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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.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Humanos , Ratones , Vacuna BCG , Antígenos CD40 , Células Dendríticas , Interleucina-12/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/metabolismo , Ratones Endogámicos C57BLRESUMEN
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.
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Macrófagos/microbiología , Mycobacterium bovis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Niacinamida/farmacología , Complejo Vitamínico B/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Citocinas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Niacina/farmacología , Niacinamida/administración & dosificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
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.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Monocitos/metabolismo , Sitios de Carácter Cuantitativo , Tuberculosis/genética , Citocinas/metabolismoRESUMEN
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.
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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 6âh 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.
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Infecciones por VIH , Tuberculosis Latente , Mycobacterium tuberculosis , Humanos , Ensayos de Liberación de Interferón gamma , Prueba de Tuberculina , Monocitos , Tuberculosis Latente/diagnóstico , Leucocitos Mononucleares , Infecciones por VIH/complicacionesRESUMEN
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.
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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.
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Infección Latente , Tuberculosis Latente , Mycobacterium tuberculosis , Humanos , Ensayos de Liberación de Interferón gamma/métodos , Prueba de Tuberculina/métodos , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/genética , Tuberculosis Latente/complicaciones , FenotipoRESUMEN
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.
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Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Humanos , Ensayos de Liberación de Interferón gamma/métodos , Tuberculosis Latente/diagnóstico , Monocitos , Prueba de Tuberculina/métodos , Tuberculosis/diagnósticoRESUMEN
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.
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Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Virus Sindbis/patogenicidad , Animales , Chlorocebus aethiops , Regulación hacia Abajo , Interferón Tipo I , Interferón gamma , Ratones , Fosforilación , Especificidad de la Especie , Células Vero , VirulenciaRESUMEN
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.
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Mycobacterium tuberculosis , Tuberculosis Pulmonar , Tuberculosis , Método Doble Ciego , Doxiciclina/farmacología , Humanos , Tuberculosis/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológicoRESUMEN
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.
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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.
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Acrilamidas/farmacología , Antituberculosos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Fenilendiaminas/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Adolescente , Adulto , Células Cultivadas , Citocinas/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/inmunología , Transducción de Señal , Tuberculosis Pulmonar/enzimología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
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.
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Proteínas Quinasas Activadas por AMP , Ensayos de Liberación de Interferón gamma , Tuberculosis Latente , Monocitos/metabolismo , Mycobacterium tuberculosis/metabolismo , Polimorfismo de Nucleótido Simple , Transcripción Genética , Prueba de Tuberculina , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Humanos , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/metabolismo , Masculino , Persona de Mediana Edad , Células U937RESUMEN
Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-beta and IFN-gamma. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-beta treatment but did inhibit Jak1 and Jak2 activation in response to IFN-gamma, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.
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Virus de la Encefalitis Equina Venezolana/patogenicidad , Factor de Transcripción STAT1/antagonistas & inhibidores , Transducción de Señal , Animales , Chlorocebus aethiops , Cricetinae , Células HeLa , Humanos , Interferón beta/antagonistas & inhibidores , Interferón beta/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/inmunología , Fosforilación , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiología , Tirosina/metabolismo , Células VeroRESUMEN
Alphaviruses such as Ross River virus (RRV) and chikungunya virus are mosquito-transmitted viruses that cause explosive epidemics of debilitating arthritis and myositis affecting millions of humans worldwide. Previous studies using a mouse model of RRV-induced disease demonstrated that viral infection results in a severe inflammatory arthritis and myositis and that complement component 3 (C3) contributes to the destructive phase of the inflammatory disease but not the recruitment of cellular infiltrates to the sites of RRV-induced inflammation. Here, we demonstrate that mice deficient in complement receptor 3 (CR3) (CD11b(-/-)), a signaling receptor activated by multiple ligands including the C3 cleavage fragment iC3b, develop less-severe disease signs and decreased tissue destruction compared to RRV-infected wild-type mice. CR3 deficiency had no effect on viral replication, nor did it diminish the magnitude, kinetics, and composition of the cellular infiltrates at the sites of inflammation. However, the genetic absence of CR3 diminished the expression of specific proinflammatory and cytotoxic effectors, including S100A9/S100A8 and interleukin-6, within the inflamed tissues, suggesting that CR3-dependent signaling at the sites of inflammation contributes to tissue damage and severe disease.
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Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/patología , Antígeno de Macrófago-1/inmunología , Virus del Río Ross/inmunología , Animales , Calgranulina B/biosíntesis , Interleucina-6/biosíntesis , Articulaciones/patología , Articulaciones/virología , Antígeno de Macrófago-1/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/patología , Músculo Esquelético/virología , Índice de Severidad de la Enfermedad , Carga ViralRESUMEN
Legionella pneumophila (Lp) is a flagellated, intracellular bacterium that can cause Legionnaires' disease (LD). Lp activates multiple innate immune receptors, and TOLLIP dampens MyD88-dependent signaling and may influence susceptibility to LD. We evaluated the effect of TOLLIP on innate immunity, pneumonia severity, and LD susceptibility in mouse lungs and human populations. To accomplish this, we evaluated the effect of TOLLIP on lung-specific Lp control and immune response and associated a common functional TOLLIP variant with Lp-induced innate immune responses and LD susceptibility in humans. After aerosol Lp infection, Tollip-/- mice demonstrated significantly fewer bacterial colony-forming unit and increased cytokine responses from BAL fluid. Tollip-/- macrophages also suppressed intracellular Lp replication in a flagellin-independent manner. The presence of a previously characterized, functionally active SNP associated with decreased TOLLIP mRNA transcript in monocytes was associated with increased TNF and IL-6 secretion after Lp stimulation of PBMC ex vivo. This genotype was separately associated with decreased LD susceptibility (309 controls, 88 cases, p = 0.008, OR 0.36, 95% CI 0.16-0.76) in a candidate gene association study. These results suggest that TOLLIP decreases lung-specific TLR responses to increase LD susceptibility in human populations. Better understanding of TOLLIP may lead to novel immunomodulatory therapies.
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Péptidos y Proteínas de Señalización Intracelular/deficiencia , Legionella pneumophila/patogenicidad , Enfermedad de los Legionarios/metabolismo , Pulmón/metabolismo , Adulto , Anciano , Animales , Carga Bacteriana , Estudios de Casos y Controles , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Legionella pneumophila/crecimiento & desarrollo , Legionella pneumophila/inmunología , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/inmunología , Enfermedad de los Legionarios/microbiología , Pulmón/inmunología , Pulmón/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Transducción de SeñalRESUMEN
The non-invasive, quick, and safe collection of exhaled breath condensate makes it a candidate as a diagnostic matrix in personalized health monitoring devices. The lack of standardization in collection methods and sample analysis is a persistent limitation preventing its practical use. The collection method and hardware design are recognized to significantly affect the metabolomic content of EBC samples, but this has not been systematically studied. Here, we completed a series of experiments to determine the sole effect of collection temperature on the metabolomic content of EBC. Temperature is a likely parameter that can be controlled to standardize among different devices. The study considered six temperature levels covering two physical phases of the sample; liquid and solid. The use of a single device in our study allowed keeping saliva filtering and collector surface effects as constant parameters and the temperature as a controlled variable; the physiological differences were minimized by averaging samples from a group of volunteers and a period of time. After EBC collection, we used an organic solvent rinse to collect the non-water-soluble compounds from the condenser surface. This additional matrix enhanced metabolites recovery, was less dependent on temperature changes, and may possibly serve as an additional pointer to standardize EBC sampling methodologies. The collected EBC samples were analyzed with a set of mass spectrometry methods to provide an overview of the compounds and their concentrations present at each temperature level. The total number of volatile and polar non-volatile compounds slightly increased in each physical phase as the collection temperature was lowered to minimum, 0⯰C for liquid and -30, -56⯰C for solid. The low-polarity non-volatile compounds showed a weak dependence on the collection temperature. The metabolomic content of EBC samples may not be solely dependent on temperature but may be influenced by other phenomena such as greater sample dilution due to condensation from the ambient air at colder temperatures, or due to adhesion properties of the collector surface and occurring chemical reactions. The relative importance of other design parameters such as condenser coating versus temperature requires further investigation.
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Artefactos , Pruebas Respiratorias/instrumentación , Pruebas Respiratorias/métodos , Diseño de Equipo , Espiración , Metabolómica , Temperatura , Humanos , Espectrometría de Masas/instrumentación , Metabolómica/instrumentación , Metabolómica/métodosRESUMEN
Mycobacterium tuberculosis is a leading cause of mortality worldwide and establishes a long-lived latent infection in a substantial proportion of the human population. Multiple lines of evidence suggest that some individuals are resistant to latent M. tuberculosis infection despite long-term and intense exposure, and we term these individuals 'resisters'. In this Review, we discuss the epidemiological and genetic data that support the existence of resisters and propose criteria to optimally define and characterize the resister phenotype. We review recent insights into the immune mechanisms of M. tuberculosis clearance, including responses mediated by macrophages, T cells and B cells. Understanding the cellular mechanisms that underlie resistance to M. tuberculosis infection may reveal immune correlates of protection that could be utilized for improved diagnostics, vaccine development and novel host-directed therapeutic strategies.
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Tuberculosis Latente/inmunología , Tuberculosis Latente/prevención & control , Linfocitos B/inmunología , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Humanos , Inmunidad Humoral , Inmunidad Innata , Fenómenos Inmunogenéticos , Tuberculosis Latente/genética , Macrófagos/inmunología , Modelos Inmunológicos , Mycobacterium tuberculosis/inmunología , Fenotipo , Linfocitos T/inmunologíaRESUMEN
Volatile organic compounds (VOCs) are off-gassed from all living organisms and represent end products of metabolic pathways within the system. In agricultural systems, these VOCs can provide important information on plant health and can ordinarily be measured non-invasively without harvesting tissue from the plants. Previously we reported a portable gas chromatography/differential mobility spectrometry (GC/DMS) system that could distinguish VOC profiles of pathogen-infected citrus from healthy trees before visual symptoms of disease were present. These measurements were taken directly from canopies in the field, but the sampling and analysis protocol did not readily transfer to a controlled greenhouse study where the ambient background air was saturated with volatiles contained in the facility. In this study, we describe for the first time a branch enclosure uniquely coupled with GC/DMS to isolate and measure plant volatiles. To test our system, we sought to replicate our field experiment within a contained greenhouse and distinguish the VOC profiles of healthy versus citrus infected with Candidatus Liberibacter asiaticus. We indeed confirm the ability to track infection-related trace biogenic VOCs using our sampling system and method and we now show this difference in Lisbon lemons (Citrus×limon L. Burm. f.), a varietal not previously reported. Furthermore, the system differentiates the volatile profiles of Lisbon lemons from Washington navels [Citrus sinensis (L.) Osbeck] and also from Tango mandarins (Citrus reticulata Blanco). Based on this evidence, we believe this enclosure-GC/DMS system is adaptable to other volatile-based investigations of plant diseases in greenhouses or other contained settings, and this system may be helpful for basic science research studies of infection mechanisms.