Johanneke Kleinnijenhuis

Bacille Calmette-Guérin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes

Adaptive features of innate immunity, recently described as “trained immunity,” have been documented in plants, invertebrate animals, and mice, but not yet in humans. Here we show that bacille Calmette-Guérin (BCG) vaccination in healthy volunteers led not only to a four- to sevenfold increase in the production of IFN-γ, but also to a twofold enhanced release of monocyte-derived cytokines, such as TNF and IL-1β, in response to unrelated bacterial and fungal pathogens. The enhanced function of circulating monocytes persisted for at least 3 mo after vaccination and was accompanied by increased expression of activation markers such as CD11b and Toll-like receptor 4. These training effects were induced through the NOD2 receptor and mediated by increased histone 3 lysine 4 trimethylation. In experimental studies, BCG vaccination induced T- and B-lymphocyte–independent protection of severe combined immunodeficiency SCID mice from disseminated candidiasis (100% survival in BCG-vaccinated mice vs. 30% in control mice). In conclusion, BCG induces trained immunity and nonspecific protection from infections through epigenetic reprogramming of innate immune cells.

Innate immune recognition of Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a major health problem, with 10 million new cases diagnosed each year. Innate immunity plays an important role in the host defense against M. tuberculosis, and the first step in this process is recognition of MTB by cells of the innate immune system. Several classes of pattern recognition receptors (PPRs) are involved in the recognition of M. tuberculosis, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and Nod-like receptors (NLRs). Among the TLR family, TLR2, TLR4, and TLR9 and their adaptor molecule MyD88 play the most prominent roles in the initiation of the immune response against tuberculosis. In addition to TLRs, other PRRs such as NOD2, Dectin-1, Mannose receptor, and DC-SIGN are also involved in the recognition of M. tuberculosis. Human epidemiological studies revealed that genetic variation in genes encoding for PRRs and downstream signaling products influence disease susceptibility, severity, and outcome. More insight into PRRs and the recognition of mycobacteria, combined with immunogenetic studies in TB patients, does not only lead to a better understanding of the pathogenesis of tuberculosis but also may contribute to the design of novel immunotherapeutic strategies.

Mycobacterium tuberculosis induces IL-17A responses through TLR4 and dectin-1 and is critically dependent on endogenous IL-1

In the present study, we dissected the pathways that trigger the IL‐17A responses by MTB. Dectin‐1 and TLR4 were shown to be involved in MTB‐induced IL‐17A production, and blockade of the NOD2, TLR2, or MR had no effect on IL‐17A. The MAPK Erk, known to mediate transcription of IL‐1β mRNA, was strongly involved in the IL‐17A production induced by MTB. The intracellular enzymes caspase‐1 and serine proteases, which process pro‐IL‐1β into the active IL‐1β, were also crucial for the induction of IL‐17A. Lastly, the MTB‐induced IL‐17A response was strongly dependent on signaling through the IL‐1R but not the IL‐6R pathway. In conclusion, the MTB‐induced IL‐17A response relies strongly on the endogenous IL‐1 pathway and IL‐1R signaling. TLR4 and dectin‐1 are the main receptors responsible for mediating the signals responsible for IL‐17A production by MTB. These findings contribute to a better understanding of the host response to mycobacteria and provide the opportunity to explore potential, novel, therapeutic strategies against TB.

Transcriptional and inflammasome-mediated pathways for the induction of IL-1beta production by Mycobacterium tuberculosis

Proinflammatory cytokines of the IL‐1 family play an important role for the anti‐mycobacterial host defense mechanisms. In the present study we have deciphered the pathways leading from recognition of Mycobacterium tuberculosis to the production and release of IL‐1β, the most important member of the IL‐1 family. By stimulating cells defective in various pattern recognition receptors, we could demonstrate that IL‐1β production is induced by M. tuberculosis through pathways involving TLR2/TLR6 and NOD2 receptors. In contrast, TLR4, TLR9 and TLR1 receptors are not involved in IL‐1β induction. Recognition of M. tuberculosis by TLR and NOD2 leads to transcription of proIL‐1β through mechanisms involving ERK, p38 and Rip2, but not JNK. Interestingly, although caspase‐1 is necessary for the processing of proIL‐1β, activation of caspase‐1 is not dependent on the stimulation of cells by M. tuberculosis. Monocytes expressed constitutively active caspase‐1. The secretion of IL‐1β is dependent on the activation of P2X7‐induced pathways by endogenously released ATP. In conclusion, we have dissected the molecular mechanisms responsible for IL‐1β production by M. tuberculosis, and that may contribute to a deeper knowledge of the mechanisms of cell activation by M. tuberculosis.

