RESUMO
Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs.
Assuntos
Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Lectinas Tipo C/imunologia , Tuberculose/imunologia , Animais , Feminino , Lectinas Tipo C/genética , Macaca mulatta , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT1/imunologia , Transdução de SinaisRESUMO
Clinical trials of novel tuberculosis (TB) vaccines are expensive, while global resources for TB vaccine development are limited. Therefore, there is a need for robust and predictive preclinical data to support advancement of candidate vaccines into clinical trials. Here, we provide a rationale for using the nonhuman primate as an essential component of these efforts, as well as guidance to the TB community for standardizing experimental design and aligning endpoints to facilitate development of new TB vaccines.
Assuntos
Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/isolamento & purificação , Tuberculose/prevenção & controle , Animais , PrimatasRESUMO
Five acute-phase reactants-serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin, albumin, and iron-were measured using commercially available assays in 110 healthy rhesus macaques (Macaca mulatta), and reference intervals were established for future use in health monitoring of this species. Reference intervals established were as follows: SAA, 29.5-87.7 mg/L; CRP, 0-17.5 mg/L; haptoglobin, 354.3-2,414.7 mg/ L; albumin, 36.1-53.0 g/L; and iron, 13.3-40.2 micromol/L. Furthermore, changes in the acute-phase reactants were studied in two additional groups of animals: eight rhesus macaques suffering from acute traumatic injuries and nine rhesus macaques experimentally infected with Mycobacterium tuberculosis reflecting a chronic active inflammation. In animals with inflammation, SAA and haptoglobin concentrations were moderately increased, while CRP increased more than 200-fold. In addition, marked decreases in albumin and iron concentrations were observed. These results show that SAA, CRP, and haptoglobin are positive acute-phase proteins, whereas albumin and iron are negative acute-phase reactants in rhesus macaques.
Assuntos
Reação de Fase Aguda/patologia , Macaca mulatta , Doenças dos Macacos/sangue , Envelhecimento , Albuminas/metabolismo , Animais , Biomarcadores , Proteína C-Reativa/metabolismo , Feminino , Haptoglobinas/metabolismo , Ferro/sangue , Masculino , Doenças dos Macacos/metabolismo , Valores de Referência , Proteína Amiloide A Sérica/metabolismo , Caracteres SexuaisRESUMO
Host genetic factors are important in determining the outcome of infections caused by intracellular pathogens, including mycobacteria and salmonellae, but until now have been poorly characterized. Recently, some individuals with severe infections due to otherwise weakly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis bacille Calmette-Guérin) or Salmonella species have been shown to be unable to produce or respond to interferon-gamma. This inability results from mutations in any of five genes encoding essential proteins of the type 1 cytokine cascade: interleukin-12p40, interleukin-12R beta 1, interferon-gamma R1, interferon-gamma R2 or STAT1. Ten syndromes have thus far been identified. Recent insights in genetically controlled host defense and susceptibility to mycobacterial disease are discussed.
Assuntos
Citocinas/genética , Infecções por Mycobacterium/genética , Infecções por Salmonella/genética , Citocinas/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Predisposição Genética para Doença , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Subunidade p40 da Interleucina-12 , Infecções por Mycobacterium/imunologia , Fenótipo , Subunidades Proteicas , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina-12 , Fator de Transcrição STAT1 , Infecções por Salmonella/imunologia , Transativadores/genética , Transativadores/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor de Interferon gamaRESUMO
Tuberculosis (TB) is a prevalent disease causing an estimated 1.6 million deaths and 10.6 million new cases annually. Discriminating TB disease from differential diagnoses can be complex, particularly in the field. Increased levels of complement component C1q in serum have been identified as a specific and accessible biomarker for TB disease but the source of C1q in circulation has not been identified. Here, data and samples previously collected from human cohorts, a clinical trial and a non-human primate study were used to identify cells producing C1q in circulation. Cell subset frequencies were correlated with serum C1q levels and combined with single cell RNA sequencing and flow cytometry analyses. This identified monocytes as C1q producers in circulation, with a pronounced expression of C1q in classical and intermediate monocytes and variable expression in non-classical monocytes.
