Arjan van Laarhoven

Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

Summary The interaction of microbes with pattern recognition receptors (PRRs) is essential for protective immunity. While many PRRs that recognize mycobacteria have been identified, none is essentially required for host defense in vivo. Here, we have identified the C-type lectin receptor CLECSF8 (CLEC4D, MCL) as a key molecule in anti-mycobacterial host defense. Clecsf8−/− mice exhibit higher bacterial burdens and increased mortality upon M. tuberculosis infection. Additionally, Clecsf8 deficiency is associated with exacerbated pulmonary inflammation, characterized by enhanced neutrophil recruitment. Clecsf8−/− mice show reduced mycobacterial uptake by pulmonary leukocytes, but infection with opsonized bacteria can restore this phagocytic defect as well as decrease bacterial burdens. Notably, a CLECSF8 polymorphism identified in humans is associated with an increased susceptibility to pulmonary tuberculosis. We conclude that CLECSF8 plays a non-redundant role in anti-mycobacterial immunity in mouse and in man.

Immunity Against Heterosubtypic Influenza Virus Induced By Adenovirus And MVA Expressing Nucleoprotein And Matrix Protein-1

Alternate prime/boost vaccination regimens employing recombinant replication-deficient adenovirus or MVA, expressing Influenza A virus nucleoprotein and matrix protein 1, induced antigen-specific T cell responses in intradermally (ID) vaccinated mice; with the strongest responses resulting from Ad/MVA immunization. In BALB/C mice the immunodominant response was shifted from the previously identified immunodominant epitope to a novel epitope when the antigen was derived from A/Panama/2007/1999 rather than A/PR/8. Alternate immunization routes did not affect the magnitude of antigen-specific systemic IFN-γ response, but higher CD8+ T-cell IFN-γ immune responses were seen in the bronchoalveolar lavage following intransal (IN) boosting after intramuscular (IM) priming, whilst higher splenic antigen-specific CD8+ T cell IFN-γ was seen following IM boosting. Partial protection against heterologous influenza virus challenge was achieved following either IM/IM or IM/IN but not ID/ID immunization. These data may be of relevance for the design of optimal immunization regimens for human influenza vaccines, especially for influenza-naïve infants.

Improved adjuvanting of seasonal influenza vaccines: Preclinical studies of MVA‐NP+M1 coadministration with inactivated influenza vaccine

Licensed seasonal influenza vaccines induce antibody (Ab) responses against influenza hemagglutinin (HA) that are limited in their ability to protect against different strains of influenza. Cytotoxic T lymphocytes recognizing the conserved internal nucleoprotein (NP) and matrix protein (M1) are capable of mediating a cross‐subtype immune response against influenza. Modified vaccinia Ankara (MVA) virus encoding NP and M1 (MVA‐NP+M1) is designed to boost preexisting T‐cell responses in adults in order to elicit a cross‐protective immune response. We examined the coadministration of HA protein formulations and candidate MVA‐NP+M1 influenza vaccines in murine, avian, and swine models. Ab responses postimmunization were measured by ELISA and pseudotype neutralization assays. Here, we demonstrate that MVA‐NP+M1 can act as an adjuvant enhancing Ab responses to HA while simultaneously inducing potent T‐cell responses to conserved internal Ags. We show that this regimen leads to the induction of cytophilic Ab isotypes that are capable of inhibiting hemagglutination and in the context of H5 exhibit cross‐clade neutralization. The simultaneous induction of T cells and Ab responses has the potential to improve seasonal vaccine performance and could be employed in pandemic situations.

Low Induction of Proinflammatory Cytokines Parallels Evolutionary Success of Modern Strains within the Mycobacterium tuberculosis Beijing Genotype

ABSTRACT One of the most widespread clades of Mycobacterium tuberculosis worldwide, the Beijing genotype family, consists of ancient (atypical) and modern (typical) strains. Modern Beijing strains outcompete ancient strains in terms of prevalence, while reserving a higher degree of genetic conservation. We hypothesize that their selective advantage lies in eliciting a different host immune response. Bead-disrupted lysates of a collection of different M. tuberculosis strains of the modern (n = 7) or ancient (n = 7) Beijing genotype, as well as the Euro-American lineage (n = 6), were used for induction of ex vivo cytokine production in peripheral blood mononuclear cells (PBMCs) from 10 healthy individuals. Hierarchical clustering and multivariate regression analyses were used to study possible differences in production of nine cytokines. Modern and ancient M. tuberculosis Beijing genotypes induced different cytokine signatures. Overall induction of interleukin-1β (IL-1β), gamma interferon (IFN-γ), and IL-22 was 38 to 40% lower after stimulation with modern Beijing strains (corrected P values of <0.0001, 0.0288, and 0.0002, respectively). Euro-American reactivation strains induced 2-fold more TNF-α production than both types of Beijing strains. The observed differences in cytokine induction point to a reduction in proinflammatory cytokine response as a possible contributing factor to the evolutionary success of modern Beijing strains.

Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells

Cells in homeostasis metabolize glucose mainly through the tricarboxylic acid cycle and oxidative phosphorylation, while activated cells switch their basal metabolism to aerobic glycolysis. In this study, we examined whether metabolic reprogramming toward aerobic glycolysis is important for the host response to Mycobacterium tuberculosis (Mtb). Through transcriptional and metabolite analysis we show that Mtb induces a switch in host cellular metabolism toward aerobic glycolysis in human peripheral blood mononuclear cells (PBMCs). The metabolic switch is TLR2 dependent but NOD2 independent, and is mediated in part through activation of the AKT‐mTOR (mammalian target of rapamycin) pathway. We show that pharmacological inhibition of the AKT/mTOR pathway inhibits cellular responses to Mtb both in vitro in human PBMCs, and in vivo in a model of murine tuberculosis. Our findings reveal a novel regulatory layer of host responses to Mtb that will aid understanding of host susceptibility to Mtb, and which may be exploited for host‐directed therapy.

Cerebral tryptophan metabolism and outcome of tuberculous meningitis: an observational cohort study

BACKGROUND Immunopathology contributes to the high mortality of tuberculous meningitis, but the biological pathways involved are mostly unknown. We aimed to compare cerebrospinal fluid (CSF) and serum metabolomes of patients with tuberculous meningitis with that of controls without tuberculous meningitis, and assess the link between metabolite concentrations and mortality. METHODS In this observational cohort study at the Hasan Sadikin Hospital (Bandung, Indonesia) we measured 425 metabolites using liquid chromatography-mass spectrometry in CSF and serum from 33 HIV-negative Indonesian patients with confirmed or probable tuberculous meningitis and 22 control participants with complete clinical data between March 12, 2009, and Oct 27, 2013. Associations of metabolite concentrations with survival were validated in a second cohort of 101 patients from the same centre. Genome-wide single nucleotide polymorphism typing was used to identify tryptophan quantitative trait loci, which were used for survival analysis in a third cohort of 285 patients. FINDINGS Concentrations of 250 (70%) of 351 metabolites detected in CSF were higher in patients with tuberculous meningitis than in controls, especially in those who died during follow-up. Only five (1%) of the 390 metobolites detected in serum differed between patients with tuberculous meningitis and controls. CSF tryptophan concentrations showed a pattern different from most other CSF metabolites; concentrations were lower in patients who survived compared with patients who died (9-times) and to controls (31-times). The association of low CSF tryptophan with patient survival was confirmed in the validation cohort (hazard ratio 0·73; 95% CI 0·64-0·83; p<0·0001; per each halving). 11 genetic loci predictive for CSF tryptophan concentrations in tuberculous meningitis were identified (p<0·00001). These quantitative trait loci predicted survival in a third cohort of 285 HIV-negative patients in a prognostic index including age and sex, also after correction for possible confounders (p=0·0083). INTERPRETATION Cerebral tryptophan metabolism, which is known to affect Mycobacterium tuberculosis growth and CNS inflammation, is important for the outcome of tuberculous meningitis. CSF tryptophan concentrations in tuberculous meningitis are under strong genetic influence, probably contributing to the variable outcomes of tuberculous meningitis. Interventions targeting tryptophan metabolism could improve outcomes of tuberculous meningitis. FUNDING Royal Dutch Academy of Arts and Sciences; Netherlands Foundation for Scientific Research; Radboud University; National Academy of Sciences; Ministry of Research, Technology, and Higher Education, Indonesia; European Research Council; and PEER-Health.

