Tom H. M. Ottenhoff

Innate Immunity to Mycobacterium tuberculosis

SUMMARY The different manifestations of infection with Mycobacterium tuberculosis reflect the balance between the bacillus and host defense mechanisms. Traditionally, protective immunity to tuberculosis has been ascribed to T-cell-mediated immunity, with CD4+ T cells playing a crucial role. Recent immunological and genetic studies support the long-standing notion that innate immunity is also relevant in tuberculosis. In this review, emphasis is on these natural, innate host defense mechanisms, referring to experimental data (e.g., studies in gene knockout mice) and epidemiological, immunological, and genetic studies in human tuberculosis. The first step in the innate host defense is cellular uptake of M. tuberculosis, which involves different cellular receptors and humoral factors. Toll-like receptors seem to play a crucial role in immune recognition of M. tuberculosis, which is the next step. The subsequent inflammatory response is regulated by production of pro- and anti-inflammatory cytokines and chemokines. Different natural effector mechanisms for killing of M. tuberculosis have now been identified. Finally, the innate host response is necessary for induction of adaptive immunity to M. tuberculosis. These basic mechanisms augment our understanding of disease pathogenesis and clinical course and will be of help in designing adjunctive treatment strategies.

Vaccines against Tuberculosis: Where Are We and Where Do We Need to Go?

In this review we discuss recent progress in the development, testing, and clinical evaluation of new vaccines against tuberculosis (TB). Over the last 20 years, tremendous progress has been made in TB vaccine research and development: from a pipeline virtually empty of new TB candidate vaccines in the early 1990s, to an era in which a dozen novel TB vaccine candidates have been and are being evaluated in human clinical trials. In addition, innovative approaches are being pursued to further improve existing vaccines, as well as discover new ones. Thus, there is good reason for optimism in the field of TB vaccines that it will be possible to develop better vaccines than BCG, which is still the only vaccine available against TB.

Novel human immunodeficiencies reveal the essential role of type-1 cytokines in immunity to intracellular bacteria

Abstract Studies of patients with severe infections arising from poorly pathogenic mycobacterium or Salmonella species have revealed genetic mutations in type-1 cytokine (IL-12p40) or type-1 cytokine receptor (IFN-γR1, IFN-γR2, IL-12Rβ1) genes. This article summarizes the findings and discusses their implications.

NOD2 and toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis is one of the leading causes of death worldwide. Recognition of M. tuberculosis by pattern recognition receptors is crucial for activation of both innate and adaptive immune responses. In the present study, we demonstrate that nucleotide-binding oligomerization domain 2 (NOD2) and Toll-like receptors (TLRs) are two nonredundant recognition mechanisms of M. tuberculosis. CHO cell lines transfected with human TLR2 or TLR4 were responsive to M. tuberculosis. TLR2 knock-out mice displayed more than 50% defective cytokine production after stimulation with mycobacteria, whereas TLR4-defective mice also released 30% less cytokines compared to controls. Similarly, HEK293T cells transfected with NOD2 responded to stimulation with M. tuberculosis. The important role of NOD2 for the recognition of M. tuberculosis was demonstrated in mononuclear cells of individuals homozygous for the 3020insC NOD2 mutation, who showed an 80% defective cytokine response after stimulation with M. tuberculosis. Finally, the mycobacterial TLR2 ligand 19-kDa lipoprotein and the NOD2 ligand muramyl dipeptide synergized for the induction of cytokines, and this synergism was lost in cells defective in either TLR2 or NOD2. Together, these results demonstrate that NOD2 and TLR pathways are nonredundant recognition mechanisms of M. tuberculosis that synergize for the induction of proinflammatory cytokines.

Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFN-γ- and CD40L-mediated costimulation

