RESUMO
Humanized mice harboring human immune systems (HIS) represent a platform to study immune responses against pathogens and to screen vaccine candidates and novel immunotherapeutics. Innate and adaptive immune responses are suboptimal in HIS mice, possibly due to poor reconstitution of human antigen-presenting cells, including dendritic cells (DCs). DC homeostasis is regulated by cytokine availability, and Flt3-ligand (Flt3L) is one factor that conditions this process. Mouse myelopoiesis is essentially normal in most current HIS models. As such, developing mouse myeloid cells may limit human DC reconstitution by reducing available Flt3L and by cellular competition for specific "niches." To address these issues, we created a novel HIS model that compromises host myeloid cell development via deficiency in the receptor tyrosine kinase Flk2/Flt3. In Balb/c Rag2(-/-) Il2rg(-/-) Flt3(-/-) (BRGF) recipients, human conventional DCs and plasmacytoid DCs develop from hCD34(+) precursors and can be specifically boosted with exogenous Flt3L. Human DCs that develop in this context normally respond to TLR stimulation, and improved human DC homeostasis is associated with increased numbers of human NK and T cells. This new HIS-DC model should provide a means to dissect human DC differentiation and represents a novel platform to screen immune adjuvants and DC targeting therapies.
Assuntos
Diferenciação Celular , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Tirosina Quinase 3 Semelhante a fms/deficiência , Adjuvantes Imunológicos , Animais , Homeostase , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/fisiologia , Células T Matadoras Naturais/fisiologia , Linfócitos T/fisiologia , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
The contribution of lymphotoxin (LT)α in the host immune response to virulent Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin infections was investigated. Despite their ability to induce Th1 cytokine, IFN-γ, and IL-12 pulmonary response, "conventional" LTα(-/-) mice succumb rapidly to virulent M. tuberculosis aerosol infection, with uncontrolled bacilli growth, defective granuloma formation, necrosis, and reduced pulmonary inducible NO synthase expression, similar to TNF(-/-) mice. Contributions from developmental lymphoid abnormalities in LTα(-/-) mice were excluded because hematopoietic reconstitution with conventional LTα(-/-) bone marrow conferred enhanced susceptibility to wild-type mice, comparable to conventional LTα(-/-) control mice. However, conventional LTα(-/-) mice produced reduced levels of TNF after M. bovis bacillus Calmette-Guérin infection, and their lack of control of mycobacterial infection could be due to a defective contribution of either LTα or TNF, or both, to the host immune response. To address this point, the response of "neo-free" LTα(-/-) mice with unperturbed intrinsic TNF expression to M. tuberculosis infection was investigated in a direct comparative study with conventional LTα(-/-) mice. Strikingly, although conventional LTα(-/-) mice were highly sensitive, similar to TNF(-/-) mice, neo-free LTα(-/-) mice controlled acute M. tuberculosis infection essentially as wild-type mice. Pulmonary bacterial burden and inflammation was, however, slightly increased in neo-free LTα(-/-) mice 4-5 mo postinfection, but importantly, they did not succumb to infection. Our findings revise the notion that LTα might have a critical role in host defense to acute mycobacterial infection, independent of TNF, but suggest a contribution of LTα in the control of chronic M. tuberculosis infection.
Assuntos
Linfotoxina-alfa/imunologia , Tuberculose/imunologia , Animais , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Linfotoxina-alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/imunologia , Tuberculose/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Mycobacterium tuberculosis is recognized by multiple pattern recognition receptors involved in innate immune defense, but their direct role in tuberculosis pathogenesis remains unknown. Beyond TLRs, scavenger receptors (SRs) and C-type lectins may play a crucial role in the sensing and signaling of pathogen motifs, as well as contribute to M. tuberculosis immune evasion. In this study, we addressed the relative role and potential redundancy of these receptors in the host response and resistance to M. tuberculosis infection using mice deficient for representative SR, C-type lectin receptor, or seven transmembrane receptor families. We show that a single deficiency in the class A SR, macrophage receptor with collagenous structure, CD36, mannose receptor, specific ICAM-3 grabbing nonintegrin-related, or F4/80 did not impair the host resistance to acute or chronic M. tuberculosis infection in terms of survival, control of bacterial clearance, lung inflammation, granuloma formation, and cytokine and chemokine expression. Double deficiency for the SRs class A SR types I and II plus CD36 or for the C-type lectins mannose receptor plus specific ICAM-3 grabbing nonintegrin-related had a limited effect on macrophage uptake of mycobacteria and TNF response and on the long-term control of M. tuberculosis infection. By contrast, mice deficient in the TNF, IL-1, or IFN-gamma pathway were unable to control acute M. tuberculosis infection. In conclusion, we document a functional redundancy in the pattern recognition receptors, which might cooperate in a coordinated response to sustain the full immune control of M. tuberculosis infection, in sharp contrast with the nonredundant, essential role of the TNF, IL-1, or IFN-gamma pathway for host resistance to M. tuberculosis.
