Polarization of Human Monocyte-Derived Cells With Vitamin D Promotes Control of Mycobacterium tuberculosis Infection

Abstract

Background: Understanding macrophage behavior is key to decipher Mycobacterium tuberculosis (Mtb) pathogenesis. We studied the phenotype and ability of human monocyte-derived cells polarized with active vitamin D [1,25(OH) 2 D 3 ] to control intracellular Mtb infection compared with polarization of conventional subsets, classical M1 or alternative M2. Methods: Human blood-derived monocytes were treated with active vitamin D or different cytokines to obtain 1,25(OH) 2 D 3-polarized as well as M1-and M2-like cells or fully polarized M1 and M2 subsets. We used an in vitro macrophage Mtb infection model to assess both phenotype and functional markers i.e., inhibitory and scavenger receptors, costimulatory molecules, cytokines, chemokines, and effector molecules using flow cytometry and quantitative mRNA analysis. Intracellular uptake of bacilli and Mtb growth was monitored using flow cytometry and colony forming units. Results: Uninfected M1 subsets typically expressed higher levels of CCR7, TLR2, and CD86, while M2 subsets expressed higher CD163, CD200R, and CD206. Most of the investigated markers were up-regulated in all subsets after Mtb infection, generating a mixed M1/M2 phenotype, while the expression of CD206, HLADR, and CD80 was specifically up-regulated (P < 0.05) on 1,25(OH) 2 D 3-polarized macrophages. Consistent with the pro-inflammatory features of M1 cells, Mtb uptake and intracellular Mtb growth was significantly (P < 0.01-0.001 and P < 0.05-0.01) lower in the M1 (19.3%) compared with the M2 (82.7%) subsets 4 h post-infection. However, infectivity rapidly and gradually increased in M1 cells at 24-72 h. 1,25(OH) 2 D 3-polarized monocyte-derived cells was the most potent subset to inhibit Mtb growth at both 4 and 72 h (P < 0.05-0.01) post-Mtb infection. This ability was associated with high mRNA levels of pro-inflammatory cytokines and the antimicrobial peptide LL-37 but also anti-inflammatory IL-10, while expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase) remained low in Mtb-infected 1,25(OH) 2 D 3-polarized cells compared with the other subsets. Rao Muvva et al. Vitamin D-Mediated Macrophage Polarization in Tuberculosis Conclusions: Mtb infection promoted a mixed M1/M2 macrophage activation, and 1,25(OH) 2 D 3-polarized monocyte-derived cells expressing LL-37 but not IDO, were most effective to control intracellular Mtb growth. Macrophage polarization in the presence of vitamin D may provide the capacity to mount an antimicrobial response against Mtb and simultaneously prevent expression of inhibitory molecules that could accelerate local immunosuppression in the microenvironment of infected tissue.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon <em>Mycobacterium tuberculosis</em> Infection

“…NOTE: For differentiation and polarization of monocytederived cells, a protocol that we previously established for M0, M1-like and M2-like cells as well as fully M1 and M2 polarized cells was followed 25 . For simplicity, only fully polarized M1 and M2 macrophages are described here.…”

“…Furthermore, the viability stain revealed an enhanced cell death (17-22%) among the Mtb-infected M1 and M2 cells at a MOI of 5, compared to uninfected M0 cells (99%) (Figure 2A-C To characterize the efficacy of M1/M2 polarization in uninfected monocyte-derived cells, dot plots were used to identify M1 cells that were double-positive for CD64 and CD86 (CD64 + CD86 + ) and M2 cells that were double-positive for CD163 and CD200R (CD163 + CD200R + ; Figure 3A,B). The selection of M1/M2 markers was primarily made based on the results from our previous work 25 but also from other studies 26 , 27 , 28 , 29 . The quadrants for the stained cells, were set using corresponding gates for unstained M1/M2 cells ( Figure 3A).…”

A DNA methylome biosignature in alveolar macrophages from TB-exposed individuals predicts exposure to mycobacteria

“…VEGF-release (P00056) by macrophages has been shown to recruit immune cells during granuloma formation 37 . Further, vitamin D has been shown to strengthen the anti-mycobacterial activity of macrophages 17, 38 , and upregulation of components of the vitamin D pathway is linked to the production of anti-microbial peptides 18 , providing a possible effector mechanism for mycobacterial control. Recent literature on immune regulation through T cell-derived acetylcholine 39, 40 attributes relevance to the acetylcholine receptor pathway identified among the HLA-DR pathways.…”

Androgen and Androgen Receptors as Regulators of Monocyte and Macrophage Biology in the Healthy and Diseased Lung

“…While the M1/M2 designations are useful for in vitro studies with stimulation with defined cytokines, the in vivo phenotype of macrophages exists on a spectrum somewhere in between these two well-defined opposing phenotypes or does not fit the paradigm at all. For example, M1 and M2 markers can exist simultaneously within the same cell in some cases ( 162 – 164 ). The key factors dictating the macrophage phenotype or activation state are the stage of the immune response and the soluble factors and interactions in a particular tissue microenvironment.…”