs. For RNAseq, a transcript change consistent in all the three replicates was considered as significant at the statistic level if the q-value was less than 0.05.

3. Results

3.1. DNA from different M.tuberculosis phylogenetic lineages elicits differential inflammatory response in human macrophages

We first aimed to evaluate TLR9 activation by DNA from well characterized Mtb species belonging to different genotypes [18] (available on ATCC) on human macrophages. Utilizing endosomal delivery of Mtb DNA as a nanocomplex with PEI [19, 20], we evaluated differences in pro-inflammatory cytokines and interferon induction in THP-1 cells by nucleic acids belonging to distinct Mtb genotypes. We observed that different genotypes are able to elicit different NF-κB and different IRF-mediated responses after stimulation with the distinct Mtb DNAs (Figure 1, white bars).

Figure 1. DNA from different M.tuberculosis phylogenetic lineages elicits differential inflammatory response in human macrophages.

Figure 1.

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Dual THP-1 cells were stimulated for 24 hours with 100ng of DNA from several different phylogenetic lineages of Mtb. Response from IRF (A) or an NF-kB (B) reporters are shown. Pretreatment of cells with Vitamin D (gray bars) show increase the response to DNA from HN878 and virulent strain H37Rv (A). * p<0.05 Unpaired t test.

No apparent relationship was observed between genetic distance between lineages and the strength of the elicited NF-kB or IRF-mediated-responses. East-African-Indian (Lineage 3), and Indo-Oceanic (Lineage 1), show the highest response (over 15 and 50 fold, compared to unstimulated). Despite their relative closeness to Lineage 3 [18], DNA from HN878 (Lineage 2), and H37Rv (Lineage 4), showed a relatively weak response (below 5 fold, compared to unstimulated).

3.2. Vitamin D increases the IRF-mediated response to Mtb DNA in cells that were initially poorly reactive

As macrophage activation by Vit D is important in Mtb recognition and clearance [15, 16], we then evaluated the effect of Vit D activation on the response upon Mtb-DNA stimulation. Although pre-treatment of THP-1 cells with Vit D did not substantially affect the overall NF-kB-mediated response (Figure 1B), it did increase the secretion of TNF-α (Figure 2). With respect to the IRF-mediated response, Vit D increased the IRF-mediated response to Mtb-DNA of HN878 and H37Rv (Fig. 1A, gray bars). A two way ANOVA analysis was performed to assess the interaction of Vit D with the responses upon stimulation with the different Mtb DNAs. Vit D treatment constituted a factor that impacted the IRF-mediated responses to Mtb DNA stimuli (p= 0.01). This did not occur in respect to NF-κB-mediated responses (p=0.37). Vit D also increased the IRF-mediated response to other TLR ligands (Figure 3A). In fact, Vit D was the only condition that increased this response. Neither IFN-γ nor IFN-γ + LPS showed any difference compared to untreated or PMA-treated cells (Suppl 1). Overall, treatment of THP-1 cells with Vit D induced upregulation of genes involved in inflammatory immune response to infection, immune cell activation, recruitment of effector cells to site of inflammation, antigen presentation and recognition, Type I IFN regulation, and regulation of inflammation (Suppl 2 and Table 2).

Figure 2. Pre-treatment of human macrophages with Vitamin D increases secretion of TNF-α after stimulation with DNA from different M.tuberculosis phylogenetic lineages.

Figure 2.

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Dual THP-1 cells were pre-treated (or not) with Vitamin (VD) for 72 hours, and then stimulated for 24 hours with DNA from several different phylogenetic lineages of Mtb. Two way ANOVA p<0.05

Figure 3. Vitamin D promotes upregulation of TLR9 splicing variants in THP-1 cells THP-1 cells were pre-treated with Vit D or PMA for 72 hrs, this latter with or without LPS plus IFN-γ for 8 hrs.

Figure 3.

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Heatmap shows upregulation of a set of TLR genes by Vit D that includes specific TLR9 splicing variants. Data for A and B excludes and includes, respectively, the data with PMA followed by LPS and IFN-γ treatment. Each of the three columns per condition indicate a biological replicate (different cell preparation). PMA and LPS/IFN-γ treatments induced upregulation of other TLRs that do not include TLR9.

a Comparison between THP-1 vs. PMA

b Comparison between THP-1 vs. Vit D

c Comparison between PMA vs. LPS/IFN-γ

Table 2.

