Interferon-γ, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages

Abstract

Mycobacterium tuberculosis (MTB) remains a leading infectious threat to human health. Macrophages are the cells targeted for infection by the bacterium as well as key effector cells for clearance of the pathogen. Interleukin (IL)-27 opposes macrophage-mediated control of MTB because supplying IL-12 and blocking the activity of IL-27 limits bacterial growth in primary human macrophages. The purpose of this study was to determine the immunological regulators of this macrophage mechanism to restrict MTB growth. Interferon (IFN)-γ, TNF-α, and IL-18 were all demonstrated to be important to the environment that limits bacterial growth when IL-12 is supplied and IL-27 is neutralized. We find IL-18 works in conjunction with IL-12 to achieve optimal IFN-γ production in this system. We also demonstrate novel interactions between these cytokines to influence the expression or responsiveness to one another. Quantitative assays show that IFN-γ enhances expression of the IL-18 receptor signaling chain, as well as TNF expression and secretion. In turn, TNF-α augments expression of the receptor for IFN-γ, the amount at the cell surface, and the extent of IFN-γ -induced signaling. We further define how the cytokine environment supports an enhanced state of classical macrophage activation. Collectively, these results describe novel immunological mechanisms that provide additional insights into the effects of IL-12 and IL-27 on macrophage regulation during MTB infection.

Introduction

Mycobacterium tuberculosis (MTB) is an intracellular pathogen and the causative agent of tuberculosis. This organism is among the most successful human pathogens and estimated to have infected one-third of the world’s population. Globally in 2009, there were an estimated 9.4 million new cases of tuberculosis [1]. However, in the face of this large incidence of infection, primary tuberculosis only occurs in approximately 5-10% of those infected [2]. This observation suggests that both the innate and specific immune functions of most infected individuals contain the bacterium. Yet despite this containment, they are typically not sufficient to eliminate the pathogen. A greater understanding of immune mechanisms that function to control the bacterium is a necessary prerequisite to development of therapeutic strategies aimed at more effective clearance of mycobacteria.

T lymphocytes are of unquestionable importance to host protection during tuberculosis [3]. They are essential for proper granuloma formation and maintenance [4], [5], [6]. Activated Th1 cells are also a dominant source of protective cytokines such as IFN-γ and TNF. As such, significant attention is applied to understanding and improving Th1 responses during tuberculosis. However, host macrophages represent the target cell for MTB in the lungs and the effector immune cell at the heart of the granuloma. MTB is able to subvert phagosome maturation and survive within professional phagocytes. Thus, marked improvement can also be made in understanding how innate cells may better control intracellular bacterial growth and cooperate with adaptive responses to reduce the bacterial burden during infection.

Although macrophages and dendritic cells are prototypical producers of IL-12, macrophages do not produce adequate quantities of IL-12 in response to MTB [7], [8]. In contrast, both EBI3 and p28 subunits of the structurally related heterodimeric cytokine, IL-27, are strongly induced by MTB in human macrophages [9]. The immunobiology of IL-27 has become more appreciated over the last decade, and this cytokine is somewhat paradoxical in that it has both proinflammatory and anti-inflammatory activity towards lymphocytes. IL-27 cooperates with IL-12 to initiate Th1 responses and also augments IFN-γ production by these cells [10], [11], [12], [13]. However, the primary function of IL-27 may be to limit inflammatory responses in other circumstances. In experimental models of tuberculosis, mice deficient in IL-27 signaling exhibited improved control of bacteria, but uncontrolled inflammatory responses reduced animal survival over time [14], [15]. This was accompanied by an increase in CD4+ T cell proliferation in the lungs [14], [15], as well as increased proinflammatory cytokine production, including IFN-γ [15]. T cell hyperactivity is also reported in Toxoplasma gondii-infected mice that do not express WSX-1, the ligand binding component of the IL-27 receptor [16]. Other studies have shown direct involvement for IL-27 in negatively regulating T_H-17 cells associated with inflammatory responses [17], [18] and in inducing IL-10-producing suppressive T cells [19].

What has been less well understood is the effect of IL-27 on the innate cells responsible for its production. We have previously demonstrated that supplementing IL-12 along with a soluble receptor to neutralize IL-27 (sIL-27R) reduces MTB recovered from infected human macrophages [9]. We have extended that study in an effort to better understand the immunological parameters and mechanism involved in this response. Our results demonstrate that production of IFN-γ, TNF-α, and IL-18 are most important for reducing the mycobacterial burden in human macrophages when IL-12 is supplied and IL-27 signaling is blocked. Furthermore, IFN-γ, TNF-α, and IL-18 cooperate in a network such that they influence the expression and/or responsiveness to one another. Finally, this study further highlights the inhibitory nature of IL-27 toward human macrophages. In the case of chronic infections such as tuberculosis, this activity may compromise control of the bacteria to prevent overactive immunological responses.