Longitudinal soluble marker profiles reveal strong association between cytokine storms resulting from macrophage activation and disease severity in COVID-19 disease.

SARS-CoV2 infection results in a range of disease severities, but the underlying differential pathogenesis is still not completely understood. At presentation it remains difficult to estimate and predict severity, in particular, identify individuals at greatest risk of progression towards the most severe disease-states. Here we used advanced models with circulating serum analytes as variables in combination with daily assessment of disease severity using the SCODA-score, not only at single time points but also during the course of disease, to correlate analyte levels and disease severity. We identified a remarkably strong pro-inflammatory cytokine/chemokine profile with high levels for sCD163, CCL20, HGF, CHintinase3like1 and Pentraxin3 in serum which correlated with COVID-19 disease severity and overall outcome. Although precise analyte levels differed, resulting biomarker profiles were highly similar at early and late disease stages, and even during convalescence similar biomarkers were elevated and further included CXCL3, CXCL6 and Osteopontin. Taken together, strong pro-inflammatory marker profiles were identified in patients with COVID-19 disease which correlated with overall outcome and disease severity.

Whole blood RNA signatures in tuberculosis patients receiving H56:IC31 vaccine as adjunctive therapy.

Introduction: Therapeutic vaccination in tuberculosis (TB) represents a Host Directed Therapy strategy which enhances immune responses in order to improve clinical outcomes and shorten TB treatment. Previously, we have shown that the subunit H56:IC31 vaccine induced both humoral and cellular immune responses when administered to TB patients adjunctive to standard TB treatment (TBCOX2 study, NCT02503839). Here we present the longitudinal whole blood gene expression patterns in H56:IC31 vaccinated TB patients compared to controls receiving standard TB treatment only. Methods: The H56:IC31 group (N=11) and Control group (N=7) underwent first-line TB treatment for 182 days. The H56:IC31 group received 5 micrograms of the H56:IC31 vaccine (Statens Serum Institut; SSI, Valneva Austria GmbH) intramuscularly at day 84 and day 140. Total RNA was extracted from whole blood samples collected in PAXgene tubes on days 0, 84, 98, 140, 154, 182 and 238. The expression level of 183 immune-related genes was measured by high-throughput microfluidic qPCR (Biomark HD system, Standard BioTools). Results: The targeted gene expression profiling unveiled the upregulation of modules such as interferon (IFN) signalling genes, pattern recognition receptors and small nucleotide guanosine triphosphate (GTP)-ases in the vaccinated group compared to controls two weeks after administration of the first H56:IC31 vaccine. Additionally, the longitudinal analysis of the Adolescent Cohort Study-Correlation of Risk (ACS-COR) signature showed a progressive downregulation in both study arms towards the end of TB treatment, in congruence with reported treatment responses and clinical improvements. Still, two months after the end of TB treatment, vaccinated patients, and especially those developing both cellular and humoral vaccine responses, showed a lower expression of the ACS-COR genes compared to controls. Discussion: Our data report gene expression patterns following H56:IC31 vaccination which might be interpreted as a lower risk of relapse in therapeutically vaccinated patients. Further studies are needed to conclude if these gene expression patterns could be used as prognostic biosignatures for therapeutic TB vaccine responses.

Positive mood induction does not reduce return of fear: A virtual reality exposure study for public speaking anxiety.

Previous laboratory work has shown that induction of positive mood prior to fear extinction decreases the negative valence of the conditional stimulus (CS) and reduces reinstatement of fear. Before translating these insights to clinical practice, it is important to test this strategy in anxious individuals. Students with a high fear of public speaking (N = 62) were randomized to either a positive mood induction, a negative mood induction, or no induction control group. All participants performed two weekly sessions of virtual reality exposure and a 1-week follow-up test including a spontaneous recovery test and reinstatement test after a social rejection (unconditional stimulus). We used self-reported fear measures and skin conductance responses. We expected that the positive group, compared to the other groups, would evaluate the CS (i.e., speaking in front of an audience) as less negative following exposure and would show less spontaneous recovery and reinstatement of fear following a social rejection. Although mood was successfully manipulated, there were no group differences in CS valence following exposure. In all conditions, VR exposure successfully reduced public speaking fear, and these effects were stable at follow-up. In contrast with expectations, the positive group showed more spontaneous recovery of CS negative valence than the negative group. To conclude, we found no evidence that positive mood induction prior to exposure optimizes exposure effects for anxious individuals.

