Tuberculosis severity associates with variants and eQTLs related to vascular biology and infection-induced inflammation.

BACKGROUND: Tuberculosis (TB) remains a major public health problem globally, even compared to COVID-19. Genome-wide studies have failed to discover genes that explain a large proportion of genetic risk for adult pulmonary TB, and even fewer have examined genetic factors underlying TB severity, an intermediate trait impacting disease experience, quality of life, and risk of mortality. No prior severity analyses used a genome-wide approach. METHODS AND FINDINGS: As part of our ongoing household contact study in Kampala, Uganda, we conducted a genome-wide association study (GWAS) of TB severity measured by TBScore, in two independent cohorts of culture-confirmed adult TB cases (n = 149 and n = 179). We identified 3 SNPs (P<1.0 x 10-7) including one on chromosome 5, rs1848553, that was GWAS significant (meta-analysis p = 2.97x10-8). All three SNPs are in introns of RGS7BP and have effect sizes corresponding to clinically meaningful reductions in disease severity. RGS7BP is highly expressed in blood vessels and plays a role in infectious disease pathogenesis. Other genes with suggestive associations defined gene sets involved in platelet homeostasis and transport of organic anions. To explore functional implications of the TB severity-associated variants, we conducted eQTL analyses using expression data from Mtb-stimulated monocyte-derived macrophages. A single variant (rs2976562) associated with monocyte SLA expression (p = 0.03) and subsequent analyses indicated that SLA downregulation following MTB stimulation associated with increased TB severity. Src Like Adaptor (SLAP-1), encoded by SLA, is highly expressed in immune cells and negatively regulates T cell receptor signaling, providing a potential mechanistic link to TB severity. CONCLUSIONS: These analyses reveal new insights into the genetics of TB severity with regulation of platelet homeostasis and vascular biology being central to consequences for active TB patients. This analysis also reveals genes that regulate inflammation can lead to differences in severity. Our findings provide an important step in improving TB patient outcomes.

TOLLIP Optimizes Dendritic Cell Maturation to Lipopolysaccharide and Mycobacterium tuberculosis.

TOLLIP is a central regulator of multiple innate immune signaling pathways, including TLR2, TLR4, IL-1R, and STING. Human TOLLIP deficiency, regulated by single-nucleotide polymorphism rs5743854, is associated with increased tuberculosis risk and diminished frequency of bacillus Calmette-Guérin vaccine-specific CD4+ T cells in infants. How TOLLIP influences adaptive immune responses remains poorly understood. To understand the mechanistic relationship between TOLLIP and adaptive immune responses, we used human genetic and murine models to evaluate the role of TOLLIP in dendritic cell (DC) function. In healthy volunteers, TOLLIP single-nucleotide polymorphism rs5743854 G allele was associated with decreased TOLLIP mRNA and protein expression in DCs, along with LPS-induced IL-12 secretion in peripheral blood DCs. As in human cells, LPS-stimulated Tollip -/- bone marrow-derived murine DCs secreted less IL-12 and expressed less CD40. Tollip was required in lung and lymph node-resident DCs for optimal induction of MHC class II and CD40 expression during the first 28 d of Mycobacterium tuberculosis infection in mixed bone marrow chimeric mice. Tollip -/- mice developed fewer M. tuberculosis-specific CD4+ T cells after 28 d of infection and diminished responses to bacillus Calmette-Guérin vaccination. Furthermore, Tollip -/- DCs were unable to optimally induce T cell proliferation. Taken together, these data support a model where TOLLIP-deficient DCs undergo suboptimal maturation after M. tuberculosis infection, impairing T cell activation and contributing to tuberculosis susceptibility.

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

Metabolite content profiling of bottlenose dolphin exhaled breath.

Changing ocean health and the potential impact on marine mammal health are gaining global attention. Direct health assessments of wild marine mammals, however, is inherently difficult. Breath analysis metabolomics is a very attractive assessment tool due to its noninvasive nature, but it is analytically challenging. It has never been attempted in cetaceans for comprehensive metabolite profiling. We have developed a method to reproducibly sample breath from small cetaceans, specifically Atlantic bottlenose dolphins (Tursiops truncatus). We describe the analysis workflow to profile exhaled breath metabolites and provide here a first library of volatile and nonvolatile compounds in cetacean exhaled breath. The described analytical methodology enabled us to document baseline compounds in exhaled breath of healthy animals and to study changes in metabolic content of dolphin breath with regard to a variety of factors. The method of breath analysis may provide a very valuable tool in future wildlife conservation efforts as well as deepen our understanding of marine mammals biology and physiology.