Autophagy modulates the Mycobacterium tuberculosis‐induced cytokine response

Both autophagy and pro‐inflammatory cytokines are involved in the host defence against mycobacteria, but little is known regarding the effect of autophagy on Mycobacterium tuberculosis (MTB)‐induced cytokine production. In the present study, we assessed the effect of autophagy on production of monocyte‐derived and T‐cell‐derived cytokines, and examined whether two functional polymorphisms in autophagy genes led to altered cytokine production. Blocking autophagy inhibited tumour necrosis factor‐α (TNF‐α) production, while enhancing interleukin‐1β (IL‐1β) production in peripheral blood mononuclear cells stimulated with MTB. Induction of autophagy by starvation or interferon‐γ (IFN‐γ) had the opposite effect. The modulation of both TNF‐α and IL‐1β production by autophagy was induced at the level of gene transcription. Functional polymorphisms in the autophagy genes ATG16L1 and IRGM did not have a major impact on MTB‐induced cytokine production in healthy volunteers, although a moderate effect was observed on IFN‐γ production by the ATG16L1 T300A polymorphism. These data demonstrate the interplay between autophagy and inflammation during host defence against mycobacteria, and future studies to investigate the clinical implications of these effects for the susceptibility to tuberculosis are warranted.

Immunometabolic Pathways in BCG-Induced Trained Immunity

Summary The protective effects of the tuberculosis vaccine Bacillus Calmette-Guerin (BCG) on unrelated infections are thought to be mediated by long-term metabolic changes and chromatin remodeling through histone modifications in innate immune cells such as monocytes, a process termed trained immunity. Here, we show that BCG induction of trained immunity in monocytes is accompanied by a strong increase in glycolysis and, to a lesser extent, glutamine metabolism, both in an in-vitro model and after vaccination of mice and humans. Pharmacological and genetic modulation of rate-limiting glycolysis enzymes inhibits trained immunity, changes that are reflected by the effects on the histone marks (H3K4me3 and H3K9me3) underlying BCG-induced trained immunity. These data demonstrate that a shift of the glucose metabolism toward glycolysis is crucial for the induction of the histone modifications and functional changes underlying BCG-induced trained immunity. The identification of these pathways may be a first step toward vaccines that combine immunological and metabolic stimulation.

Autophagy Controls BCG-Induced Trained Immunity and the Response to Intravesical BCG Therapy for Bladder Cancer

The anti-tuberculosis-vaccine Bacillus Calmette-Guérin (BCG) is the most widely used vaccine in the world. In addition to its effects against tuberculosis, BCG vaccination also induces non-specific beneficial effects against certain forms of malignancy and against infections with unrelated pathogens. It has been recently proposed that the non-specific effects of BCG are mediated through epigenetic reprogramming of monocytes, a process called trained immunity. In the present study we demonstrate that autophagy contributes to trained immunity induced by BCG. Pharmacologic inhibition of autophagy blocked trained immunity induced in vitro by stimuli such as β–glucans or BCG. Single nucleotide polymorphisms (SNPs) in the autophagy genes ATG2B (rs3759601) and ATG5 (rs2245214) influenced both the in vitro and in vivo training effect of BCG upon restimulation with unrelated bacterial or fungal stimuli. Furthermore, pharmacologic or genetic inhibition of autophagy blocked epigenetic reprogramming of monocytes at the level of H3K4 trimethylation. Finally, we demonstrate that rs3759601 in ATG2B correlates with progression and recurrence of bladder cancer after BCG intravesical instillation therapy. These findings identify a key role of autophagy for the nonspecific protective effects of BCG.

The role of autophagy in host defence against Mycobacterium tuberculosis infection

Autophagy is a vital homeostatic process triggered by starvation and other cellular stresses, in which cytoplasmatic cargo is targeted for degradation in specialized structures termed autophagosomes. Autophagy is involved in nutrient regeneration, protein and organelle degradation, but also in clearance of intracellular pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis. Recent studies suggest that induction of autophagy in macrophages is an effective mechanism to enhance intracellular killing of M. tuberculosis, and that the ability of the pathogen to inhibit this process is of paramount importance for its survival. Patient studies have shown genetic associations between tuberculosis and the autophagy gene IRGM, as well as with several genes indirectly involved in autophagy. In this review we will discuss the complex interplay between M. tuberculosis and autophagy, as well as the effect of polymorphisms in autophagy-related genes on susceptibility to tuberculosis.

Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose (600 mg twice daily), ritonavir also inhibits CYP2D6. The effect of low‐dose ritonavir on CYP2D6 is unknown and was investigated in this study.

Polymorphisms in autophagy genes and susceptibility to tuberculosis.

Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02) and MTOR (p = 0.02) and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04). All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.