Transmissible Mycobacterium tuberculosis Strains Share Genetic Markers and Immune Phenotypes

Rationale: Successful transmission of tuberculosis depends on the interplay of human behavior, host immune responses, and Mycobacterium tuberculosis virulence factors. Previous studies have been focused on identifying host risk factors associated with increased transmission, but the contribution of specific genetic variations in mycobacterial strains themselves are still unknown. Objectives: To identify mycobacterial genetic markers associated with increased transmissibility and to examine whether these markers lead to altered in vitro immune responses. Methods: Using a comprehensive tuberculosis registry (n = 10,389) and strain collection in the Netherlands, we identified a set of 100 M. tuberculosis strains either least or most likely to be transmitted after controlling for host factors. We subjected these strains to whole‐genome sequencing and evolutionary convergence analysis, and we repeated this analysis in an independent validation cohort. We then performed immunological experiments to measure in vitro cytokine production and neutrophil responses to a subset of the original strains with or without the identified mutations associated with increased transmissibility. Measurements and Main Results: We identified the loci espE, PE‐PGRS56, Rv0197, Rv2813‐2814c, and Rv2815‐2816c as targets of convergent evolution among transmissible strains. We validated four of these regions in an independent set of strains, and we demonstrated that mutations in these targets affected in vitro monocyte and T‐cell cytokine production, neutrophil reactive oxygen species release, and apoptosis. Conclusions: In this study, we identified genetic markers in convergent evolution of M. tuberculosis toward enhanced transmissibility in vivo that are associated with altered immune responses in vitro.

MST1R mutation as a genetic cause of Lady Windermere syndrome

The prevalence of pulmonary nontuberculous mycobacterial (pNTM) disease is increasing [1]. The most commonly isolated disease-causing NTMs belong to the Mycobacterium avium complex [1]. Susceptibility to and clinical manifestation of NTM disease are largely governed by the immune status of a person. Disseminated or extrapulmonary NTM infections are strongly associated with severe immunosuppression, such as those with frank defects in the interferon (IFN)-γ–interleukin (IL)-12 axis [2]. Isolated pNTM is strongly associated with certain underlying conditions, such as cystic fibrosis, chronic obstructive pulmonary disease and primary ciliary dyskinesia [3, 4]. However, substantial numbers of pNTM patients have no apparent risk factors, and a significant proportion of them exhibit a body morphotype characterised by lifelong slender body habitus, pectus excavatum, scoliosis and mitral valve prolapse [5, 6], also called the Lady Windermere syndrome. A modest reduction in IFN-γ production and an increase in transforming growth factor (TGF)-β levels have been described [7–10]. Fowler et al. [11] quantified ciliary beat frequency of 58 pNTM patients and 40 controls and found reduced ciliary beat frequency in the pNTM patients. Szymanski et al. [12] performed whole-exome sequencing on patients with pNTM, their unaffected family members and a control group and concluded that pNTM is a multigenic disease, encompassing potential defects in proteins encoded by cilia genes, the cystic fibrosis transmembrane conductance regulator gene, connective tissue genes and certain immune-related genes. Susceptibility to NTM infections in patients with Lady Windermere syndrome is associated with MST1R variants http://ow.ly/xKgX304uhse

Presentation, etiology, and outcome of brain infections in an Indonesian hospital: A cohort study

Background Little detailed knowledge is available regarding the etiology and outcome of CNS infection, particularly in HIV-infected individuals, in low-resource settings. Methods From January 2015 to April 2016, we prospectively included all adults with suspected CNS infection in a referral hospital in Jakarta, Indonesia. Systematic screening included HIV testing, CSF examination, and neuroimaging. Results A total of 274 patients with suspected CNS infection (median age 26 years) presented after a median of 14 days with headache (77%), fever (78%), seizures (27%), or loss of consciousness (71%). HIV coinfection was common (54%), mostly newly diagnosed (30%) and advanced (median CD4 cell count 30/µL). Diagnosis was established in 167 participants (65%), including definite tuberculous meningitis (TBM) (n = 44), probable TBM (n = 48), cerebral toxoplasmosis (n = 48), cryptococcal meningitis (n = 14), herpes simplex virus/varicella-zoster virus/cytomegalovirus encephalitis (n = 10), cerebral lymphoma (n = 1), neurosyphilis (n = 1), and mucormycosis (n = 1). In-hospital mortality was 32%; 6-month mortality was 57%. The remaining survivors had either moderate or severe disability (36%) according to Glasgow Outcome Scale. Conclusion In this setting, patients with CNS infections present late with severe disease and often associated with advanced HIV infection. Tuberculosis, toxoplasmosis, and cryptococcosis are common. High mortality and long-term morbidity underline the need for service improvements and further study.