Assuntos
Monócitos , Tuberculose , Animais , Humanos , Monócitos/metabolismo , Complemento C1q/metabolismo , Tuberculose/diagnóstico , Tuberculose/metabolismo , Primatas , Biomarcadores/metabolismoRESUMO
Background: Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods: In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results: Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions: The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.
Assuntos
Pesquisa Biomédica , Vacinas , Comércio , Fatores SocioeconômicosRESUMO
It has recently become clear that spatial organization contributes to cellular function and that expanding our knowledge on cellular organization is essential to further our understanding of processes in health and disease. Imaging mass cytometry enables high dimensional imaging of tissue while preserving spatial context and is therefore a suitable tool to unravel spatial relationships between cells. As availability of human tissue collected over the course of disease or infection is limited, preclinical models are a valuable source of such material. Non-human primate models are used for translational research as their anatomy, physiology and immune system closely resemble those of humans due to close evolutionary proximity. Tissue from non-human primate studies is often preserved large archives encompassing a range of conditions and organs. However, knowledge on antibody clones suitable for FFPE tissue of non-human primate origin is very limited. Here, we present an imaging mass cytometry panel development pipeline which enables the selection and incorporation of antibodies for imaging of non-human primate tissue. This has resulted in an 18-marker backbone panel which enables visualization of a broad range of leukocyte subsets in rhesus and cynomolgus macaque tissues. This high-dimensional imaging mass cytometry panel can be used to increase our knowledge of cellular organization within tissues and its effect on outcome of disease.
Assuntos
Citometria por Imagem , Sistema Imunitário , Animais , Imunofenotipagem , Macaca fascicularis , Macaca mulattaRESUMO
Vaccine development for Plasmodium vivax, an important human relapsing malaria, is lagging behind. In the case of the most deadly human malaria P. falciparum, unprecedented high levels of protection have been obtained by immunization with live sporozoites under accompanying chemoprophylaxis, which prevents the onset of blood-stage malaria. Such an approach has not been fully evaluated for relapsing malaria. Here, in the P. cynomolgi-rhesus macaque model for relapsing malaria, we employ the parasites' natural relapsing phenotype to self-boost the immune response against liver-stage parasites, following a single-shot high-dose live sporozoite vaccination. This approach resulted in sterile protection against homologous sporozoite challenge in three out of four animals in the group that was also exposed for several days to blood stages during primary infection and relapses. One out of four animals in the group that received continuous chemoprophylaxis to abort blood-stage exposure was also protected from sporozoite challenge. Although obtained in a small number of animals as part of a Proof-of-Concept study, these results suggest that limited blood-stage parasite exposure may augment protection in this model. We anticipate our data are a starting point for further research into correlates of protection and extrapolation of the single-shot approach to develop efficacious malaria vaccines against relapsing human malaria.
RESUMO
Despite the possibilities of routine clinical measures and assays on readily accessible bio-samples, it is not always essential in animals to investigate the dynamics of disease longitudinally. In this regard, minimally invasive imaging methods provide powerful tools in preclinical research. They can contribute to the ethical principle of gathering as much relevant information per animal as possible. Besides, with an obvious parallel to clinical diagnostic practice, such imaging platforms are potent and valuable instruments leading to a more refined use of animals from a welfare perspective. Non-human primates comprise highly relevant species for preclinical research to enhance our understanding of disease mechanisms and/or the development of improved prophylactic or therapeutic regimen for various human diseases. In this paper, we describe parameters that critically affect the quality of integrated positron emission tomography and computed tomography (PET-CT) in non-human primates. Lessons learned are exemplified by results from imaging experimental infectious respiratory disease in macaques; specifically tuberculosis, influenza, and SARS-CoV-2 infection. We focus on the thorax and use of 18F-fluorodeoxyglucose as a PET tracer. Recommendations are provided to guide various stages of PET-CT-supported research in non-human primates, from animal selection, scan preparation, and operation, to processing and analysis of imaging data.
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The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a 'challenge' experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as 'Moderate' in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.
Assuntos
Mycobacterium tuberculosis , Vacinas contra a Tuberculose , Tuberculose , Animais , Cobaias , Leucócitos Mononucleares , Camundongos , Primatas , Tuberculose/prevenção & controleRESUMO
To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis-derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies.
Assuntos
Vacina BCG/administração & dosagem , Imunidade nas Mucosas , Mycobacterium tuberculosis/imunologia , Mucosa Respiratória/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/prevenção & controle , Administração Intranasal , Animais , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Feminino , Regulação da Expressão Gênica , Injeções Intradérmicas , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Macaca mulatta , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Mucosa Respiratória/microbiologia , Células Th1/imunologia , Células Th1/microbiologia , Células Th17/imunologia , Células Th17/microbiologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.
RESUMO
BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces.
Assuntos
Vacina BCG/administração & dosagem , Imunidade nas Mucosas , Mycobacterium tuberculosis/imunologia , Mucosa Respiratória/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/prevenção & controle , Acetilação , Administração Intranasal , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Medula Óssea/microbiologia , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Feminino , Regulação da Expressão Gênica , Histonas/genética , Histonas/imunologia , Injeções Intravenosas , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Macaca mulatta , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Mucosa Respiratória/microbiologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tuberculosis (TB) causes 1.6 million deaths annually. Early differential diagnosis of active TB infection is essential in optimizing treatment and reducing TB mortality, but is hampered by a lack of accurate and accessible diagnostics. Previously, we reported on complement component C1q, measured in serum by ELISA, as a candidate biomarker for active tuberculosis. In this work we further examine the dynamics of C1q as a marker of progressive TB disease in non-human primates (NHP). We assessed systemic and pulmonary C1q levels after experimental infection using high or low single dose as well as repeated limiting dose Mycobacterium tuberculosis (Mtb) challenge of macaques. We show that increasing C1q levels, either peripherally or locally, correlate with progressive TB disease, assessed by PET-CT imaging or post-mortem evaluation. Upregulation of C1q did not precede detection of Mtb infection by a conventional interferon-gamma release assay, confirming its association with disease progression. Finally, pulmonary vaccination with Bacillus Calmette Guérin also increased local production of C1q, which might contribute to the generation of pulmonary protective immunity. Our data demonstrate that NHP modelling of TB can be utilized to study the role of C1q as a liquid biomarker in TB protection and disease, complementing findings in TB patients.
Assuntos
Complemento C1q/metabolismo , Tuberculose Pulmonar/diagnóstico , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Progressão da Doença , MacacaRESUMO
Vaccination through the natural route of infection represents an attractive immunization strategy in vaccinology. In the case of tuberculosis, vaccine delivery by the respiratory route has regained interest in recent years, showing efficacy in different animal models. In this context, respiratory vaccination triggers lung immunological mechanisms which are omitted when vaccines are administered by parenteral route. However, contribution of mucosal antibodies to vaccine- induced protection has been poorly studied. In the present study, we evaluated in mice and non-human primates (NHP) a novel whole cell inactivated vaccine (MTBVAC HK), by mucosal administration. MTBVAC HK given by intranasal route to BCG-primed mice substantially improved the protective efficacy conferred by subcutaneous BCG only. Interestingly, this improved protection was absent in mice lacking polymeric Ig receptor (pIgR), suggesting a crucial role of mucosal secretory immunoglobulins in protective immunity. Our study in NHP confirmed the ability of MTBVAC HK to trigger mucosal immunoglobulins. Importantly, in vitro assays demonstrated the functionality of these immunoglobulins to induce M. tuberculosis opsonization in the presence of human macrophages. Altogether, our results suggest that mucosal immunoglobulins can be induced by vaccination to improve protection against tuberculosis and therefore, they represent a promising target for next generation tuberculosis vaccines.
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Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.
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Tuberculosis (TB) remains the deadliest infectious disease1, and the widely used Bacillus Calmette-Guérin (BCG) vaccine fails to curb the epidemic. An improved vaccination strategy could provide a cost-effective intervention to break the transmission cycle and prevent antimicrobial resistance2,3. Limited knowledge of the host responses critically involved in protective immunity hampers the development of improved TB vaccination regimens. Therefore, assessment of new strategies in preclinical models to select the best candidate vaccines before clinical vaccine testing remains indispensable. We have previously established in rhesus macaques (Macaca mulatta) that pulmonary mucosal BCG delivery reduces TB disease where standard intradermal injection fails4,5. Here, we show that pulmonary BCG prevents infection by using a repeated limiting-dose Mycobacterium tuberculosis challenge model and identify polyfunctional T-helper type 17 (TH17) cells, interleukin-10 and immunoglobulin A as correlates of local protective immunity. These findings warrant further research into mucosal immunization strategies and their translation to clinical application to more effectively prevent the spread of TB.
Assuntos
Vacina BCG/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Carga Bacteriana , Relação Dose-Resposta Imunológica , Imunidade Humoral , Interferon gama/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macaca mulatta , Masculino , Mucosa/imunologia , VacinaçãoRESUMO
While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.
Assuntos
Macaca fascicularis/imunologia , Macaca mulatta/imunologia , Mycobacterium tuberculosis , Tuberculose Pulmonar/imunologia , Animais , Citocinas/imunologia , Imunidade Inata , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
DC-SIGN (CD209/CLEC4L) is a C-type lectin receptor (CLR) that serves as a reliable cell-surface marker of interleukin 4 (IL-4)-activated human macrophages [M(IL-4)], which historically represent the most studied subset within the M2 spectrum of macrophage activation. Although DC-SIGN plays important roles in Mycobacterium tuberculosis (Mtb) interactions with dendritic cells, its contribution to the Mtb-macrophage interaction remains poorly understood. Since high levels of IL-4 are correlated with tuberculosis (TB) susceptibility and progression, we investigated the role of DC-SIGN in M(IL-4) macrophages in the TB context. First, we demonstrate that DC-SIGN expression is present both in CD68+ macrophages found in tuberculous pulmonary lesions of non-human primates, and in the CD14+ cell population isolated from pleural effusions obtained from TB patients (TB-PE). Likewise, we show that DC-SIGN expression is accentuated in M(IL-4) macrophages derived from peripheral blood CD14+ monocytes isolated from TB patients, or in macrophages stimulated with acellular TB-PE, arguing for the pertinence of DC-SIGN-expressing macrophages in TB. Second, using a siRNA-mediated gene silencing approach, we performed a transcriptomic analysis of DC-SIGN-depleted M(IL-4) macrophages and revealed the upregulation of pro-inflammatory signals in response to challenge with Mtb, as compared to control cells. This pro-inflammatory gene signature was confirmed by RT-qPCR, cytokine/chemokine-based protein array, and ELISA analyses. We also found that inactivation of DC-SIGN renders M(IL-4) macrophages less permissive to Mtb intracellular growth compared to control cells, despite the equal level of bacteria uptake. Last, at the molecular level, we show that DC-SIGN interferes negatively with the pro-inflammatory response and control of Mtb intracellular growth mediated by another CLR, Dectin-1 (CLEC7A). Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology associated to TB.
Assuntos
Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/imunologia , Receptores de Superfície Celular/metabolismo , Tuberculose/imunologia , Tuberculose/metabolismo , Animais , Moléculas de Adesão Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citocinas/metabolismo , Feminino , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/genética , Macaca mulatta , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose/imunologia , Receptores de Superfície Celular/genética , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI. Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls. Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis, increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection. Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB.