Constance J. Martin

Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis

Two different forms of death are commonly observed when Mycobacterium tuberculosis (Mtb)-infected macrophages die: (i) necrosis, a death modality defined by cell lysis and (ii) apoptosis, a form of death that maintains an intact plasma membrane. Necrosis is a mechanism used by bacteria to exit the macrophage, evade host defenses, and spread. In contrast, apoptosis of infected macrophages is associated with diminished pathogen viability. Apoptosis occurs when tumor necrosis factor activates the extrinsic death domain pathway, leading to caspase-8 activation. In addition, mitochondrial outer membrane permeabilization leading to activation of the intrinsic apoptotic pathway is required. Both pathways lead to caspase-3 activation, which results in apoptosis. We have recently demonstrated that during mycobacterial infection, cell death is regulated by the eicosanoids, prostaglandin E2 (proapoptotic) and lipoxin (LX)A4 (pronecrotic). Although PGE2 protects against necrosis, virulent Mtb induces LXA4 and inhibits PGE2 production. Under such conditions, mitochondrial inner membrane damage leads to macrophage necrosis. Thus, virulent Mtb subverts eicosanoid regulation of cell death to foil innate defense mechanisms of the macrophage.

Macrophages clean up: efferocytosis and microbial control

Phagocytic leukocytes, predominantly macrophages, not only ingest and destroy invading pathogens, but are charged with clearing dead and dying host cells. The process of engulfing apoptotic cells is called efferocytosis and has long been appreciated for its role in the resolution of inflammation. New evidence is emerging that efferocytosis represents a double-edged sword in microbial immunity. Although efferocytosis of influenza and Mycobacterium tuberculosis-infected cells results in pathogen destruction, efferocytosis of Leishmania-infected neutrophils may promote infection. Understanding how macrophages, dendritic cells (DC) and neutrophils process pathogens encased within a dying cell could lead to the development of novel therapeutics that simultaneously suppress inflammation and promote pathogen clearance.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Lipids, apoptosis, and cross-presentation: links in the chain of host defense against Mycobacterium tuberculosis

Eicosanoids regulate whether human and murine macrophages infected with Mycobacterium tuberculosis die by apoptosis or necrosis. The death modality is important since apoptosis is associated with diminished pathogen viability and should be viewed as a form of innate immunity. Apoptotic vesicles derived from infected macrophages are also an important source of bacterial antigens that can be acquired by dendritic cells to prime antigen-specific T cells. This review integrates in vitro and in vivo data on how apoptosis of infected macrophages is linked to development of T cell immunity against M. tuberculosis.

A bug’s life in the granuloma

The granuloma is the defining feature of the host response to infection with Mycobacterium tuberculosis (Mtb). Despite knowing of its existence for centuries, much remains unclear regarding the host and bacterial factors that contribute to granuloma formation, heterogeneity of presentation, and the forces at play within. Mtb is highly adapted to life within the granuloma and employs many unique strategies to both create a niche within the host as well as survive the stresses imposed upon it. Adding to the complexity of the granuloma is the vast range of pathology observed, often within the same individual. Here, we explore some of the many ways in which Mtb crafts the immune response to its liking and builds a variety of granuloma features that contribute to its survival. We also consider the multitude of ways that Mtb is adapted to life in the granuloma and how variability in the deployment of these strategies may result in different fates for both the bacterium and the host. It is through better understanding of these complex interactions that we may begin to strategize novel approaches for tuberculosis treatments.

Critical role for Invariant chain in CD1d-mediated selection and maturation of Vα14-invariant NKT cells

The development and maturation of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated lipid antigen presentation in the thymus and the periphery. Cortical thymocytes mediate positive selection, while professional APCs are involved in thymic negative selection and in terminal maturation of iNKT cells in the periphery. CD1d requires entry in the endosomal pathway to allow antigen acquisition for assembly as lipid/CD1d complexes for display to iNKT cells. This process involves tyrosine-based sorting motifs in the CD1d cytoplasmic tail and invariant chain (Ii) that CD1d associates with in the endoplasmic reticulum. The function of Ii in iNKT cell thymic development and peripheral maturation had not been fully understood. Using mice deficient in Ii and the Ii-processing enzyme cathepsin S (catS), we addressed this question. Ii(-/-) mice but not catS(-/-) mice developed significantly fewer iNKT cells in thymus, that were less mature as measured by CD44 and NK1.1 expression. Ii(-/-) mice but not catS(-/-) mice developed fewer Vβ7(+) cells in their iNKT TCR repertoire than WT counterparts, indicative of a change in endogenous glycolipid antigen/CD1d-mediated iNKT cell selection. Finally, using a Mycobacterium tuberculosis infection model in macrophages, we show that iNKT developed in Ii(-/-) but not catS(-/-) mice have defective effector function. Our data support a role for professional APCs expressing Ii, but no role for catS in the thymic development and peripheral terminal maturation of iNKT cells.

Requirement for invariant chain in macrophages for Mycobacterium tuberculosis replication and CD1d antigen presentation

ABSTRACT Mycobacterium tuberculosis is an intracellular bacterium that persists in phagosomes of myeloid cells. M. tuberculosis-encoded factors support pathogen survival and reduce fusion of phagosomes with bactericidal lysosomal compartments. It is, however, not entirely understood if host factors that mediate endosomal fusion affect M. tuberculosis intracellular localization and survival. Neither is it known if endosomal fusion influences induction of host immune reactivity by M. tuberculosis-infected cells. Lysosomal degradation of M. tuberculosis appears to be pivotal for making available lipid substrates for assembly into lipid-CD1d complexes to allow activation of CD1d-restricted invariant natural killer T (iNKT) cells. To clarify the role for endosomal fusion in M. tuberculosis survival and induction of host CD1d-mediated immune defense, we focused our studies on the invariant chain (Ii). Ii regulates endosome docking and fusion and thereby controls endosomal transport. Through direct binding, Ii also directs intracellular transport of the class II major histocompatibility complex and CD1d. Our findings demonstrate that upon infection of Ii-knockout (Ii−/−) macrophages, M. tuberculosis is initially retained in early endosomal antigen 1-positive lysosomal-associated membrane protein 1-negative phagosomes, which results in slightly impaired pathogen replication. The absence of Ii did not affect the ability of uninfected and infected macrophages to produce nitric oxide, tumor necrosis factor alpha, or interleukin-12. However, induction of cell surface CD1d was impaired in infected Ii−/− macrophages, and CD1d-restricted iNKT cells were unable to suppress bacterial replication when they were cocultured with M. tuberculosis-infected Ii−/− macrophages. Thus, while the host factor Ii is not essential for the formation of the M. tuberculosis-containing vacuole, its presence is crucial for iNKT cell recognition of infected macrophages.

Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques

For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immunity in the lung, which may inform TB vaccine design and development.