Airway epithelial cells mount an early response to mycobacterial infection.

Lung epithelial cells represent the first line of host defence against foreign inhaled components, including respiratory pathogens. Their responses to these exposures may direct subsequent immune activation to these pathogens. The epithelial response to mycobacterial infections is not well characterized and may provide clues to why some mycobacterial infections are cleared, while others are persistent and pathogenic. We have utilized an air-liquid interface model of human primary bronchial epithelial cells (ALI-PBEC) to investigate the epithelial response to infection with a variety of mycobacteria: Mycobacterium tuberculosis (Mtb), M. bovis (BCG), M. avium, and M. smegmatis. Airway epithelial cells were found to be infected by all four species, albeit at low frequencies. The proportion of infected epithelial cells was lowest for Mtb and highest for M. avium. Differential gene expression analysis revealed a common epithelial host response to mycobacteria, including upregulation of BIRC3, S100A8 and DEFB4, and downregulation of BPIFB1 at 48 h post infection. Apical secretions contained predominantly pro-inflammatory cytokines, while basal secretions contained tissue growth factors and chemokines. Finally, we show that neutrophils were attracted to both apical and basal secretions of infected ALI-PBEC. Neutrophils were attracted in high numbers to apical secretions from PBEC infected with all mycobacteria, with the exception of secretions from M. avium-infected ALI-PBEC. Taken together, our results show that airway epithelial cells are differentially infected by mycobacteria, and react rapidly by upregulation of antimicrobials, and increased secretion of inflammatory cytokines and chemokines which directly attract neutrophils. Thus, the airway epithelium may be an important immunological component in controlling and regulating mycobacterial infections.

Impaired resolution of blood transcriptomes through tuberculosis treatment with diabetes comorbidity.

BACKGROUND: People with diabetes are more likely to develop tuberculosis (TB) and to have poor TB-treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at diagnosis. It is unknown whether this persists through treatment and contributes to the adverse outcomes. METHODS: Pulmonary TB patients recruited in South Africa, Indonesia and Romania were classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or TB-only, based on glycated haemoglobin concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood at diagnosis and intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification. Transcriptomic data were analysed by longitudinal mixed-model regression to identify whether genes were differentially expressed between clinical groups through time. Predictive models of TB-treatment response across groups were developed and cross-tested. RESULTS: Gene expression differed between TB and TB-DM patients at diagnosis and was modulated by TB treatment in all clinical groups but to different extents, such that differences remained in TB-DM relative to TB-only throughout. Expression of some genes increased through TB treatment, whereas others decreased: some were persistently more highly expressed in TB-DM and others in TB-only patients. Genes involved in innate immune responses, anti-microbial immunity and inflammation were significantly upregulated in people with TB-DM throughout treatment. The overall pattern of change was similar across clinical groups irrespective of diabetes status, permitting models predictive of TB treatment to be developed. CONCLUSIONS: Exacerbated transcriptome changes in TB-DM take longer to resolve during TB treatment, meaning they remain different from those in uncomplicated TB after treatment completion. This may indicate a prolonged inflammatory response in TB-DM, requiring prolonged treatment or host-directed therapy for complete cure. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes for use across populations.

Omitting continuous memory recall from dual-task interventions does not reduce intervention effectiveness.

In Eye Movement Desensitization and Reprocessing a patient recalls a traumatic memory, while simultaneously performing a dual-task (e.g., making horizontal eye movements, tapping a pattern). Earlier lab studies show that increasing the load of a dual-task -and leaving fewer resources for memory recall-results in larger decreases in memory vividness and emotionality compared to control conditions. Therefore, we investigated whether it is necessary to continuously and deliberately recall the memory next to performing high taxing dual-tasks. In two online experiments, participants (N = 172, N = 198) recalled a negative autobiographical memory and were randomly assigned to (1) Memory Recall + Dual-Tasks, (2) Dual-Tasks Only, or (3) No Intervention Control. The dual-tasks were complex pattern tapping and spelling out loud. Before and after the intervention the memory was rated on vividness, emotionality, and accessibility. High taxing dual-tasks, regardless of whether there was continuous memory recall, resulted in the largest reductions in all dependent variables compared to control. Unexpectedly, there was no evidence that the addition of continuous memory recall added to these reductions. These results suggest that continuous memory recall might not, or only minimally needed for the beneficial effects of the dual-task procedure. We discuss the necessity of memory (re)activation, alternative explanations, and implications for practice.

Differences in Inflammatory Pathways Between Dutch South Asians vs Dutch Europids With Type 2 Diabetes.

CONTEXT: South Asian individuals are more prone to develop type 2 diabetes (T2D) coinciding with earlier complications than Europids. While inflammation plays a central role in the development and progression of T2D, this factor is still underexplored in South Asians. OBJECTIVE: This work aimed to study whether circulating messenger RNA (mRNA) transcripts of immune genes are different between South Asian compared with Europid patients with T2D. METHODS: A secondary analysis was conducted of 2 randomized controlled trials of Dutch South Asian (n = 45; age: 55 ± 10 years, body mass index [BMI]: 29 ± 4 kg/m2) and Dutch Europid (n = 44; age: 60 ± 7 years, BMI: 32 ± 4 kg/m2) patients with T2D. Main outcome measures included mRNA transcripts of 182 immune genes (microfluidic quantitative polymerase chain reaction; Fluidigm Inc) in fasted whole-blood, ingenuity pathway analyses (Qiagen). RESULTS: South Asians, compared to Europids, had higher mRNA levels of B-cell markers (CD19, CD79A, CD79B, CR2, CXCR5, IGHD, MS4A1, PAX5; all fold change > 1.3, false discovery rate [FDR] < 0.008) and interferon (IFN)-signaling genes (CD274, GBP1, GBP2, GBP5, FCGR1A/B/CP, IFI16, IFIT3, IFITM1, IFITM3, TAP1; all FC > 1.2, FDR < 0.05). In South Asians, the IFN signaling pathway was the top canonical pathway (z score 2.6; P < .001) and this was accompanied by higher plasma IFN-γ levels (FC = 1.5, FDR = 0.01). Notably, the ethnic difference in gene expression was larger for women (20/182 [11%]) than men (2/182 [1%]). CONCLUSION: South Asian patients with T2D show a more activated IFN-signaling pathway compared to Europid patients with T2D, which is more pronounced in women than men. We speculate that a more activated IFN-signaling pathway may contribute to the more rapid progression of T2D in South Asian compared with Europid individuals.

Global blood miRNA profiling unravels early signatures of immunogenicity of Ebola vaccine rVSVΔG-ZEBOV-GP.

The vectored Ebola vaccine rVSVΔG-ZEBOV-GP elicits protection against Ebola Virus Disease (EVD). In a study of forty-eight healthy adult volunteers who received either the rVSVΔG-ZEBOV-GP vaccine or placebo, we profiled intracellular microRNAs (miRNAs) from whole blood cells (WB) and circulating miRNAs from serum-derived extracellular vesicles (EV) at baseline and longitudinally following vaccination. Further, we identified early miRNA signatures associated with ZEBOV-specific IgG antibody responses at baseline and up to one year post-vaccination, and pinpointed target mRNA transcripts and pathways correlated to miRNAs whose expression was altered after vaccination by using systems biology approaches. Several miRNAs were differentially expressed (DE) and miRNA signatures predicted high or low IgG ZEBOV-specific antibody levels with high classification performance. The top miRNA discriminators were WB-miR-6810, EV-miR-7151-3p, and EV-miR-4426. An eight-miRNA antibody predictive signature was associated with immune-related target mRNAs and pathways. These findings provide valuable insights into early blood biomarkers associated with rVSVΔG-ZEBOV-GP vaccine-induced IgG antibody responses.

Blood transcriptional profiles distinguish different clinical stages of cutaneous leishmaniasis in humans.

