Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

doi:10.1016/j.cellimm.2011.02.006

Cellular Immunology

Volume 268, Issue 2, 2011, Pages 60-67

https://doi.org/10.1016/j.cellimm.2011.02.006 Get rights and content

Abstract

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.

Introduction

Macrophages play an important role in granulomatous responses against Mycobacterium tuberculosis infection [1], [2]. Histological evidence in human tissue has indicated that granulomas mainly consist of infected macrophages surrounded by recruited mononuclear cells, these being mainly monocytes, lymphocytes and fibroblasts [2], [3]. Although it is difficult to study the natural history of granuloma formation in humans, studies in the zebrafish model of Mycobacterium marinum infection [4] have provided evidence for a similar structure to that of human granulomas developed as a response to M. tuberculosis infection [5], [6]. Interestingly, it has been demonstrated that alterations in granuloma organization may promote mycobacterial dissemination rather than contention [6].

The role of newly-recruited monocytes in mycobacterial control inside a granuloma is still not fully understood. Monocytes could come into contact with mycobacterial soluble products and become infected with M. tuberculosis before completing their differentiation. Innate and adaptive responses against bacilli infection may thus become compromised. It is currently unknown whether M. tuberculosis-infected monocytes are able to differentiate into macrophages.

Freshly isolated human monocytes were infected with M. tuberculosis H37Rv at low multiplicity of infection and allowed to differentiate into macrophages in the present study. It was observed that M. tuberculosis infection altered several events related to monocyte to macrophage differentiation and function, including expression of key cell markers, cytokine release, anti-mycobacterial activity and T-cell proliferation.

Section snippets

Mononuclear phagocyte differentiation

Peripheral blood mononuclear cells (PBMC) from healthy individuals containing 2.5 × 10⁵ CD14⁺ cells were plated into 48-well plates (Corning Incorporated Life Science, Lowell, MA) using 1 ml of RPMI-1640 (Gibco-BRL, Grand Island, NY) plus 0.5% pooled human serum (PHS), for 4 h at 37 °C. Wells were extensively washed with pre-warmed phosphate-buffered saline (PBS, Gibco-BRL) plus 0.5% Fetal bovine serum (FBS, Gibco-BRL) to remove non-adherent cells (this was defined as differentiation time zero).

M. tuberculosis infection prevented differentiation marker up-regulation in mononuclear phagocytes

Our previous observations have shown that MDM (120 h differentiation) are more granular, adherent and larger, having increased cytoplasmic projections and increased CD36, CD86, CD68 and HLA-II expression compared to non-differentiated mononuclear phagocytes and freshly isolated cells (Castaño et al., submitted for publication). The percentage of M. tuberculosis-FDA-associated cells was thus determined before differentiation to evaluate the effect of M. tuberculosis on monocyte differentiation

Discussion

Infected macrophages attract immune cells through chemokine production [14]. Newly-recruited monocytes can be derived from the circulation or tissue reservoirs [6], [15] and may be either infected or exposed to mycobacterial or cellular products before becoming fully differentiated into macrophages; they would then not be able to properly execute some effector mechanisms. Our results showed that mononuclear phagocytes infected with live and virulent M. tuberculosis H37Rv did not completely

Conflicts of interest

The authors have neither financial nor personal conflicts of interest in the present study.

Acknowledgments

This study was financed by COLCIENCIAS (Bogotá, Colombia) Grants RC431-2004 (“Centro Colombiano de Investigación en Tuberculosis”) and 111540520270 and Programa de Sostenibilidad 2009–2010 grant from the Universidad de Antioquia. Diana Castaño was awarded a PhD scholarship by COLCIENCIAS. The authors would like to thank the healthy individuals who participated in this study.

References (21)

J. Pieters
Mycobacterium tuberculosis and the macrophage: maintaining a balance
Cell Host Microbe
(2008)
D.O. Co et al.
Mycobacterial granulomas: keys to a long-lasting host–pathogen relationship
Clin. Immunol.
(2004)
R. Lesley et al.
Insights into early mycobacterial pathogenesis from the zebrafish
Curr. Opin. Microbiol.
(2008)
J.M. Davis et al.
The role of the granuloma in expansion and dissemination of early tuberculous infection
Cell
(2009)
M.D. Sánchez et al.
Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment
Microbes Infect.
(2006)
A. Zelenin
Acridine orange as a probe for cell and molecular biology
C.M. Rueda et al.
Characterization of CD4 and CD8 T cells producing IFN-gamma in human latent and active tuberculosis
Tuberculosis (Edinb)
(2010)
M.L. Arcila et al.
Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2
Cell Immunol.
(2007)
W. Peters et al.
Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis
Microbes Infect.
(2003)
R. Doffinger et al.
Host genetic factors and mycobacterial infections: lessons from single gene disorders affecting innate and adaptive immunity
Microbes Infect.
(2006)

There are more references available in the full text version of this article.

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Cellular Immunology

Abstract

Introduction

Section snippets

Mononuclear phagocyte differentiation

M. tuberculosis infection prevented differentiation marker up-regulation in mononuclear phagocytes

Discussion

Conflicts of interest

Acknowledgments

References (21)

Mycobacterium tuberculosis and the macrophage: maintaining a balance

Cell Host Microbe

Mycobacterial granulomas: keys to a long-lasting host–pathogen relationship

Clin. Immunol.

Insights into early mycobacterial pathogenesis from the zebrafish

Curr. Opin. Microbiol.

The role of the granuloma in expansion and dissemination of early tuberculous infection

Cell

Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment

Microbes Infect.

Acridine orange as a probe for cell and molecular biology

Characterization of CD4 and CD8 T cells producing IFN-gamma in human latent and active tuberculosis

Tuberculosis (Edinb)

Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2

Cell Immunol.

Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis

Microbes Infect.

Host genetic factors and mycobacterial infections: lessons from single gene disorders affecting innate and adaptive immunity

Microbes Infect.

Cited by (26)

The role of low molecular weight thiols in Mycobacterium tuberculosis

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The morphological changes of monocytes in peripheral blood as a potential indicator for predicting active pulmonary tuberculosis

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Differentiation of human mononuclear phagocytes increases their innate response to Mycobacterium tuberculosis infection

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Rapid CommunicationMycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

Abstract

Introduction

Section snippets

Mononuclear phagocyte differentiation

M. tuberculosis infection prevented differentiation marker up-regulation in mononuclear phagocytes

Discussion

Conflicts of interest

Acknowledgments

Cell Host Microbe

Clin. Immunol.

Curr. Opin. Microbiol.

Cell

Microbes Infect.

Tuberculosis (Edinb)

Cell Immunol.

Microbes Infect.

Microbes Infect.

Rapid Communication
Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro