RESUMO
Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.
Assuntos
Mycobacterium tuberculosis , Fibrose Pulmonar , Tuberculose , Animais , Ecossistema , Granuloma , Pulmão , Macaca fascicularis , Fibrose Pulmonar/patologiaRESUMO
Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.
Assuntos
Administração Intravenosa , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Tuberculose/prevenção & controle , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Macaca mulatta , Tuberculose/imunologia , Vacinação/normasRESUMO
Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection . Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, positron emission tomography-computed tomography (PET CT) imaging, flow cytometry, and cytokine profiling, we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination, or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the seven antibiotic-treated animals demonstrated sterilizing immunity against reinfection, while four of the seven non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic-treated macaques.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Camundongos , Reinfecção , Tuberculose/tratamento farmacológico , Macaca fascicularis , Antibacterianos/farmacologia , Antibacterianos/uso terapêuticoRESUMO
Tuberculosis (TB) continues to be one of the deadliest infectious diseases in the world, causing ~1.5 million deaths every year. The World Health Organization initiated an End TB Strategy that aims to reduce TB-related deaths in 2035 by 95%. Recent research goals have focused on discovering more effective and more patient-friendly antibiotic drug regimens to increase patient compliance and decrease emergence of resistant TB. Moxifloxacin is one promising antibiotic that may improve the current standard regimen by shortening treatment time. Clinical trials and in vivo mouse studies suggest that regimens containing moxifloxacin have better bactericidal activity. However, testing every possible combination regimen with moxifloxacin either in vivo or clinically is not feasible due to experimental and clinical limitations. To identify better regimens more systematically, we simulated pharmacokinetics/pharmacodynamics of various regimens (with and without moxifloxacin) to evaluate efficacies, and then compared our predictions to both clinical trials and nonhuman primate studies performed herein. We used GranSim, our well-established hybrid agent-based model that simulates granuloma formation and antibiotic treatment, for this task. In addition, we established a multiple-objective optimization pipeline using GranSim to discover optimized regimens based on treatment objectives of interest, i.e., minimizing total drug dosage and lowering time needed to sterilize granulomas. Our approach can efficiently test many regimens and successfully identify optimal regimens to inform pre-clinical studies or clinical trials and ultimately accelerate the TB regimen discovery process.
Assuntos
Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Animais , Camundongos , Antituberculosos , Moxifloxacina/uso terapêutico , Tuberculose/tratamento farmacológicoRESUMO
Mycobacterium tuberculosis infection outcomes have been described as active tuberculosis or latent infection but a spectrum of outcomes is now recognized. We used a nonhuman primate model, which recapitulates human infection, to characterize the clinical, microbiologic, and radiographic patterns associated with developing latent M. tuberculosis infection. Four patterns were identified. "Controllers" had normal erythrocyte sedimentation rate (ESR) without M. tuberculosis growth in bronchoalveolar lavage or gastric aspirate (BAL/GA). "Early subclinicals" showed transient ESR elevation and/or M. tuberculosis growth on BAL/GA for 60 days postinfection, "mid subclinicals" were positive for 90 days, and "late subclinicals" were positive intermittently, despite the absence of clinical disease. Variability was noted regarding granuloma formation, lung/lymph node metabolic activity, lung/lymph node bacterial burden, gross pathology, and extrapulmonary disease. Like human M. tuberculosis infection, this highlights the heterogeneity associated with the establishment of latent infection, underscoring the need to understand the clinical spectrum and risk factors associated with severe disease.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Tuberculose Latente/diagnóstico por imagem , Tuberculose Latente/microbiologia , Pulmão/patologia , MacacaRESUMO
Pre-existing HIV infection increases tuberculosis (TB) risk in children. Antiretroviral therapy (ART) reduces, but does not abolish, this risk in children with HIV. The immunologic mechanisms involved in TB progression in both HIV-naive and HIV-infected children have not been explored. Much of our current understanding is based on human studies in adults and adult animal models. In this study, we sought to model childhood HIV/Mycobacterium tuberculosis (Mtb) coinfection in the setting of ART and characterize T cells during TB progression. Macaques equivalent to 4 to 8 year-old children were intravenously infected with SIVmac239M, treated with ART 3 months later, and coinfected with Mtb 3 months after initiating ART. SIV-naive macaques were similarly infected with Mtb alone. TB pathology and total Mtb burden did not differ between SIV-infected, ART-treated and SIV-naive macaques, although lung Mtb burden was lower in SIV-infected, ART-treated macaques. No major differences in frequencies of CD4+ and CD8+ T cells and unconventional T cell subsets (Vγ9+ γδ T cells, MAIT cells, and NKT cells) in airways were observed between SIV-infected, ART-treated and SIV-naive macaques over the course of Mtb infection, with the exception of CCR5+ CD4+ and CD8+ T cells which were slightly lower. CD4+ and CD8+ T cell frequencies did not differ in the lung granulomas. Immune checkpoint marker levels were similar, although ki-67 levels in CD8+ T cells were elevated. Thus, ART treatment of juvenile macaques, 3 months after SIV infection, resulted in similar progression of Mtb and T cell responses compared to Mtb in SIV-naive macaques.
Assuntos
Antirretrovirais , Modelos Animais de Doenças , Macaca , Mycobacterium tuberculosis , Vírus da Imunodeficiência Símia , Tuberculose , Humanos , Pré-Escolar , Criança , Animais , Tuberculose/complicações , Tuberculose/imunologia , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Linfócitos T/imunologia , Antirretrovirais/administração & dosagem , Mycobacterium tuberculosis/fisiologiaRESUMO
BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and kills more than 1.5 million people each year. METHODS: We examine the frequency and function of NK cells in the blood and airways over the course of Mtb infection in a TB macaque model and demonstrate differences in NK marker expression between the two compartments. Flow cytometry and intracellular cytokine staining were utilized to identify NK cell subsets (expressing NKG2A, CD56, or CD16) and function (IL-10, TNF, IL-2, IFN-g, IL-17, and CD107a). RESULTS: Blood and airway NK cell frequencies were similar during infection though there were differences in subset populations between blood and airway. Increased functional (cytokine/CD107a) parameters were observed in airway NK cells during the course of infection while none were seen in the blood. CONCLUSIONS: This study suggests that NK cells in the airway may play an important role in TB host response.
Assuntos
Células Matadoras Naturais , Tuberculose Latente , Pulmão , Mycobacterium tuberculosis , Tuberculose Pulmonar , Animais , Citocinas/metabolismo , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Macaca , Mycobacterium tuberculosis/imunologia , Modelos Animais de Doenças , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/imunologia , Tuberculose Latente/sangue , Tuberculose Latente/imunologia , Pulmão/imunologiaRESUMO
Tuberculosis (TB) is the leading infectious cause of death among people living with HIV. People living with HIV are more susceptible to contracting Mycobacterium tuberculosis and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on M. tuberculosis coinfection is critical in combating this global health epidemic. We previously showed in a model of SIV and M. tuberculosis coinfection in Mauritian cynomolgus macaques (MCM) that SIV/M. tuberculosis-coinfected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to M. tuberculosis infection. We infected MCM with SIVmac239, followed by coinfection with M. tuberculosis Erdman 6 mo later. Although similar, TB progression was observed in both SIV+ and SIV-naive animals at 6 wk post-M. tuberculosis infection; longitudinal sampling of the blood (PBMC) and airways (bronchoalveolar lavage) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways of SIV+ animals. At sites of M. tuberculosis infection (i.e., granulomas), SIV/M. tuberculosis-coinfected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ T cells in granulomas of SIV/M. tuberculosis-coinfected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of M. tuberculosis replication soon postinfection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection, which promotes TB progression.
Assuntos
Granuloma/imunologia , Mycobacterium tuberculosis/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Linfócitos T/imunologia , Tuberculose/imunologia , Fatores de Necrose Tumoral/imunologia , Animais , Biomarcadores/análise , Linfócitos T CD8-Positivos/imunologia , Macaca , Vírus da Imunodeficiência Símia/imunologiaRESUMO
Mucosal-associated invariant T (MAIT) cells can recognize and respond to some bacterially infected cells. Several in vitro and in vivo models of Mycobacterium tuberculosis (Mtb) infection suggest that MAIT cells can contribute to control of Mtb, but these studies are often cross-sectional and use peripheral blood cells. Whether MAIT cells are recruited to Mtb-affected granulomas and lymph nodes (LNs) during early Mtb infection and what purpose they might serve there is less well understood. Furthermore, whether HIV/SIV infection impairs MAIT cell frequency or function at the sites of Mtb replication has not been determined. Using Mauritian cynomolgus macaques (MCM), we phenotyped MAIT cells in the peripheral blood and bronchoalveolar lavage (BAL) before and during infection with SIVmac239. To test the hypothesis that SIV co-infection impairs MAIT cell frequency and function within granulomas, SIV+ and -naïve MCM were infected with a low dose of Mtb Erdman, and necropsied at 6 weeks post Mtb-challenge. MAIT cell frequency and function were examined within the peripheral blood, BAL, and Mtb-affected lymph nodes (LN) and granulomas. MAIT cells did not express markers indicative of T cell activation in response to Mtb in vivo within granulomas in animals infected with Mtb alone. SIV and Mtb co-infection led to increased expression of the activation/exhaustion markers PD-1 and TIGIT, and decreased ability to secrete TNFα when compared to SIV-naïve MCM. Our study provides evidence that SIV infection does not prohibit the recruitment of MAIT cells to sites of Mtb infection, but does functionally impair those MAIT cells. Their impaired function could have impacts, either direct or indirect, on the long-term containment of TB disease.
Assuntos
Coinfecção/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Mycobacterium tuberculosis/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Tuberculose Pulmonar/imunologia , Animais , Coinfecção/patologia , Granuloma/imunologia , Granuloma/patologia , Linfonodos/imunologia , Linfonodos/patologia , Macaca fascicularis , Células T Invariantes Associadas à Mucosa/patologia , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Tuberculose Pulmonar/patologiaRESUMO
Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.
Assuntos
Coinfecção/imunologia , Tuberculose Latente/imunologia , Tuberculose Latente/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Ativação Viral/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Granuloma/virologia , Hospedeiro Imunocomprometido/imunologia , Macaca fascicularis , Mycobacterium tuberculosis/imunologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.
Assuntos
Granuloma/imunologia , Inflamação/imunologia , Interleucina-10/metabolismo , Pulmão/patologia , Linfonodos/imunologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunidade , Pulmão/imunologia , Macaca fascicularis , Fibrose PulmonarRESUMO
Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli.
Assuntos
Linfonodos/imunologia , Macaca/imunologia , Tuberculose/imunologia , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças/patologia , Granuloma/patologia , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Linfonodos/microbiologia , Macaca/microbiologia , Mycobacterium tuberculosis/patogenicidade , Tomografia por Emissão de PósitronsRESUMO
For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immunity in the lung, which may inform TB vaccine design and development.
Assuntos
Coinfecção/imunologia , Mycobacterium tuberculosis/imunologia , Pneumonia/prevenção & controle , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/prevenção & controle , Animais , Macaca , Pneumonia/imunologia , Pneumonia/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , VacinaçãoRESUMO
BACKGROUND: Tuberculosis (TB) kills millions of people every year. CD4 and CD8 T cells are critical in the immune response against TB. T cells expressing both CD4 and CD8 (CD4CD8 T cells) are functionally active and have not been examined in the context of TB. METHODS: We examine peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BAL) and lung granulomas from 28 cynomolgus macaques during Mycobacterium tuberculosis (Mtb) infection. RESULTS: CD4CD8 T cells increase in frequency during early Mtb infection in PBMC and BAL from pre-infection. Peripheral, airway, and lung granuloma CD4CD8 T cells have distinct patterns and greater cytokine production than CD4 or CD8 T cells. CONCLUSION: Our data suggest that CD4CD8 T cells transient the blood and airways early during infection to reach the granulomas where they are involved directly in the host response to Mtb.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Macaca fascicularis , Tuberculose/imunologia , Animais , Modelos Animais de Doenças , Granuloma/imunologia , Leucócitos/imunologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologiaRESUMO
Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium-antigen murine inflammation model and macaques with TB to identify [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4ß1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and ß1, further supporting that macrophages and T cells drive [64Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.
Assuntos
Glucose/metabolismo , Granuloma/imunologia , Integrina alfa4beta1/genética , Tuberculose/diagnóstico por imagem , Tuberculose/imunologia , Animais , Diferenciação Celular , Movimento Celular , Granuloma/diagnóstico por imagem , Granuloma/metabolismo , Granuloma/fisiopatologia , Compostos Heterocíclicos com 2 Anéis/química , Integrina alfa4beta1/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Macaca , Macrófagos/imunologia , Neutrófilos/imunologia , Organofosfonatos/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos , Linfócitos T/imunologia , Tuberculose/diagnóstico , Tuberculose/microbiologiaRESUMO
Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and IFN-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.
Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/virologia , Alvéolos Pulmonares/virologia , Síndrome do Desconforto Respiratório/virologia , Replicação Viral , Aerossóis , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Modelos Animais de Doenças , Imunidade Inata/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Pulmão/imunologia , Pulmão/virologia , Macaca fascicularis , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Macrófagos Alveolares/virologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/fisiopatologia , Pneumonia Viral/imunologia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/fisiopatologiaRESUMO
In the past 2 decades, it has become increasingly clear that nonhuman primates, specifically macaques, are useful models for human tuberculosis (TB). Several macaque species have been used for TB studies, and questions remain about the similarities and differences in TB pathogenesis among macaque species, which can complicate decisions about the best species for a specific experiment. Here we provide a quantitative assessment, using serial positron emission tomography and computed tomography (PET-CT) imaging and precise quantitative determination of bacterial burdens of low-dose Mycobacterium tuberculosis infection in cynomolgus macaques of Chinese origin, rhesus macaques of Chinese origin, and Mauritian cynomolgus macaques. This comprehensive study demonstrates that there is substantial variability in the outcome of infection within and among species. Overall, rhesus macaques have higher rates of disease progression, more lung, lymph node, and extrapulmonary involvement, and higher bacterial burdens than Chinese cynomolgus macaques. The small cohort of Mauritian cynomolgus macaques assessed here indicates that this species is more similar to rhesus macaques than to Chinese cynomolgus macaques in terms of M. tuberculosis infection outcome. These data provide insights into the differences among species, providing valuable data to the field for assessing macaque studies of TB.