Cutaneous leishmaniasis (CL) is a neglected tropical disease with severe morbidity and socioeconomic sequelae. A better understanding of underlying immune mechanisms that lead to different clinical outcomes of CL could inform the rational design of intervention measures. While transcriptomic analyses of CL lesions were recently reported by us and others, there is a dearth of information on the expression of immune-related genes in the blood of CL patients. Herein, we investigated immune-related gene expression in whole blood samples collected from individuals with different clinical stages of CL along with healthy volunteers in an endemic CL region where Leishmania (L.) tropica is prevalent. Study participants were categorized into asymptomatic (LST+) and healthy uninfected (LST-) groups based on their leishmanin skin test (LST). Whole blood PAXgene samples were collected from volunteers, who had healed CL lesions, and patients with active L. tropica cutaneous lesions. Quality RNA extracted from 57 blood samples were subjected to Dual-color reverse-transcription multiplex ligation-dependent probe amplification (dcRT-MLPA) assay for profiling 144 immune-related genes. Results show significant changes in the expression of genes involved in interferon signaling pathway in the blood of active CL patients, asymptomatics and healed individuals. Nonetheless, distinct profiles for several immune-related genes were identified in the healed, the asymptomatic, and the CL patients compared to the healthy controls. Among others, IFI16 and CCL11 were found as immune transcript signatures for the healed and the asymptomatic individuals, respectively. These results warrant further exploration to pinpoint novel blood biomarkers for different clinical stages of CL.

Transcriptional profiles predict treatment outcome in patients with tuberculosis and diabetes at diagnosis and at two weeks after initiation of anti-tuberculosis treatment.

BACKGROUND: Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control. METHODS: Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n  =  94) and Indonesia (n  =  81), who had concomitant DM (n  =  59), intermediate hyperglycaemia (n  =  79) or normal glycaemia/no DM (n  =  37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA. FINDINGS: We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0·815) or two weeks (AUC = 0·834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0·749). INTERPRETATION: These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM. FUNDING: The research leading to these results, as part of the TANDEM Consortium, received funding from the European Community's Seventh Framework Programme (FP7/2007-2013 Grant Agreement No. 305279) and the Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038). The research leading to the results presented in the Indian validation cohort was supported by Research Council of Norway Global Health and Vaccination Research (GLOBVAC) projects: RCN 179342, 192534, and 248042, the University of Bergen (Norway).

Recombinant BCG-LTAK63 Vaccine Candidate for Tuberculosis Induces an Inflammatory Profile in Human Macrophages.

Tuberculosis (TB) is one of the top 10 leading causes of death worldwide. The recombinant BCG strain expressing the genetically detoxified A subunit of the thermolabile toxin from Escherichia coli (LTAK63) adjuvant (rBCG-LTAK63) has previously been shown to confer superior protection and immunogenicity compared to BCG in a murine TB infection model. To further investigate the immunological mechanisms induced by rBCG-LTAK63, we evaluated the immune responses induced by rBCG-LTAK63, BCG, and Mycobacterium tuberculosis (Mtb) H37Rv strains in experimental infections of primary human M1 and M2 macrophages at the transcriptomic and cytokine secretion levels. The rBCG-LTAK63-infected M1 macrophages more profoundly upregulated interferon-inducible genes such as IFIT3, OAS3, and antimicrobial gene CXCL9 compared to BCG, and induced higher levels of inflammatory cytokines such as IL-12(p70), TNF-ß, and IL-15. The rBCG-LTAK63-infected M2 macrophages more extensively upregulated transcripts of inflammation-related genes, TAP1, GBP1, SLAMF7, TNIP1, and IL6, and induced higher levels of cytokines related to inflammation and tissue repair, MCP-3 and EGF, as compared to BCG. Thus, our data revealed an important signature of immune responses induced in human macrophages by rBCG-LTAK63 associated with increased inflammation, activation, and tissue repair, which may be correlated with a protective immune response against TB.

The effect of cold exposure on circulating transcript levels of immune genes in Dutch South Asian and Dutch Europid men.