Novel interactive effects of darkness and retinoid signaling in the ability to form long-term memory following aversive operant conditioning.

The vitamin A metabolite, retinoic acid, is important for memory formation and hippocampal synaptic plasticity in vertebrate species. In our studies in the mollusc Lymnaea stagnalis, we have shown that retinoic acid plays a role in memory formation following operant conditioning of the aerial respiratory behaviour. Inhibition of either retinaldehyde dehydrogenase (RALDH) or the retinoid receptors prevents long-term memory (LTM) formation, whereas synthetic retinoid receptor agonists promote memory formation by converting intermediate-term memory (ITM) into LTM. In this study, animals were exposed to constant darkness in order to test whether light-sensitive retinoic acid would promote memory formation. However, we found that exposure to constant darkness alone (in the absence of retinoic acid) enhanced memory formation. To determine whether the memory-promoting effects of darkness could override the memory-inhibiting effects of the retinoid signaling inhibitors, we exposed snails to RALDH inhibitors or a retinoid receptor antagonist in constant darkness. We found that darkness overcame the inhibitory effects of RALDH inhibition, but did not overcome the inhibitory effects of the retinoid receptor antagonist. We also tested whether constant darkness and training affected the mRNA levels of the retinoid metabolic enzymes RALDH and Cyp26, or the mRNA levels of the retinoid receptors, but found no significant effect. Overall, these data demonstrate an interaction between environmental light conditions and the retinoid signaling pathway, which influence long-term memory formation in a mollusc.

Mycobacterium tuberculosis-dependent monocyte expression quantitative trait loci, cytokine production, and TB pathogenesis.

Introduction: The heterogeneity of outcomes after Mycobacterium tuberculosis (Mtb) exposure is a conundrum associated with millennia of host-pathogen co-evolution. We hypothesized that human myeloid cells contain genetically encoded, Mtb-specific responses that regulate critical steps in tuberculosis (TB) pathogenesis. Methods: We mapped genome-wide expression quantitative trait loci (eQTLs) in Mtb-infected monocytes with RNAseq from 80 Ugandan household contacts of pulmonary TB cases to identify monocyte-specific, Mtb-dependent eQTLs and their association with cytokine expression and clinical resistance to tuberculin skin test (TST) and interferon-γ release assay (IGRA) conversion. Results: cis-eQTLs (n=1,567) were identified in Mtb-infected monocytes (FDR<0.01), including 29 eQTLs in 16 genes which were Mtb-dependent (significant for Mtb:genotype interaction [FDR<0.1], but not classified as eQTL in uninfected condition [FDR≥0.01]). A subset of eQTLs were associated with Mtb-induced cytokine expression (n=8) and/or clinical resistance to TST/IGRA conversion (n=1). Expression of BMP6, an Mtb-dependent eQTL gene, was associated with IFNB1 induction in Mtb-infected and DNA ligand-induced cells. Network and enrichment analyses identified fatty acid metabolism as a pathway associated with eQTL genes. Discussion: These findings suggest that monocyte genes contain Mtb-dependent eQTLs, including a subset associated with cytokine expression and/or clinical resistance to TST/IGRA conversion, providing insight into immunogenetic pathways regulating susceptibility to Mtb infection and TB pathogenesis.

Epigenetic programming of host lipid metabolism associates with resistance to TST/IGRA conversion after exposure to Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure leads to a range of outcomes including clearance, latent TB infection (LTBI), and pulmonary tuberculosis (TB). Some heavily exposed individuals resist tuberculin skin test (TST) and interferon gamma release assay (IGRA) conversion (RSTR), which suggests that they employ IFNγ-independent mechanisms of Mtb control. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. Chromatin accessibility did not differ between uninfected RSTR and LTBI monocytes. In contrast, methylation significantly differed at 174 CpG sites and across 63 genomic regions. Consistent with previous transcriptional findings in this cohort, differential methylation was enriched in lipid and cholesterol associated pathways including in the genes APOC3, KCNQ1, and PLA2G3. In addition, methylation was enriched in Hippo signaling, which is associated with cholesterol homeostasis and includes CIT and SHANK2. Lipid export and Hippo signaling pathways were also associated with gene expression in response to Mtb in RSTR as well as IFN stimulation in monocyte-derived macrophages (MDMs) from an independent healthy donor cohort. Moreover, serum-derived HDL from RSTR had elevated ABCA1-mediated cholesterol efflux capacity (CEC) compared to LTBI. Our findings suggest that resistance to TST/IGRA conversion is linked to regulation of lipid accumulation in monocytes, which could facilitate early Mtb clearance among RSTR subjects through IFNγ-independent mechanisms.