Assuntos
Lectinas Tipo C/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Receptores Depuradores/imunologia , Tuberculose/imunologia , Animais , Antígenos CD36/genética , Antígenos CD36/imunologia , Antígenos CD36/metabolismo , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-1/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Mycobacterium tuberculosis/imunologia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tuberculose/genética , Tuberculose/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TNF is essential to control Mycobacterium tuberculosis infection and cannot be replaced by other proinflammatory cytokines. Overproduction of TNF may cause immunopathology, while defective TNF production results in uncontrolled infection. The critical role of TNF in the control of tuberculosis has been illustrated recently by primary and reactivation of latent infection in some patients under pharmacological anti-TNF therapy for rheumatoid arthritis or Crohn's disease. In this review, we discuss results of recent studies aimed at better understanding of molecular, cellular and kinetic aspects of TNF-mediated regulation of host-mycobacteria interactions. In particular, recent data using either mutant mice expressing solely membrane TNF or specific inhibitor sparing membrane TNF demonstrated that membrane TNF is sufficient to control acute M. tuberculosis infection. This is opening the way to selective TNF neutralization that might retain the desired anti-inflammatory effect but reduce the infectious risk.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Neutralizantes/efeitos adversos , Humanos , Inflamação/imunologia , Ativação Linfocitária , Ativação de Macrófagos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Imunológicos , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
TNF and IL-1 are major mediators involved in severe inflammatory diseases against which therapeutic neutralizing antibodies are developed. However, both TNF and IL-1 receptor pathways are essential for the control of Mycobacterium tuberculosis infection, and it is critical to assess the respective role of IL-1α, IL-1ß, and TNF. Using gene-targeted mice we show that absence of both IL-1α and IL-1ß recapitulates the uncontrolled M. tuberculosis infection with increased bacterial burden, exacerbated lung inflammation, high IFNγ, reduced IL-23 p19 and rapid death seen in IL-1R1-deficient mice. However, presence of either IL-1α or IL-1ß in single-deficient mice is sufficient to control acute M. tuberculosis infection, with restrained bacterial burden and lung pathology, in conditions where TNF deficient mice succumbed within 4 weeks with overwhelming infection. Systemic infection by attenuated M. bovis BCG was controlled in the absence of functional IL-1 pathway, but not in the absence of TNF. Therefore, although both IL-1α and IL-1ß are required for a full host response to virulent M. tuberculosis, the presence of either IL-1α or IL-1ß allows some control of acute M. tuberculosis infection, and IL-1 pathway is dispensable for controlling M. bovis BCG acute infection. This is in sharp contrast with TNF, which is essential for host response to both attenuated and virulent mycobacteria and may have implications for anti-inflammatory therapy with IL-1ß neutralizing antibodies.
RESUMO
Tumour Necrosis Factor (TNF) is critical for host control of M. tuberculosis, but the relative contribution of TNF from innate and adaptive immune responses during tuberculosis infection is unclear. Myeloid versus T-cell-derived TNF function in tuberculosis was investigated using cell type-specific TNF deletion. Mice deficient for TNF expression in macrophages/neutrophils displayed early, transient susceptibility to M. tuberculosis but recruited activated, TNF-producing CD4(+) and CD8(+) T-cells and controlled chronic infection. Strikingly, deficient TNF expression in T-cells resulted in early control but susceptibility and eventual mortality during chronic infection with increased pulmonary pathology. TNF inactivation in both myeloid and T-cells rendered mice critically susceptible to infection with a phenotype resembling complete TNF deficient mice, indicating that myeloid and T-cells are the primary TNF sources collaborating for host control of tuberculosis. Thus, while TNF from myeloid cells mediates early immune function, T-cell derived TNF is essential to sustain protection during chronic tuberculosis infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Pulmão/imunologia , Mycobacterium tuberculosis/imunologia , Células Mieloides/imunologia , Pneumonia/etiologia , Tuberculose/prevenção & controle , Fator de Necrose Tumoral alfa/fisiologia , Animais , Western Blotting , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Integrases/metabolismo , Pulmão/citologia , Pulmão/microbiologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/microbiologia , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Pneumonia/metabolismo , Pneumonia/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB(4). However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-α and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-γ. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NFκB but was not dependent on peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-IκBα formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.
