Elsevier

Human Immunology

Volume 74, Issue 6, June 2013, Pages 722-729
Human Immunology

Mycobacterium tuberculosis may escape helper T cell recognition by infecting human fibroblasts

https://doi.org/10.1016/j.humimm.2013.02.005Get rights and content

Abstract

The host immune response can limit Mycobacterium tuberculosis (Mtb) spreading in primary tuberculosis (TB) without eradicating all bacilli, which can persist causing latent TB infection and are responsible for reactivation TB. Persistent Mtb is confined to granulomas within phagocytes, but it is also found in other non-immune cells. We focused on fibroblasts since these cells participate to the granuloma formation and were shown to be infected in latent TB infections.

We show that in vitro both Mtb and Bacille Calmette-Guérin actively replicate in human fibroblasts. Mycobacterial infection of fibroblasts causes a significant inhibition of interferon (IFN)-γ induced membrane expression of major histocompatibility complex class II molecules in these cells. The functional consequence of in vitro infection is a significant reduction of the fibroblast capacity to present peptides and soluble proteins to autologous specific CD4+ T cell clones. Moreover, fibroblasts are capable of presenting antigen derived from the processing of heat-killed Mtb, but not from viable Mtb. Data indicate that IFN-γ treated fibroblasts are capable of presenting antigens derived from the processing of whole bacteria in addition to the capacity to present peptides and isolated proteins. Interestingly, Mtb infected fibroblasts lose this capacity, suggesting that Mtb may evade T helper immune surveillance by infecting fibroblasts.

Introduction

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and is able to infect antigen presenting cells (APCs), including macrophages and dendritic cells (DCs) [1]. In these cells, Mtb survives in modified phagosomes and uses multiple mechanisms to evade both innate and adaptive host immunity, including inhibition of phagosome maturation, resistance to innate microbicidal mechanisms and cytokine-mediated host defenses, as well as inhibition of antigen presentation [2], [3]. Since TB is mainly transmitted via airborne droplets from people with active respiratory disease to susceptible individuals, Mtb most commonly affects the lungs. Thus, it is generally accepted that the first cell types confronting Mtb during primary infection are alveolar macrophages and type II pneumocytes [4], [5]. However, this microorganism can enter a variety of other cell types in vitro, as shown by several authors following the early observations by Shepard, [6] who demonstrated that Mtb can enter monolayers of HeLa, monkey kidney and human amnion cells [6], [7]. More recently, Mtb has been shown to infect lung epithelial and endothelial cells [8], [9], [10], [11] as well as adipocytes [12] and fibroblasts [13], [14], in addition to professional APCs, such as monocytes and DCs, even if the capacity of Mtb to replicate within these different cell types may vary significantly [15], [16]. The capacity of Mtb to infect cells different from macrophages has also been proven by ex vivo experiments showing Mtb or its DNA in different cell types in both TB patients and subjects with latent TB infection (LTBI), corroborating the significance of in vitro experiments [12], [17]. In particular, Mtb was shown to persist intracellularly in lung tissue without histological evidence of tuberculous lesions and Mtb DNA was shown to be situated not only in macrophages but also in other non-professional APCs [17]. The capacity of Mtb to infect and replicate within different cell types may be dependent on both the capacity of cells to internalize and host Mtb and/or to the strength of the immune response, which may force Mtb to colonize alternative cell types possibly in relation to the TB stage. This is relevant since internalization in non-professional APCs may represent an important pathogenic feature of mycobacterial infection. In these cells major histocompatibility complex (MHC) class I expression is constitutive but class II is not expressed in normal conditions, although it may be induced by interferon (IFN)-γ release [18]. Non-professional APCs are not capable of priming antigen specific T cells, but they can present antigen to memory CD4+ T cells in inflammatory microenvironments where they are induced to express MHC class II molecules by locally released IFN-γ. The capacity of mycobacteria to infect, survive and interfere with antigen processing and presentation in these cells is far from being elucidated [19]. Moreover the role of non-professional APCs in TB is intriguing in the light of their possible contribution in maintaining the infection latent and possibly representing the mycobacterial reservoir in LTBI [17]. In this paper we investigated whether the known capacity of Mtb to interfere with MHC class II expression and the consequent impairment of antigen presentation could be observed in non professional APCs such as fibroblasts. In fact, fibroblasts are recruited [20] and proliferate into TB lesions where they are involved in tissue remodeling and granuloma formation, thus representing possible target for Mtb infection.

Section snippets

Ethics statement

This study has been approved by the Istituto Superiore di Sanità review board and written informed consent has been obtained by the healthy volunteers who participated to the study.

Bacterial cultures

Mtb H37Rv (ATCC 27294) and Bacillus Calmette-Guérin (BCG), (ATCC 27291) were grown with gentle agitation (80 r.p.m.) in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.05% Tween 80 (Sigma Chemical Company, St. Louis, MO) and 10% Middlebrook ADC enrichment (Becton Dickinson, Mountain View,

Mycobacteria infect human fibroblasts

The human fibroblasts cell line MRC-5 and RNFib were infected with Mtb or BCG at MOI of 3:1 or 10:1 respectively. After 2 days of incubation cells were fixed and stained by Kinyoun method and microscopically observed to evaluate the cell-mycobacteria interactions. Fig. 1 reports Kinyoun staining of MRC-5 fibroblasts cultures after BCG (Fig. 1A) or Mtb (Fig. 1B) infection. Mycobacteria are associated to fibroblasts and only rare mycobacterial cells are found in the intercellular spaces. In

Discussion

In this work we confirm that in vitro Mtb infects human fibroblasts. Infection seems not to be related to the virulence of Mtb, since also the vaccine strain BCG is endowed with the same capacity to infect and grow within fibroblasts. These observations are in line with previous published data demonstrating the capacity of Mtb, as well as attenuated or non-pathogenic mycobacteria such as M. smegmatis, to infect non-phagocytic cells [8], [9], [10], [11] by macropinocytosis through lamellipodia

Acknowledgments

This work was partially supported by the 7th EC Framework Programme Project “NEWTBVac” Grant #241745.

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