Epigenetic programming of host lipid metabolism associated with resistance to TST/IGRA conversion after exposure to Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure leads to a range of outcomes including clearance, latent TB infection (LTBI), and pulmonary tuberculosis (TB). Some heavily exposed individuals resist tuberculin skin test (TST) and interferon-gamma (IFNγ) release assay (IGRA) conversion (RSTR), which suggests that they employ IFNγ-independent mechanisms of Mtb control. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. Chromatin accessibility did not differ between uninfected RSTR and LTBI monocytes. By contrast, methylation significantly differed at 174 CpG sites and across 63 genomic regions. Consistent with previous transcriptional findings in this cohort, differential methylation was enriched in lipid- and cholesterol-associated pathways including the genes APOC3, KCNQ1, and PLA2G3. In addition, methylation was enriched in Hippo signaling, which is associated with cholesterol homeostasis and includes CIT and SHANK2. Lipid export and Hippo signaling pathways were also associated with gene expression in response to Mtb in RSTR as well as IFN stimulation in monocyte-derived macrophages (MDMs) from an independent healthy donor cohort. Moreover, serum-derived high-density lipoprotein from RSTR had elevated ABCA1-mediated cholesterol efflux capacity (CEC) compared to LTBI. Our findings suggest that resistance to TST/IGRA conversion is linked to regulation of lipid accumulation in monocytes, which could facilitate early Mtb clearance among RSTR subjects through IFNγ-independent mechanisms.IMPORTANCETuberculosis (TB) remains an enduring global health challenge with millions of deaths and new cases each year. Despite recent advances in TB treatment, we lack an effective vaccine or a durable cure. While heavy exposure to Mycobacterium tuberculosis often results in latent TB latent infection (LTBI), subpopulations exist that are either resistant to infection or contain Mtb with interferon-gamma (IFNγ)-independent mechanisms not indicative of LTBI. These resisters provide an opportunity to investigate the mechanisms of TB disease and discover novel therapeutic targets. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. We identify methylation signatures in host lipid and cholesterol pathways with potential relevance to early TB clearance before the sustained IFN responses indicative of LTBI. This adds to a growing body of literature linking TB disease outcomes to host lipids.

Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.

Many insect species exhibit pesticide-resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real-time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.

Mycobacterium tuberculosis -induced monocyte transcriptional responses associated with resistance to tuberculin skin test/interferon-γ release assay conversion in people with HIV.

OBJECTIVE: To determine whether Mycobacterium tuberculosis (Mtb)-induced monocyte transcriptional responses differ in people with HIV (PWH) who do (RSTR) or do not (LTBI) resist tuberculin skin test/interferon-γ (IFN-γ) release assay (TST/IGRA) conversion after exposure. DESIGN: We compared ex-vivo Mtb-induced monocyte transcriptional responses in a Ugandan tuberculosis (TB) household contact study of RSTR and LTBI individuals among PWH. METHODS: Monocytes were isolated from peripheral blood mononuclear cells from 19 household contacts of pulmonary TB patients, and their transcriptional profiles were measured with RNA-Seq after a 6 h infection with Mtb (H37Rv) or media. Differentially expressed genes (DEGs) were identified by a linear mixed effects model and pathways by gene set enrichment analysis that compared RSTR and LTBI phenotypes with and without Mtb stimulation. RESULTS: Among PWH, we identified 8341 DEGs that were dependent on Mtb stimulation [false discovery rate (FDR) <0.01]. Of these, 350 were not significant (FDR >0.2) in individuals without HIV. Additionally, we found 26 genes that were differentially expressed between RSTR and LTBI monocytes in PWH, including 20 which were Mtb-dependent (FDR <0.2). In unstimulated monocytes, several gene sets [TGF-ß signaling, TNF-α signaling via NF-κB, NOTCH signaling, coagulation, and epithelial mesenchymal transition (EMT)] were enriched in RSTR relative to LTBI monocytes (FDR <0.1). These patterns were not observed in individuals without HIV. CONCLUSION: RSTR monocytes in PWH show different gene expressions in response to Mtb infection when compared with those with LTBI and RSTR without HIV. These differential expression patterns are enriched in inflammatory pathways.

Mycobacterium tuberculosis-dependent Monocyte Expression Quantitative Trait Loci and Tuberculosis Pathogenesis.

The heterogeneity of outcomes after Mycobacterium tuberculosis (Mtb) exposure is a conundrum associated with millennia of host-pathogen co-evolution. We hypothesized that human myeloid cells contain genetically encoded, Mtb-specific responses that regulate critical steps in tuberculosis (TB) pathogenesis. We mapped genome-wide expression quantitative trait loci (eQTLs) in Mtb-infected monocytes with RNAseq from 80 Ugandan household contacts of pulmonary TB cases to identify monocyte-specific, Mtb-dependent eQTLs and their association with cytokine expression and clinical resistance to tuberculin skin test (TST) and interferon-γ release assay (IGRA) conversion. cis-eQTLs (n=1,567) were identified in Mtb-infected monocytes (FDR<0.01), including 29 eQTLs in 16 genes which were Mtb-dependent (significant for Mtb:genotype interaction [FDR<0.1], but not classified as eQTL in media condition [FDR≥0.01]). A subset of eQTLs were associated with Mtb-induced cytokine expression (n=8) and/or clinical resistance to TST/IGRA conversion (n=1). Expression of BMP6, an Mtb-dependent eQTL gene, was associated with IFNB1 induction in Mtb-infected and DNA ligand-induced cells. Network and enrichment analyses identified fatty acid metabolism as a pathway associated with eQTL genes. These findings suggest that monocyte genes contain Mtb-dependent eQTLs, including a subset associated with cytokine expression and/or clinical resistance to TST/IGRA conversion, providing insight into immunogenetic pathways regulating susceptibility to Mtb infection and TB pathogenesis.

Differentially expressed transcript isoforms associate with resistance to tuberculin skin test and interferon gamma release assay conversion.

BACKGROUND: A mechanistic understanding of uncommon immune outcomes such as resistance to infection has led to the development of novel therapies. Using gene level analytic methods, we previously found distinct monocyte transcriptional responses associated with resistance to Mycobacterium tuberculosis (Mtb) infection defined as persistently negative tuberculin skin test (TST) and interferon gamma release assay (IGRA) reactivity among highly exposed contacts (RSTR phenotype). OBJECTIVE: Using transcript isoform analyses, we aimed to identify novel RSTR-associated genes hypothesizing that previous gene-level differential expression analysis obscures isoform-specific differences that contribute to phenotype. MATERIALS AND METHODS: Monocytes from 49 RSTR versus 52 subjects with latent Mtb infection (LTBI) were infected with M. tuberculosis (H37Rv) or left unstimulated (media) prior to RNA isolation and sequencing. RSTR-associated gene expression was then identified using differential transcript isoform analysis. RESULTS: We identified 81 differentially expressed transcripts (DETs) in 70 genes (FDR <0.05) comparing RSTR and LTBI phenotypes with the majority (n = 79 DETs) identified under Mtb-stimulated conditions. Seventeen of these genes were previously identified with gene-level bulk RNAseq analyses including genes in the IFNγ response that had increased expression among LTBI subjects, findings consistent with a clinical phenotype based on IGRA reactivity. Among the subset of 23 genes with positive differential expression among Mtb-infected RSTR monocytes, 13 were not previously identified. These novel DET genes included PDE4A and ZEB2, which each had multiple DETs with higher expression among RSTR subjects, and ACSL4 and GAPDH that each had a single transcript isoform associated with RSTR. CONCLUSION AND LIMITATIONS: Transcript isoform-specific analyses identify transcriptional associations, such as those associated with resistance to TST/IGRA conversion, that are obscured when using gene-level approaches. These findings should be validated with additional RSTR cohorts and whether the newly identified candidate resistance genes directly influence the monocyte Mtb response requires functional study.

Monocyte Transcriptional Responses to Mycobacterium tuberculosis Associate with Resistance to Tuberculin Skin Test and Interferon Gamma Release Assay Conversion.

Heavy exposure to Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) and among the top infectious killers worldwide, results in infection that is cleared, contained, or progresses to disease. Some heavily exposed tuberculosis contacts show no evidence of infection using the tuberculin skin test (TST) and interferon gamma release assay (IGRA); yet the mechanisms underlying this "resister" (RSTR) phenotype are unclear. To identify transcriptional responses that distinguish RSTR monocytes, we performed transcriptome sequencing (RNA-seq) on monocytes isolated from heavily exposed household contacts in Uganda and gold miners in South Africa after ex vivo M. tuberculosis infection. Gene set enrichment analysis (GSEA) revealed several gene pathways that were consistently enriched in response to M. tuberculosis among RSTR subjects compared to controls with positive TST/IGRA testing (latent TB infection [LTBI]) across Uganda and South Africa. The most significantly enriched gene set in which expression was increased in RSTR relative to LTBI M. tuberculosis-infected monocytes was the tumor necrosis factor alpha (TNF-α) signaling pathway whose core enrichment (leading edge) substantially overlapped across RSTR populations. These leading-edge genes included candidate resistance genes (ABCA1 and DUSP2) with significantly increased expression among Uganda RSTRs (false-discovery rate [FDR], <0.1). The distinct monocyte transcriptional response to M. tuberculosis among RSTR subjects, including increased expression of the TNF signaling pathway, highlights genes and inflammatory pathways that may mediate resistance to TST/IGRA conversion and provides therapeutic targets to enhance host restriction of M. tuberculosis intracellular infection. IMPORTANCE After heavy M. tuberculosis exposure, the events that determine why some individuals resist TST/IGRA conversion are poorly defined. Enrichment of the TNF signaling gene set among RSTR monocytes from multiple distinct cohorts suggests an important role for the monocyte TNF response in determining this alternative immune outcome. These TNF responses to M. tuberculosis among RSTRs may contribute to antimicrobial programs that result in early clearance or the priming of alternative (gamma interferon-independent) cellular responses.

A determinant of Sindbis virus neurovirulence enables efficient disruption of Jak/STAT signaling.

Previous studies with Venezuelan equine encephalitis virus and Sindbis virus (SINV) indicate that alphaviruses are capable of suppressing the cellular response to type I and type II interferons (IFNs) by disrupting Jak/STAT signaling; however, the relevance of this signaling inhibition toward pathogenesis has not been investigated. The relative abilities of neurovirulent and nonneurovirulent SINV strains to downregulate Jak/STAT signaling were compared to determine whether the ability to inhibit IFN signaling correlates with virulence potential. The adult mouse neurovirulent strain AR86 was found to rapidly and robustly inhibit tyrosine phosphorylation of STAT1 and STAT2 in response to IFN-γ and/or IFN-ß. In contrast, the closely related SINV strains Girdwood and TR339, which do not cause detectable disease in adult mice, were relatively inefficient inhibitors of STAT1/2 activation. Decreased STAT activation in AR86-infected cells was associated with decreased activation of the IFN receptor-associated tyrosine kinases Tyk2, Jak1, and Jak2. To identify the viral factor(s) involved, we infected cells with several panels of AR86/Girdwood chimeric viruses. Surprisingly, we found that a single amino acid determinant, threonine at nsP1 position 538, which is required for AR86 virulence, was also required for efficient disruption of STAT1 activation, and this determinant fully restored STAT1 inhibition when it was introduced into the avirulent Girdwood background. These data indicate that a key virulence determinant plays a critical role in downregulating the response to type I and type II IFNs, which suggests that the ability of alphaviruses to inhibit Jak/STAT signaling relates to their in vivo virulence potential.

Remodeling the matrix: doxycycline modulates tuberculosis immunopathology.

Pulmonary cavitation is a hallmark of Mycobacterium tuberculosis (Mtb) infection that provides an immune-privileged niche for extracellular bacillary replication, which associates with increased transmission rates, drug resistance, and chronic lung dysfunction following antituberculous therapy (ATT). Inhibitors of matrix metalloproteinases (MMPs), which are induced by Mtb infection, have shown efficacy in preclinical models and improved microbiologic and immunopathologic outcomes. In this issue of the JCI, Hao Miow et al. performed a double-blind, randomized controlled trial exploring host-directed effects of the MMP inhibitor doxycycline versus placebo when added to standard ATT for pulmonary tuberculosis. Doxycycline treatment over two weeks durably modulated host blood transcription profiles, including the resolution of inflammatory gene programs. Reduced immunopathology markers in doxycycline-treated participants also included improved lung cavity volumes and lower MMP levels in blood and sputum. These findings provide mechanistic insight and momentum for using experimental medicine trials to develop host-directed therapies for tuberculosis.

Acute Hepatitis A Viral Infection in People With HIV With Previously Documented Hepatitis A Immunity or Appropriate Vaccination: A Case Series.

We describe 4 people with HIV (PWH) who acquired acute hepatitis A (HAV) infection during recent King County, Washington, outbreaks despite documented immunity and/or vaccination. HAV revaccination may be needed in PWH with risk factors for HAV infection regardless of preexisting immunity.

