Peripheral blood mononuclear cells (PBMCs) from healthy donors (Karolinska Hospital Blood Bank, Stockholm, Sweden) were isolated from buffy coat blood by density sedimentation over Lymphoprep (GE Healthcare Life Sciences, Logan, UT). Cells were allowed to adhere in 6-well culture plates (Nunc, NY, Rochester, USA) for 2 h at 37°C in serum free media (RPMI 1640, supplemented with 2 mM L-glutamine and 5 mM HEPES (Hyclone, GE Healthcare Life Sciences). The non-adherent cells were removed by washing with phosphate buffered saline (PBS) and thereafter adherent monocytes were differentiated for 6 days in cell culture media containing 10% of fetal calf sera (FCS) (Sigma-Aldrich, St. Louis, MO). To establish an unbiased culture system that could assess the individual effects of 1,25(OH)₂D₃ compared to conventional M1 and M2 polarization methods, unstimulated control (M0) and 1,25(OH)₂D₃-polarized monocytes were differentiated in the absence of external growth factors using similar protocols as previously described (21, 22). As the maturation and activation status of macrophages differs depending on the in vitro stimulant (23), the cells obtained with the differentiation protocols used are referred to as monocyte-derived cells. Briefly, cells were left unstimulated or conditioned with 50 ng/mL human M-CSF (Life technologies, Carlsbad, CA) or 50 ng/mL human GM-CSF (ImmunoTools, Germany) for 3 days, after which the media was replaced with fresh media with or without M-CSF or GM-CSF (100 g/ml) for another 3 days of culture. To obtain 1,25(OH)₂D₃-polarized monocyte-derived cells or fully polarized M1 or M2 subsets, unstimulated monocyte cultures were treated with 10 nmol vitamin D [1,25(OH)₂D₃, Sigma-Aldrich], while GM-CSF differentiated monocytes were treated with 50 ng/mL interferon-gamma (IFN-γ) and 10 ng/mL lipopolysaccharide (LPS), and M-CSF differentiated monocytes were treated with 20 ng/mL IL-4 (ImmunoTools, Germany), 18–20 h prior to infection with Mtb. The macrophage polarization protocols (8) and nomenclature used in this study is summarized in Table 1. The morphology of monocyte-derived cell cultures was monitored with light microscopy and the viability of adherent cells after 7 days of culture was estimated to >95% using Trypan Blue (Sigma-Aldrich) staining.

We used a fixed dose of 1,25(OH)₂D₃ (10 nmol) that was in a similar range as the doses previously used by us (18, 24) and other groups (17, 19) to study the in vitro effects of 1,25(OH)₂D₃ on human immune cells. The circulating 25(OH)D₃ proform (10–100 nmol/L in serum) is converted to equivalent concentrations of the active 1,25(OH)₂D₃ form inside cells upon uptake of 25(OH)D₃ (20), which is the basis for using the active form in the nanomolar range in cellular experiments, corresponding to a physiological concentration of active vitamin D.

For infection of monocyte-derived cells, the standard laboratory Mtb strain, H37Rv, carrying the green fluorescent protein (GFP)-encoding pFPV2 plasmid (Mtb-GFP) was used (ATCC, Rockville, MD). A multidrug-resistant (MDR)-TB clinical isolate was obtained from the Mtb strain collection from the Public Health Agency of Sweden. The bacteria were stored at −80°C in Middlebrook 7H9 media supplemented with 10% glycerol (Karolinska University Hospital Solna, Sweden). Bacterial aliquots were thawed and cultured in Middlebrook 7H9 media, supplemented with 0.2% (v/v) glycerol, 10% OADC (Oleic Albumin Dextrose Catalase) enrichment and 0.05% (v/v) Tween-80 (Karolinska University Hospital Solna, Sweden) for 2–3 days. Bacteria were then sub-cultured in 50 ml tubes (dilution of bacterial stock 1:20 in new Middlebrook 7H9 media) and incubated at 37°C on a shaker for 7–10 days. Bacterial suspensions were pelleted and washed with phosphate buffer saline pH 7.4 (PBS) containing 0.05% (v/v) Tween-80 and then resuspended in 5 ml of RPMI complete media before transfer into a sterile 15 ml falcon tube. After 10 min pulse-sonication of inoculi, the optical density was measured at 600 nm. The final bacterial concentration was adjusted to ~2.5 × 10⁶ CFU/ml by adding RPMI complete media without antibiotics.