OBJECTIVES: Although cold exposure is commonly believed to be causally related to acute viral respiratory infections, its effect on the immune system is largely unexplored. In this study, we determined transcript levels of a large panel of immune genes in blood before and after cold exposure. We included both Dutch Europid and Dutch South Asian men to address whether the immune system is differently regulated in the metabolically vulnerable South Asian population. METHODS: Fasted blood samples were obtained from nonobese Dutch Europid (n = 11; mean age 26 ± 3 y) and Dutch South Asian (n = 12; mean age 28 ± 3 y) men before and directly after short-term (∼2.5 h) mild cold exposure. Transcript levels of 144 immune genes were measured using a dual-color reverse transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) assay. RESULTS: Cold exposure acutely upregulated mRNA levels of GNLY (+35%, P < 0.001) and PRF1 (+45%, P < 0.001), which encode cytotoxic proteins, and CCL4 (+8%, P < 0.01) and CCL5 (+5%, P < 0.05), both pro-inflammatory chemokines. At thermoneutrality, mRNA levels of four markers of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR)-family, involved in inflammasomes, were lower in Dutch South Asians compared to Dutch Europids, namely NLRP2 (-57%, P < 0.05), NLRP7 (-17%, P < 0.05), NLRP10 (-21%, P < 0.05), and NLRC4 (-23%, P < 0.05). CONCLUSIONS: Mild cold exposure acutely increases mRNA levels of genes involved in cytotoxicity of immune cells in blood. In addition, Dutch South Asians display lower circulating mRNA levels of inflammasome genes compared to Dutch Europids.

Transcriptomic signatures induced by the Ebola virus vaccine rVSVΔG-ZEBOV-GP in adult cohorts in Europe, Africa, and North America: a molecular biomarker study.

BACKGROUND: A recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine. METHODS: 354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe (Geneva, Switzerland [November, 2014, to January, 2015]) and North America (USA [Dec 5, 2014, to June 23, 2015]), and dose-escalation trials in Africa (Lambaréné, Gabon [November, 2014, to January, 2015], and Kilifi, Kenya [December, 2014, to January, 2015]) using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP; 3 × 105 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses (days 0, 1, 2, 3, 7, 14, and 28) were measured in whole blood using a targeted gene expression profiling platform (dual-colour reverse-transcriptase multiplex ligation-dependent probe amplification) focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity. FINDINGS: Our results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 106 pfu) of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity (common to all four cohorts) was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event (ie, arthritis). INTERPRETATION: Collectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines. FUNDING: Innovative Medicines Initiative 2 Joint Undertaking.

Stratification of COVID-19 patients based on quantitative immune-related gene expression in whole blood.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes mild symptoms in the majority of infected individuals, yet in some cases it leads to a life-threatening condition. Determination of early predictive biomarkers enabling risk stratification for coronavirus disease 2019 (COVID-19) patients can inform treatment and intervention strategies. Herein, we analyzed whole blood samples obtained from individuals infected with SARS-CoV-2, varying from mild to critical symptoms, approximately one week after symptom onset. In order to identify blood-specific markers of disease severity status, a targeted expression analysis of 143 immune-related genes was carried out by dual-color reverse transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA). The clinically well-defined subgroups of COVID-19 patients were compared with healthy controls. The transcriptional profile of the critically ill patients clearly separated from that of healthy individuals. Moreover, the number of differentially expressed genes increased by severity of COVID-19. It was also found that critically ill patients can be distinguished by reduced peripheral blood expression of several genes, which most likely reflects the lower lymphocyte counts. There was a notable predominance of IFN-associated gene expression in all subgroups of COVID-19, which was most profound in critically ill patients. Interestingly, the gene encoding one of the main TNF-receptors, TNFRS1A, had selectively lower expression in mild COVID-19 cases. This report provides added value in understanding COVID-19 disease, and shows potential of determining early immune transcript signatures in the blood of patients with different disease severity. These results can guide further explorations to uncover mechanisms underlying immunity and immunopathology in COVID-19.

[A man with currant jelly sputum]. / Een man met bessengelei-achtig sputum.

A 40-year-old man presented with acute respiratory distress and a productive cough in the past three days with 'currant jelly' sputum. X-ray showed a right upper lobe infiltrate. Culture of sputum showed Klebsiella pneumoniae, consistent with 'Friedländer' pneumonia. The patient was treated with antibiotics but died after a complicated course.

Pyruvate Dehydrogenase Kinase Inhibitor Dichloroacetate Improves Host Control of Salmonella enterica Serovar Typhimurium Infection in Human Macrophages.