Genes upregulated by Vit D treatment of THP-1 cells

Function Gene name Role in TB
CXCL8 Stimulator of phagocytosis
Mediators of inflammatory response IL1B Caspase-1 activation. Stimulator of MIP-I alpha production
CCL22 Lung inflammation through CCR4 activation
 
Transcription Factor for Type I IFNs IRF5 Necessary for Type I IFN in response to Mtb
 
Recruitment of effector immune cells to site of inflammation CXCL8 Neutrophil chemoattractant
CCR1 Receptor for CCL-3, −4, −5, and MIP-1 alpha
CD38 M1 polarization inflammatory marker in Mtb infection
 
T cell chemoattractant IL16 Increased in TB pleural effusion
CCL22 Lung inflammation through CCR4 activation
 
CD86 M1 polarization marker in Mtb infection
T cell activation CD84 Increased in B cell precursors in Mtb infection
IL27RA Involved in resolution of inflammation. Induced by Type I IFNs in Mtb infection
 
Neutrophil and NK-mediated bacterial killing CXCR1 Oxidative defense in active TB
CD244 Increased in lymphocytes of TB patients
 
Antigen presentation HLA-B,HLA-C Restricting alleles for TB CD8 antigens
 
Pathogen Recognition Receptor TLR8 Enhances the innate and adaptive immune response
TLR5 Expressed on myeloid cells in TB granulomas
P2RX7 Induces cell death and subsequent loss of intracellular bacterial viability in human macrophages infected with mycobacteria
CD14 Facilitates the uptake of non-opsonized Mtb
 
Intracellular PRR NOD2 Enhances the innate response of alveolar macrophages to Mtb
AIM1 IL-1β and IL-18 production and Th1 responses to Mtb
 
TLR adapter TIRAP Influences disease susceptibility by modulating the inflammatory response
 
Regulated by Type I IFNs and MyD88 IL10RA Anti-inflammatory cytokine expressed in chronic Mtb granulomas
 
Serine protease inhibitors SERPINB9 Inhibits apoptosis and promotes survival of Mtb-infected macrophages
SERPING1 Component of active TB signature
SERPINB1 Critical for neutrophil survival
 
Complement degradation CD46 Bactericidal activity of macrophages and granuloma formation
 
Regulation of Inflammation SIGLEC10 Increased in CD14+CD16++ monocytes upon Mtb infection
SIGLEC14,SIGLEC5 Involved in Mtb replication in monocytes
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3.3. Vitamin D promotes upregulation of TLR9 splicing variants in THP-1 cells

To address the question if the effect of Vit D was due to upregulation of TLR9 in THP-1 cells, RNAseq was carried on these cells, comparing different activation conditions. Transcriptome analysis showed that Vit D treatment of cells lead to upregulation of a cluster of TLR genes, including TLR5, TLR8, and certain splicing variants of TLR9, although the latter where not statistically significant (Fig. 3). This set of TLR genes were observed to be downregulated with the other cell treatment conditions such as PMA or IFN-γ + LPS (Figure 3B). PMA led to the known differential expression (DE) of TLR2 [29] while IFN-γ + LPS-treatment lead to upregulation of a different set of TLRs, namely TLR3, 4, 7, and 8.

Semi-quantitative PCR on Mtb-DNA stimulated cells, showed a minimal increase in the transcription of IFN-β, after stimulation with DNA of certain Mtb-strains, and an increase in TLR9 transcript in THP-1 cells pre-treated with Vit D, for all Mtb-DNA, except Indo-Oceanic (Suppl 3A-C). Even though the RT-PCR could not confirm these results, it did show a prominent increase in TLR9 expression on Vit D-treated cells after stimulation with DNA from M. bovis (Suppl 3D). To further examine the involvement of TLR-9 in the responses to Mtb DNA, we performed TLR9 inhibition utilizing a specific immunoregulatory sequence (IRS). Although a reduction was observed for the NF-kB mediated response, and not for the IRF-mediated one, this was only observed for certain Mtb lineages (Suppl 4).

4. Discussion

Little is known about nucleic acid signaling in tuberculosis or its role in susceptibility to the disease. It has been reported that stimulation of human alveolar macrophages with DNA from attenuated mycobacteria (H37Ra or BCG) vs. generates a greater TNF-α response, compared to stimulation with DNA from a virulent mycobacteria (H37Rv or M.bovis) [30]. In this study, we observed that DNA from hypervirulent HN878 strain (Lineage 2) showed both low NF-kB and IRF-mediated responses. HN878 is genetically close to virulent Mtb Beijing strain [31], which induces a diminished inflammatory production [12]. Our findings demonstrate that DNA from different Mtb phylogenetic lineages elicits differential IRF-mediated responses in human macrophages. This suggests that variations in Type I IFN responses correlate with differences in Mtb nucleic acids. This is of outmost importance as type I IFNs are mediators implicated in host resistance to TB [32].

Mtb is fundamentally a bacterium that remains in the phagosome [33], inhibiting phagosomal acidification and recruitment of lysosomal proteins in order to maintain a hospitable niche within macrophages [2]. Reports of escape of Mtb from its vacuole into the cytosol [34, 35] appear to have been generated by the experimental technique used [36, 37], and occurred in a limited percentage of cells when they are infected ex vivo [38] but not in vivo [39]. Furthermore, cytosolic Mtb-strain dependent DNA-recognizing receptor activation observed in mice, is associated with host’s DNA release, and not with that one from mycobacteria [40]. The phagosome could then be considered the platform from where components of Mtb are recognized by PRRs present at this site, as it has been shown to occur with other pathogens [41-43].

The importance of TLR signaling in the recognition of Mtb components is underlined by the fact that TLR-adaptor molecule MyD88 pathway in macrophages is needed for an adequate innate and adaptive immune response, mycobacterial clearance [44-46], and confining Mtb within phagolysosomes [38]. The subcellular localization of endosomal TLRs guarantees release of ligands, like nucleic acids, from the pathogen in a natural occurring immunity-promoting manner [47]. Endosomal TLR signaling may play an important role in the outcome of infection, as these receptors are involved in the generation of Type I IFNs. TLR9 is the endosomal TLR receptor able to recognize mycobacterial DNA in humans [4], and mice [48]. Several studies have demonstrated an association between specific endosomal TLR polymorphisms of TLR9, with susceptibility to tuberculosis [49, 50].

It is well known that TLR9-mediated recognition of the unmethylated CpG motifs can lead to distinct signaling responses depending on the sequence of oligodeoxynucleotide (ODN)s that express CpG motifs [51]. Newer and more reliable phylogenetic analysis of MTBC strains through the use of Whole Genome Sequencing (WGS) has shown that there exists more genetic diversity between strains, with genetic distance sometimes comparable to that one existing between Mtb and M. bovis [52]. So it is possible that genetic differences between Mtb strains could be sufficient to elicit different responses after PRR recognition.

We here observed that Vit D rescued low IRF-mediated response to stimulation with DNA from certain Mtb genotypes. That fact that Vit D increases the response to DNA from some of the strains that were initially “weak”, rules out the possibility that the initial differential response is due to differences in DNA preparation, as the same amount of the same DNA preparations was able to elicit an increase. In fact, Mtb DNA preparations were fairly pure, with 260/280 ratios in the range of 1.7-188 (data not shown). Also, we have previously observed that the degree of bacterial DNA fragmentation did not alter the degree of cell activation, nor the size or PEI-DNA complex particle [20].

Pretreatment of THP-1 cell with Vit D also increased secretion of TNF-α after stimulation with Mtb DNA. This is concordant with a previous study showing increased secretion of TNF-α and other cytokines upon Mtb infection of Vit D-treated THP-1 cells [53]. Vit D has also been shown to rescue impaired TNF-α response from U937 and alveolar macrophages after infection with Mtb and BCG [54]. Our transcriptome findings on Vit D activation of THP-1 cells are concordant with previous studies showing that Vit D-stimulated top-ranking genomic regions and genes related to the anti-microbial response and other immunity-related pathways [55, 56]. Only Vit D pretreatment induced upregulation of TLR9, while other forms of cell activation upregulated other TLRs. An increase in TLR 9 expression was observed upon Mtb-DNA stimulation in Vit D pre-treated cells. Besides being useful model to study Mtb infection and persistence [57], using THP-1 cell line helped to avoid the potential bias of the presence of TLR variants which could affect the amount of TLR which they generate [58].

Interaction of different TLRs appears to have important regulatory and cooperative effects against Mtb infection [48, 59]. TLR9-TLR2 crosstalk could be important in promoting vaccine enhancing responses, as the evidence of the benefits of using endosomal agonists as adjuvants for TB vaccines is increasing [60, 61].

Tissue destruction and pathogenesis during Mtb infection is not mediated by pathogens alone but induced by an immunopathological inflammatory response of the host. Differences in the inflammatory cytokine induction, especially type I and type II IFNs after recognition of Mtb nucleic acids could account for differences in the severity of the disease manifestation. Further studies could elucidate the role of the different PRRs in the response after Mtb-DNA recognition.

Supplementary Material

1

Suppl 1. Vitamin D pre-treatment activates THP-1 cells. Vitamin D increased IRF-mediated response to TLR 2 ligand stimulation. Dual THP-1 cells were pre-treated with PMA. Vit D, IFN-γ for 72 hrs., or IFN-γ for 72 hrs. followed by LPS for 8 hrs. Bars show the response ratio (compared to Unstimulated) after stimulation with TLR-2 ligand Pam3CSSNA for 24 hours.

Suppl 2. Genes differentially expressed in THP-1 cells by Vitamin D. Heatmap of transcriptome analysis of THP-1 cells pre-treated with PMA, or Vit D for 72 hrs. Each of the three columns per condition indicate a biological replicate (different cell preparation). Blue rectangle shows DE genes involved in inflammatory immune response to infection, cell adhesion, signal transduction, Type I IFN regulation, macrophage activation, pathogen recognition, pathogen DNA recognition, and recruitment of effector cells to site of inflammation.

Suppl 3. Vitamin D increases transcription of IFN-β in THP-1 cells PCR for IFN-1beta, and TLR9 of THP-1 cells untreated and pre-treated with Vit D for 72 hrs, followed by 24 hr. stimulation with genomic Mtb DNA from different lineages. A Bands show an overall increase in IFN-1beta transcription after Vit D treatment. B and C Adjusted band density values show an overall increase in TLR9 transcription in cells pretreated with Vit D (black bars), with the exception of cells stimulated with Indo-Oceaninc Mtb DNA. GAPDH is used as a housekeeping gene. Gray bars: Untreated cells. D RT-PCR for TLR9 showing an increase in the TLR9 transcript for Vit D-treated cells after stimulation with CpG or M. bovis DNA.

Suppl 4. TLR9 transcription inhibition partially affects NF-kB response to DNA from different M.tuberculosis phylogenetic lineages THP-1 cells were pre-treated with a specific immunoregulatory DNA sequence (IRS869: 5’-TCCTGGAGGGGTTGT-3’) [27], to block TLR9 signaling. IRS869 was used at a concentration of 1.4 μM, added 1 hour prior to cell stimulation. A NF-kB response was decreased after stimulation with DNA from some strains, such as Indo-Oceanic, or H37Rv, but it was increased in the case of East-Africa-Indian strain. B No change was observed in the IRF-mediated response.

Acknowledgements

Special thanks to Dr. Wendy Walker, and Olga Estrada (TTUHSC), for their assistance with RT-PCR. This work was presented in part at the Texas Tuberculosis Research Symposium (TTRS) 2018, El Paso, Texas, USA, sponsored by the Texas Tech University Health Sciences Center El Paso.

Funding

This work was supported by NIAID grants AI0901656 (JS) and CCMC Arrison and Burr Curtis Research funds (JS). XR acknowledges funding from Academic Research Fund Tier 2 MOE2013-T2-1-101 (ARC 45/13) from Singapore’s Ministry of Education. The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

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Declaration of interest: None

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Associated Data

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Supplementary Materials

1

Suppl 1. Vitamin D pre-treatment activates THP-1 cells. Vitamin D increased IRF-mediated response to TLR 2 ligand stimulation. Dual THP-1 cells were pre-treated with PMA. Vit D, IFN-γ for 72 hrs., or IFN-γ for 72 hrs. followed by LPS for 8 hrs. Bars show the response ratio (compared to Unstimulated) after stimulation with TLR-2 ligand Pam3CSSNA for 24 hours.

Suppl 2. Genes differentially expressed in THP-1 cells by Vitamin D. Heatmap of transcriptome analysis of THP-1 cells pre-treated with PMA, or Vit D for 72 hrs. Each of the three columns per condition indicate a biological replicate (different cell preparation). Blue rectangle shows DE genes involved in inflammatory immune response to infection, cell adhesion, signal transduction, Type I IFN regulation, macrophage activation, pathogen recognition, pathogen DNA recognition, and recruitment of effector cells to site of inflammation.

Suppl 3. Vitamin D increases transcription of IFN-β in THP-1 cells PCR for IFN-1beta, and TLR9 of THP-1 cells untreated and pre-treated with Vit D for 72 hrs, followed by 24 hr. stimulation with genomic Mtb DNA from different lineages. A Bands show an overall increase in IFN-1beta transcription after Vit D treatment. B and C Adjusted band density values show an overall increase in TLR9 transcription in cells pretreated with Vit D (black bars), with the exception of cells stimulated with Indo-Oceaninc Mtb DNA. GAPDH is used as a housekeeping gene. Gray bars: Untreated cells. D RT-PCR for TLR9 showing an increase in the TLR9 transcript for Vit D-treated cells after stimulation with CpG or M. bovis DNA.

Suppl 4. TLR9 transcription inhibition partially affects NF-kB response to DNA from different M.tuberculosis phylogenetic lineages THP-1 cells were pre-treated with a specific immunoregulatory DNA sequence (IRS869: 5’-TCCTGGAGGGGTTGT-3’) [27], to block TLR9 signaling. IRS869 was used at a concentration of 1.4 μM, added 1 hour prior to cell stimulation. A NF-kB response was decreased after stimulation with DNA from some strains, such as Indo-Oceanic, or H37Rv, but it was increased in the case of East-Africa-Indian strain. B No change was observed in the IRF-mediated response.

NIHMS1528528-supplement-1.docx (759KB, docx)

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