Macrophages (Mφ) comprise a heterogeneous population of cells with various immune and homeostatic functions. Recently, we have described type‐1 and type‐2 human monocyte‐derived Mφ subsets. Although both support outgrowth of intracellular mycobacteria, Mφ‐1 secretes interleukin (IL)‐23/IL‐12 and supports T helper cell type 1 (Th1) responses, whereas Mφ‐2 fails to produce IL‐23/IL‐12, predominantly secretes IL‐10, and inhibits Th1 function. Here, we further describe the phenotypic and functional profiles of Mφ‐1 and Mφ‐2 in response to microbial antigens and interferon‐γ (IFN‐γ) and CD40L as costimulatory T cell back‐talk signals. Activated IL‐23+/IL‐12+ Mφ‐1 secreted IL‐1β, IL‐18, IL‐6, and tumor necrosis factor‐α (TNF‐α), as well as IL‐8, monocyte chemoattractant protein‐1 (MCP‐1), IFN‐inducible protein 10 (IP‐10), Mφ inflammatory protein‐1β (MIP‐1β), regulated on activation, normal T expressed and secreted (RANTES), Mφ‐derived chemokine (MDC), and (low levels of) pulmonary and activation‐regulated chemokine and thymus and activation‐regulated chemokine (TARC), corroborating their proinflammatory function. Regardless of the stimulus, Mφ‐2 maintained their IL‐10+ signature cytokine profile and produced no or relatively low levels of IL‐12p40, IL‐1β, IL‐6, TNF‐α, MDC, or TARC. It is remarkable that Mφ‐2 secreted high levels of IL‐8, MCP‐1, IP‐10, MIP‐1β, and RANTES, suggesting an active role for these cells in regulating cellular immunity and homeostasis. Mφ‐1 and Mφ‐2 expressed similar levels of Toll‐like receptor and dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing nonintegrin as microbial pattern recognition receptors. Mφ‐2, unlike Mφ‐1 but like other nonclassical Mφ described previously, expressed CD163 and down‐modulated human leukocyte antigen and costimulatory molecules specifically upon activation. These findings demonstrate how Mφ‐1/Mφ‐2 polarization can differentially skew the host response toward pro‐ or anti‐inflammatory immune responses, respectively. This is likely to be relevant for host‐pathogen interactions in chronic bacterial infections and provides a model for dissecting pro‐ and anti‐inflammatory cascades.

Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4+ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4+ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4+ T‐cell subsets. Proportions of the other double or single CD4+ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4+ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4+ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1.

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome–lysosome fusion through the AS160 (also known as TBC1D4)–RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

Genetics, cytokines and human infectious disease: lessons from weakly pathogenic mycobacteria and salmonellae

Host genetic factors are important in determining the outcome of infections caused by intracellular pathogens, including mycobacteria and salmonellae, but until now have been poorly characterized. Recently, some individuals with severe infections due to otherwise weakly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis bacille Calmette-Guérin) or Salmonella species have been shown to be unable to produce or respond to interferon-γ. This inability results from mutations in any of five genes encoding essential proteins of the type 1 cytokine cascade: interleukin-12p40, interleukin-12Rβ1, interferon-γR1, interferon-γR2 or STAT1. Ten syndromes have thus far been identified. Recent insights in genetically controlled host defense and susceptibility to mycobacterial disease are discussed.

Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells.

IL‐23 is regarded as a major pro‐inflammatory mediator in autoimmune disease, a role which until recently was ascribed to its related cytokine IL‐12. IL‐23, an IL‐12p40/p19 heterodimeric protein, binds to IL‐12Rβ1/IL‐23R receptor complexes. Mice deficient for p19, p40 or IL‐12Rβ1 are resistant to experimental autoimmune encephalomyelitis or collagen‐induced arthritis. Paradoxically, however, IL‐12Rβ2‐ and IL‐12p35‐deficient mice show remarkable increases in disease susceptibility, suggesting divergent roles of IL‐23 and IL‐12 in modulating inflammatory processes. IL‐23 induces IL‐17, which mediates inflammation and tissue remodeling, but the role of IL‐12 in this respect remains unidentified. We investigated the roles of exogenous (recombinant) and endogenous (macrophage‐derived) IL‐12 and IL‐23, on IL‐17‐induction in human T‐cells. IL‐23 enhanced IL‐17 secretion, as did IL‐2, IL‐15, IL‐18 and IL‐21. In contrast, IL‐12 mediated specific inhibition of IL‐17 production. These data support the role of IL‐23 in inflammation through stimulating IL‐17 production by T lymphocytes, and importantly indicate a novel regulatory function for IL‐12 by specifically suppressing IL‐17 secretion. These data therefore extend previous reports that had indicated unique functions for IL‐23 and IL‐12 due to distinct receptor expression and signal transduction complexes, and provide novel insights into the regulation of immunity, inflammation and immunopathology.

Detection of Active Tuberculosis Infection by T Cell Responses to Early-Secreted Antigenic Target 6-kDa Protein and Culture Filtrate Protein 10

The purified protein derivative (PPD) skin test has no predictive value for tuberculosis (TB) in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals because of cross-reactive responses to nonspecific constituents of PPD. T cell responses to early-secreted antigenic target 6-kDa protein (ESAT-6) and the newly identified culture filtrate protein 10 (CFP-10), 2 proteins specifically expressed by M. tuberculosis (MTB) but not by BCG strains, were evaluated. Most TB patients responded to ESAT-6 (92%) or CFP-10 (89%). A minority of BCG-vaccinated individuals responded to both ESAT-6 and CFP-10, their history being consistent with latent infection with MTB in the presence of protective immunity. No responses were found in PPD-negative controls. The sensitivity and specificity of the assay were 84% and 100%, respectively, at a cutoff of 300 pg of interferon-gamma/mL. These data indicate that ESAT-6 and CFP-10 are promising antigens for highly specific immunodiagnosis of TB, even in BCG-vaccinated individuals.