Global increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play a key role in host control of intracellular bacterial pathogens. In this study we explored the potential of targeting host metabolic pathways for innovative host-directed therapy (HDT) against intracellular bacterial infections. Through gene expression profiling in human macrophages, pyruvate metabolism was identified as potential key pathway involved in Salmonella enterica serovar Typhimurium (Stm) infections. Next, the effect of targeting pyruvate dehydrogenase kinases (PDKs) - which are regulators of the metabolic checkpoint pyruvate dehydrogenase complex (PDC) - on macrophage function and bacterial control was studied. Chemical inhibition of PDKs by dichloroacetate (DCA) induced PDC activation and was accompanied with metabolic rewiring in classically activated macrophages (M1) but not in alternatively activated macrophages (M2), suggesting cell-type specific effects of dichloroacetate on host metabolism. Furthermore, DCA treatment had minor impact on cytokine and chemokine secretion on top of infection, but induced significant ROS production by M1 and M2. DCA markedly and rapidly reduced intracellular survival of Stm, but interestingly not Mycobacterium tuberculosis, in human macrophages in a host-directed manner. In conclusion, DCA represents a promising novel HDT compound targeting pyruvate metabolism for the treatment of Stm infections.

Bioorthogonal Correlative Light-Electron Microscopy of Mycobacterium tuberculosis in Macrophages Reveals the Effect of Antituberculosis Drugs on Subcellular Bacterial Distribution.

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis (Mtb) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (ex cellula). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution.

Functional Inhibition of Host Histone Deacetylases (HDACs) Enhances in vitro and in vivo Anti-mycobacterial Activity in Human Macrophages and in Zebrafish.

The rapid and persistent increase of drug-resistant Mycobacterium tuberculosis (Mtb) infections poses increasing global problems in combatting tuberculosis (TB), prompting for the development of alternative strategies including host-directed therapy (HDT). Since Mtb is an intracellular pathogen with a remarkable ability to manipulate host intracellular signaling pathways to escape from host defense, pharmacological reprogramming of the immune system represents a novel, potentially powerful therapeutic strategy that should be effective also against drug-resistant Mtb. Here, we found that host-pathogen interactions in Mtb-infected primary human macrophages affected host epigenetic features by modifying histone deacetylase (HDAC) transcriptomic levels. In addition, broad spectrum inhibition of HDACs enhanced the antimicrobial response of both pro-inflammatory macrophages (Mϕ1) and anti-inflammatory macrophages (Mϕ2), while selective inhibition of class IIa HDACs mainly decreased bacterial outgrowth in Mϕ2. Moreover, chemical inhibition of HDAC activity during differentiation polarized macrophages into a more bactericidal phenotype with a concomitant decrease in the secretion levels of inflammatory cytokines. Importantly, in vivo chemical inhibition of HDAC activity in Mycobacterium marinum-infected zebrafish embryos, a well-characterized animal model for tuberculosis, significantly reduced mycobacterial burden, validating our in vitro findings in primary human macrophages. Collectively, these data identify HDACs as druggable host targets for HDT against intracellular Mtb.

Linguistic dual tasking reduces emotionality, vividness and credibility of voice memories in voice-hearing individuals: Results from a controlled trial.

Dual taxation of the working memory during recall is an effective strategy to reduce the emotionality and vividness of visual intrusive memories and potentially changes dysfunctional beliefs associated with the memories. This study tested the hypothesis that dual tasking decreases emotionality, vividness and credibility of auditory intrusive images (i.e., memories of auditory hallucinations) with a two-level (time: pre and post; condition: dual tasking and recall only) within-subjects design. Thirty-seven voice-hearing participants selected two negative voice-hearing experiences. They recalled one of these experiences while performing a lingual dual task (i.e., language game on smartphone app) and recalled one memory without a dual task (in counterbalanced order). During the pre-test and post-test, emotionality and vividness of the voice-hearing memories were rated, as well as the credibility of the voice statements. There was a significantly greater decrease in emotionality, vividness and credibility during dual tasking than during recall only. This study provides proof of principle that the salience and credibility of the content of auditory hallucinations can be reduced by dual tasking; the clinical implications are also discussed.