Primary monocytes were plated in 6-well plates at a concentration of 10⁶ cells per well and allowed to differentiate for 7 days before infection with Mtb for 4 h in 37°C at a multiplicity of infection (MOI) of 5. After infection, the cells were washed vigorously three times with PBS-0.05%Tween to remove extracellular bacteria and thereafter resuspended in cell culture media without antibiotics for further experiments and analyses.

Adherent Mtb-infected monocyte-derived cells were detached at 4, 24, or 72 h post-infection using 1.5 mM EDTA (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) for 10–15 min at room temperature. Detached cells (viability <90–95%) were washed with FACS buffer [PBS containing 0.5% (v/v) FCS and 0.5 mM EDTA] and 0.5–1 × 10⁶ cells were stained for 30 min at 4°C with fluorochrome-conjugated anti-human antibodies: CD68 (PE-Cy7), TLR2 (FITC), CD200R (PE), CD163 (BV605), CD206 (APC-Cy7), CD64 (PE-Dazzle 594), HLADR (PE-Cy5), CCR7 (BV711), CD80 (BV650), and CD86 (BV421) (Biolegend, San Diego, CA). Cells were washed twice with FACS buffer and fixed with 4% formaldehyde (Sigma-Aldrich) at room temperature for 10 min. Fifty thousand cells were acquired from each well by using BD LSR Fortessa (BD Biosciences, San Jose, CA) and analyzed with FlowJo v.9. H37Rv-GFP expression in CD68-positive macrophages was visualized in the FITC channel.

On day 3 post-infection, monocyte-derived cell cultures were lysed with 0.036% SDS-lysis buffer (Sodium dodecyl sulfate from Sigma dissolved in water) for 5 min to release intracellular bacteria. Cell lysis was confirmed by fast-contrast microscopy. Standard CFU counts were determined by diluting samples 1/10 to 1/10,000 by mixing 100 μl of bacterial suspension in 900 μl PBS containing 0.05% (v/v) Tween-80 and finally plating a volume of 100 μl on Middlebrook 7H11 agar plates with 10% OADC (Karolinska University Hospital Solna, Sweden). Each dilution was performed in duplicates. The plates were incubated at 37°C for a minimum of 21 days before colonies were manually counted. As a negative control, supernatants from Mtb-infected cell cultures were collected before cell lysis and plated for CFU counts, which confirmed the absence of bacterial growth i.e., no extracellular Mtb bacilli remaining in Mtb-infected cell cultures.

Human lung tissue biopsies (TB lesion and distal sites), obtained from patients with non-cavitary TB (n = 5) as previously described (25, 26), were freeze-fixed in OCT (Tissue-TEK, Sakura) before cutting two 50 μm cryosections that were homogenized in 1 ml of TRI-reagent and processed for mRNA extraction. Similarly, RNA was extracted from uninfected or Mtb-infected monocyte-derived cells 24 h post-infection using Ribopure RNA extraction kit as described by the manufacturer (Ambion, Thermo Fisher Scientific, Waltham, MA). cDNA was synthesized using Super Script Vilo™ cDNA Synthesis Kit (Applied Biosystems, Foster City, CA). Transcripts of Arg1, Arg2, CAMP, NOS2A, CCL2, CCL22, TNFα, TGFβ, IL-10, TLR2, IL-6, IL-1β, IL-1RA, and IDO (Indoleamine 2,3-dioxygenase) (Applied Biosystems) were measured in duplicates relative to the reference gene, housekeeping 18S rRNA (18S rRNA-housekeeping gene kit, Applied Biosystems), using quantitative real-time PCT (RT-PCR) (QuantStudio 5 Real-Time PCR System, Thermo Fisher Scientific, MA, USA). The results were analyzed by using the relative standard method. Briefly, the relative expression of the target genes was calculated by relating the Ct-value for Mtb-infected to uninfected M0 monocyte-derived cells. Data are presented as fold change of mRNA.

Data are presented as the mean and standard deviation (SD) or median and interquartile range (IQR) from two or more individual experiments including monocyte-derived cells from a minimum of six healthy donors. The analyses used to calculate indicated P-values include one-way Anova, Kruskal–Wallis and Dunn's post-test as well as the Mann–Whitney or Wilcoxon-singed rank test. A P < 0.05^* was considered statistically significant. The statistical analyses were done in GraphPad Prism-6.

Initially, surface expression of selected M1 (CCR7, CD64, TLR2) or M2 (CD163, CD200R, CD206) markers as well as antigen-presenting (HLADR) and co-stimulatory (CD86, CD80) molecules was investigated on uninfected monocyte-derived cells after immune polarization in vitro. CCR7, CD64, TLR2, and CD86 were all significantly (P < 0.05–0.0001) up-regulated on M1-polarized cells, and the expression of these markers were also relatively higher on M1-polarized compared with M1-like cells (Figures 1A–E). M2-polarized cell subsets had a down-regulated expression of CCR7, CD64, and HLA-DR (P < 0.05–0.001) compared with unstimulated M0 cells (Figures 1A,B,E) and also a significantly (P < 0.01) lower expression of CD80 compared to M1-polarized cells (Figure 1I). Instead, M2-like and M2-polarized cells showed a significant up-regulation of the M2 markers CD163 (P < 0.05), CD200R (P < 0.0001), and CD206 (P < 0.05) (Figures 1F–H). 1,25(OH)₂D₃-polarized cells up-regulated TLR2, CD64 and HLADR (P < 0.05–0.01) compared with the M2 subsets (Figures 1B,C,E) and simultaneously showed a lower expression of CD163 and CD200R (P < 0.05–0.0001) (Figures 1F,G). In comparison with the M1 subsets, 1,25(OH)₂D₃-polarization resulted in a lower expression of CCR7, CD64, CD86 (P < 0.05–0.0001) (Figures 1A,B,D). Similar to M0 cells, 1,25(OH)₂D₃-polarized cells had a relatively higher expression of CD68 compared with M1 as well as M2 polarized cell subsets (Figure 1J). Thus, polarization of uninfected monocyte-derived cells toward M1 or M2 resulted in up- or down-regulation of typical M1/M2 surface markers, while 1,25(OH)₂D₃-polarization did not promote either M1 or M2 differentiation.

Next, light microscopy was used to study the morphology of immune polarized monocyte cultures (Figure 2). Unstimulated 1,25(OH)₂D₃-polarized cells showed a more circular morphology compared with M1 and M2 cells (Figures 2A–F). As expected, M1-like and M1-polarized cells had a more elongated and stretched or dendritic-like morphology compared with the other subsets (Figures 2C,D). M2 subsets were confluent but with a rounded shape, and M2-polarized cells were more circular compared with M2-like cells (Figures 2E,F). Overall, these results confirmed that M1 and M2 subsets had a phenotype and morphology consistent with classically and alternatively activated macrophages, respectively. Instead, 1,25(OH)₂D₃-polarized cells maintained the morphology of less mature macrophages, but a high surface expression of CD68, CD64, and HLADR were consistent with differentiation of monocyte-derived macrophage-like cells.

To explore if vitamin D could affect Mtb uptake and subsequently a productive infection of in vitro polarized monocyte-derived cells, expression of GFP-labeled Mtb (H37Rv) was assessed after 4 h (uptake), 24 and 72 h (productive infection) post-infection of the different subsets. At 4 h, we observed a relatively lower percentage of M1-like and M1-polarized cells expressing H37Rv-GFP compared with the other subsets (Figures 3A,B), and this difference was significant comparing the M1 subsets with the M2 subsets (P < 0.01–0.001) (Figure 3B). Mtb uptake was evident in 18–19% of M1 cells, while ~30% of unstimulated M0 as well as 1,25(OH)₂D₃-polarized cells, and about 82% of M2 cells, were H37Rv-GFP-postive at 4 h (Figures 3A,B). However, at 24 and 72 h post-Mtb infection, the level of GFP-expressing cells rapidly and gradually increased in the M1 subsets (P < 0.05), while Mtb infectivity declined in M2-like cells (P < 0.05) and remained unchanged in the other monocyte-derived cell subsets (Figure 3B). At 24 h, the level of productive Mtb infection was significantly lower in 1,25(OH)₂D₃-polarized cells compared with the M2-like and M2-polarized cells (P < 0.05) (Figure 3B).

Next, intracellular growth of Mtb was assessed at 4 and 72 h post-infection with H37Rv (Figure 3C) or a clinical MDR-TB isolate (Figure 3D) using CFU counts. At 4 h post-infection, we observed substantially elevated CFU counts in the M2 subsets compared with the 1,25(OH)₂D₃ and M1 groups (P < 0.05–0.01) (Figures 3C,D), indicating a reduced colony forming capacity of Mtb bacilli that has been taken up by 1,25(OH)₂D₃-polarized or M1 cells. This could result from enhanced bacterial killing or reduced metabolic activity (i.e., latency) of the bacteria. Despite a lower Mtb uptake in M1 cells compared with 1,25(OH)₂D₃-polarized cells (Figure 3B), the colony forming capacity was similar in these monocyte-derived cell subsets at 4 h (Figures 3C,D). Consistent with the FACS-data, significantly (P < 0.05–0.01) enhanced intracellular bacterial growth was detected in the M1- as well as M2-polarized subsets compared with 1,25(OH)₂D₃-polarized cells, 72 h post-infection (Figures 3C,D). Notably, Mtb growth in the 1,25(OH)₂D₃-polarized subset was around 40% lower compared with unstimulated M0 cells (P < 0.05), while Mtb growth was around 100–200% higher in the M1 and M2 subsets at 72 h (Figures 3C,D). Accordingly, intracellular Mtb growth was maintained at relatively lower levels in 1,25(OH)₂D₃-polarized compared with unstimulated M0 cells, while there was substantial growth in both M1 and M2 cells (Figures 3C,D).

Next, we assessed surface expression of the tested panel of M1/M2-specific markers on in vitro polarized monocyte-derived cells 4 h after infection with H37Rv-GFP. Cells in the same sample were divided into GFP-positive (Mtb-infected) and GFP-negative (uninfected) expression. Interestingly, Mtb infection altered the expression of typical M1 phenotype markers (Figures 1A–D), which were all elevated on M2 compared with M1 cell subsets (Figures 4A–D). Instead, the M2 marker CD163 was significantly up-regulated on GFP-positive M1-like (P < 0.05) and M1-polarized (P < 0.01) cells, respectively (Figure 4E). All H37Rv-GFP-positive subsets but M2, exhibited a relative up-regulation of all markers except CD200R (Figure 4). Furthermore, CD86 was significantly (P < 0.05–0.001) up-regulated on all GFP-positive subsets but unstimulated M0 cells (Figure 4D). 1,25(OH)₂D₃-polarized cells also displayed a significant (P < 0.05) up-regulation of HLA-DR (Figure 4C), the mannose receptor CD206 (Figure 4G) and CD80 (Figure 4H). Overall, these results suggest that Mtb-infected monocyte-derived cell subsets experience phenotypical alterations suggestive of a mixed M1/M2 profile.

Assessment of kinetic changes of M1/M2-specific markers on M2-like macrophages, revealed a rapid up-regulation of surface markers at 4–12 h post-Mtb infection that declined already at 24–36 h after infection (Supplementary Figure 1). At 24 and 72 h post-Mtb infection, there were no significant differences in the expression of M1 or M2 markers between GFP-positive and GFP-negative cells or comparing the different subsets (data not shown).

To obtain additional information of the functional properties of in vitro polarized monocyte-derived cell subsets, quantitative mRNA analyses were performed on Mtb-infected cells 24 h post-infection and compared with uninfected cells. Mtb infection induced production of pro-inflammatory cytokines, IL-1β and TNF-α (Figures 5A,B) or IL-6 (data not shown), in the different subsets (P < 0.05), although both IL-1β and TNF-α was relatively higher in 1,25(OH)₂D₃-polarized cells (Figures 5A,B). Moreover, Mtb enhanced expression of the monocyte chemoattractant CCL2 in 1,25(OH)₂D₃-polarized cells, but also in the M1-like subset (P < 0.05) (Figure 5C). Instead, the IL-1β antagonist, IL-1RA, was significantly induced in Mtb-infected M2-polarized subset (P < 0.0001) compared with the unstimulated control (Figure 5D). All other Mtb-infected monocyte-derived cell subsets, apart from 1,25(OH)₂D₃-polarized cells, also had a significant up-regulation of IL-1RA (P < 0.05) (Figure 5D). Similarly, Mtb infection induced a high mRNA expression of the immunosuppressive enzyme IDO, in M0, M1 as well as the M2 subsets (P < 0.05) (Figure 5E). Contrary, IDO mRNA was very low in 1,25(OH)₂D₃-polarized cells (Figure 5E). IDO mRNA was also induced in uninfected M1-polarized cells, which is consistent with the finding that IFN-γ up-regulates IDO (27). IL-10 mRNA was induced in both 1,25(OH)₂D₃ and M2-polarized uninfected cells (P < 0.001) and increased in M1-like as well as M2-like cells (P < 0.05) after Mtb infection (Figure 5F). mRNA expression of the intracellular enzymes arginase, Arg-1 (Figure 5G) and Arg-2 (data not shown), was primarily observed in M1- as well as M2-polarized subsets post-Mtb infection (P < 0.05), whereas inducible nitric oxide (iNOS) was relatively higher in uninfected as well as Mtb-infected M1 cells (P < 0.05) (Figure 5H). Mtb also induced iNOS in M2-polarized cells and to a lesser extent in 1,25(OH)₂D₃-polarized cells (Figure 5H). As expected, mRNA induction of the antimicrobial peptide, LL-37, was superior in 1,25(OH)₂D₃-polarized monocyte-derived cells (P < 0.001). Mtb infection resulted in a potent down-regulation of LL-37 expression in all subsets (P < 0.05) (Figure 5I), but was still significantly higher in 1,25(OH)₂D₃-stimulated compared with M1-like (P < 0.05) and M2-polarized (P < 0.0001) subsets (Figure 5I). LL-37 was not induced in unstimulated M0 cells (Figure 5I). Likewise, the autophagy markers LC3B as well as Atg5 and beclin-1 (data not shown) were selectively up-regulated in 1,25(OH)₂D₃-polarized cells. mRNA expression profiles in monocyte-derived cells infected with H37Rv or the clinical MDR-TB isolate were very similar and revealed no significant differences between these mycobacterial strains (data not shown).

Overall, Mtb infection resulted in enhanced or unchanged transcription of the different molecules investigated, with the exception from LL-37 mRNA expression that was significantly suppressed by Mtb infection. Corresponding mRNA profiling of lung tissue samples obtained from patients with non-cavitary TB, confirmed that most of the investigated markers were up-regulated in the TB lesions (site of Mtb infection) compared with unaffected lung parenchyma, supporting the presence of a mixed M1/M2 macrophage polarization in vivo (Supplementary Figure 2).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.