Assuntos
Suscetibilidade a Doenças , Pulmão/microbiologia , Pulmão/patologia , Macaca fascicularis , Macaca mulatta , Tuberculose/imunologia , Tuberculose/patologia , Animais , Carga Bacteriana , Modelos Animais de Doenças , Mycobacterium tuberculosis/isolamento & purificação , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tuberculose/diagnóstico por imagemRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the leading cause of death among human immunodeficiency virus (HIV)-positive patients. The precise mechanisms by which HIV impairs host resistance to a subsequent M. tuberculosis infection are unknown. We modeled this coinfection in Mauritian cynomolgus macaques (MCM) using simian immunodeficiency virus (SIV) as an HIV surrogate. We infected seven MCM with SIVmac239 intrarectally and 6 months later coinfected them via bronchoscope with â¼10 CFU of M. tuberculosis Another eight MCM were infected with M. tuberculosis alone. TB progression was monitored by clinical parameters, by culturing bacilli in gastric and bronchoalveolar lavages, and by serial [18F]fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) imaging. The eight MCM infected with M. tuberculosis alone displayed dichotomous susceptibility to TB, with four animals reaching humane endpoint within 13 weeks and four animals surviving >19 weeks after M. tuberculosis infection. In stark contrast, all seven SIV+ animals exhibited rapidly progressive TB following coinfection and all reached humane endpoint by 13 weeks. Serial PET/CT imaging confirmed dichotomous outcomes in MCM infected with M. tuberculosis alone and marked susceptibility to TB in all SIV+ MCM. Notably, imaging revealed a significant increase in TB granulomas between 4 and 8 weeks after M. tuberculosis infection in SIV+ but not in SIV-naive MCM and implies that SIV impairs the ability of animals to contain M. tuberculosis dissemination. At necropsy, animals with preexisting SIV infection had more overall pathology, increased bacterial loads, and a trend towards more extrapulmonary disease than animals infected with M. tuberculosis alone. We thus developed a tractable MCM model in which to study SIV-M. tuberculosis coinfection and demonstrate that preexisting SIV dramatically diminishes the ability to control M. tuberculosis coinfection.
Assuntos
Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Síndrome de Imunodeficiência Adquirida dos Símios/microbiologia , Tuberculose/imunologia , Tuberculose/virologia , Animais , Carga Bacteriana , Linfócitos T CD4-Positivos/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Granuloma/imunologia , Granuloma/microbiologia , Macaca fascicularis , Mycobacterium tuberculosis , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Vírus da Imunodeficiência Símia , Tuberculose/veterináriaRESUMO
Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection to active tuberculosis. Among those with latent tuberculosis, it is now recognized that there is also a spectrum of infection and this likely contributes to the variable risk of reactivation tuberculosis. Here, functional imaging with 18F-fluorodeoxygluose positron emission tomography and computed tomography (PET CT) of cynomolgus macaques with latent M. tuberculosis infection was used to characterize the features of reactivation after tumor necrosis factor (TNF) neutralization and determine which imaging characteristics before TNF neutralization distinguish reactivation risk. PET CT was performed on latently infected macaques (n = 26) before and during the course of TNF neutralization and a separate set of latently infected controls (n = 25). Reactivation occurred in 50% of the latently infected animals receiving TNF neutralizing antibody defined as development of at least one new granuloma in adjacent or distant locations including extrapulmonary sites. Increased lung inflammation measured by PET and the presence of extrapulmonary involvement before TNF neutralization predicted reactivation with 92% sensitivity and specificity. To define the biologic features associated with risk of reactivation, we used these PET CT parameters to identify latently infected animals at high risk for reactivation. High risk animals had higher cumulative lung bacterial burden and higher maximum lesional bacterial burdens, and more T cells producing IL-2, IL-10 and IL-17 in lung granulomas as compared to low risk macaques. In total, these data support that risk of reactivation is associated with lung inflammation and higher bacterial burden in macaques with latent Mtb infection.
Assuntos
Tuberculose Latente/diagnóstico por imagem , Tuberculose Latente/microbiologia , Tuberculose Latente/patologia , Ativação Viral , Latência Viral , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Processamento de Imagem Assistida por Computador , Macaca fascicularis , Mycobacterium tuberculosis , Reação em Cadeia da Polimerase , Tomografia por Emissão de Pósitrons combinada à Tomografia ComputadorizadaRESUMO
Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.