Assuntos
Dinoprostona/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inflamação/patologia , Macrófagos/patologia , Receptor 2 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Bactérias/metabolismo , Feminino , Inflamação/metabolismo , Interferon gama/farmacologia , Interleucina-10/biossíntese , Leucotrieno B4/metabolismo , Ligantes , Metabolismo dos Lipídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , PPAR gama/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Phosphatidyl myo-inositol mannosides (PIMs) are constituents of the mycobacterial cell wall and possess immunomodulatory activities. Certain PIM derivatives have immunoprotective activity and are of interest as anti-inflammatory agents. In order to identify simplified analogues of PIMs that retain this interesting activity, we have prepared a series of new analogues based either on an acyclic or on a heterocyclic scaffold that replaces the inositol moiety, and evaluated these compounds for their inhibition of LPS-induced release of NO and pro-inflammatory cytokines by macrophages. It was found that the inositol moiety can be favourably replaced by an aza-cyclitol (trihydroxy-piperidine) or an oxa-cyclitol (trihydroxy-tetrahydropyran) unit, and that the configuration of the OH-carrying carbons does not play a significant role. The biological activity is reduced if the nitrogen atom is free in the aza-cyclitol unit.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Mimetismo Molecular , Fosfatidilinositóis/química , Fosfatidilinositóis/farmacologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Inositol/química , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Relação Estrutura-AtividadeRESUMO
Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Inflamação/imunologia , Inflamação/microbiologia , Receptores de Lipopolissacarídeos/imunologia , Manosídeos/metabolismo , Mycobacterium/imunologia , Transdução de Sinais/imunologia , Acilação/efeitos dos fármacos , Animais , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Inflamação/patologia , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium/efeitos dos fármacos , Paclitaxel/química , Paclitaxel/farmacologia , Transdução de Sinais/efeitos dos fármacos , Solubilidade/efeitos dos fármacos , Tensoativos/farmacologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol anchor phosphatidyl-myo-inositol hexamannosides PIM(6) or PIM(2) exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12 (IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing adaptor protein inducing interferon beta (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced NFkappaB translocation. Synthetic PIM(1) and a PIM(2) mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM analogs represent powerful anti-inflammatory leads.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Citocinas/imunologia , Regulação para Baixo , Fator 88 de Diferenciação Mieloide/imunologia , Fosfatidilinositóis/imunologia , Receptor 4 Toll-Like/imunologia , Tuberculose/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células Cultivadas , Citocinas/genética , Expressão Gênica , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/imunologia , Fator 88 de Diferenciação Mieloide/genética , Receptor 4 Toll-Like/genética , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
4-Hydroxynonenal (HNE), a major electrophilic product of lipid peroxidation, is regarded as both a marker of oxidative stress and a mediator of oxidative damage. At subtoxic concentrations, however, this compound has been shown to be a signalling molecule that can induce the expression of various antioxidant/detoxification enzymes, including glutamate-cysteine ligase (GCL), the rate-limiting enzyme in the de novo synthesis of glutathione. GCL consists of a catalytic (GCLC) and modulatory (GCLM) subunit, which are encoded by separate genes. Here, we investigated the effect of submicromolar concentrations of HNE on the expression of the GCL genes and the transcription factors involved. We demonstrated that submicromolar concentrations of HNE (as little as 0.3 muM) could increase the expression of both GCLC and GCLM. We also found that the induction of GCL expression was abrogated by siRNA for Nrf2. Our data suggest that a submicromolar concentration of HNE, as found in human plasma under physiological conditions, can induce GCL transcription in cultured cells implying that 'basal' expression of GCL is under regulation by lipid peroxidation that occurs under physiological conditions. Moreover, this induction is mediated through the EpRE-Nrf2 signalling pathway thought to be predominantly active only during stress.