HDAC3 inhibitor RGFP966 controls bacterial growth and modulates macrophage signaling during Mycobacterium tuberculosis infection.

RATIONALE: Host-directed therapeutics for Mycobacterium tuberculosis (Mtb) offer potential strategies for combatting antibiotic resistance and for killing non-replicating bacilli. Phenylbutyrate, a partially selective histone-deacetylase (HDAC) inhibitor, was previously shown to control Mtb growth and alter macrophage inflammatory pathways at 2-4 mM concentrations. OBJECTIVE: To identify a more potent and selective HDAC inhibitor that modulates macrophage responses to mycobacteria and has direct antibacterial effects against Mtb. METHODS: We used cellular approaches to characterize the role of pharmacologic inhibition of HDAC3 on Mtb growth and Mtb-induced peripheral and alveolar macrophage immune functions. MEASUREMENTS AND MAIN RESULTS: RGFP966, an HDAC3 inhibitor, controlled Mtb, BCG and M. avium growth directly in broth culture and in human peripheral blood monocyte-derived and alveolar macrophages with an MIC50 of approximately 5-10 µM. In contrast, RGFP966 did not inhibit growth of several other intracellular and extracellular bacteria. We also found that RGFP966 modulated macrophage pro-inflammatory cytokine secretion in response to Mtb infection with decreased IL6 and TNF secretion. CONCLUSIONS: We identified a potent and selective small molecule inhibitor of HDAC3 with direct antimicrobial activity against Mtb and modulation of macrophage signaling pathways.

Clinical and Virologic Characteristics and Outcomes of Coronavirus Disease 2019 at a Cancer Center.

BACKGROUND: High morbidity and mortality have been observed in patients with cancer and coronavirus disease 2019 (COVID-19); however, there are limited data on antimicrobial use, coinfections, and viral shedding. METHODS: We conducted a retrospective cohort study of adult patients at the Seattle Cancer Care Alliance diagnosed with COVID-19 between February 28, 2020 and June 15, 2020 to characterize antimicrobial use, coinfections, viral shedding, and outcomes within 30 days after diagnosis. Cycle threshold values were used as a proxy for viral load. We determined viral clearance, defined as 2 consecutive negative results using severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction results through July 30, 2020. RESULTS: Seventy-one patients were included with a median age of 61 years; 59% had a solid tumor. Only 3 patients had documented respiratory bacterial coinfection. Empiric antibiotics for pneumonia were prescribed more frequently early in the study period (February 29-March 28, 2020; 12/34) compared to the later period (March 29-June 15, 2020; 2/36) (P = .002). The median number of days from symptom onset to viral clearance was 37 days with viral load rapidly declining in the first 7-10 days after symptom onset. Within 30 days of diagnosis, 29 (41%) patients were hospitalized and 12 (17%) died. Each additional comorbidity was associated with 45% lower odds of days alive and out of hospital in the month following diagnosis in adjusted models. CONCLUSIONS: Patients at a cancer center, particularly those with multiple comorbidities, are at increased risk of poor outcomes from COVID-19. Prolonged viral shedding is frequently observed among cancer patients, and its implications on transmission and treatment strategies warrant further study.

Monocyte metabolic transcriptional programs associate with resistance to tuberculin skin test/interferon-γ release assay conversion.

After extensive exposure to Mycobacterium tuberculosis (Mtb), most individuals acquire latent Mtb infection (LTBI) defined by a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA). To identify mechanisms of resistance to Mtb infection, we compared transcriptional profiles from highly exposed contacts who resist TST/IGRA conversion (resisters, RSTRs) and controls with LTBI using RNAseq. Gene sets related to carbon metabolism and free fatty acid (FFA) transcriptional responses enriched across 2 independent cohorts suggesting RSTR and LTBI monocytes have distinct activation states. We compared intracellular Mtb replication in macrophages treated with FFAs and found that palmitic acid (PA), but not oleic acid (OA), enhanced Mtb intracellular growth. This PA activity correlated with its inhibition of proinflammatory cytokines in Mtb-infected cells. Mtb growth restriction in PA-treated macrophages was restored by activation of AMP kinase (AMPK), a central host metabolic regulator known to be inhibited by PA. Finally, we genotyped AMPK variants and found 7 SNPs in PRKAG2, which encodes the AMPK-γ subunit, that strongly associated with RSTR status. Taken together, RSTR and LTBI phenotypes are distinguished by FFA transcriptional programs and by genetic variation in a central metabolic regulator, which suggests immunometabolic pathways regulate TST/IGRA conversion.