RESUMO
The type-III interferons (IFNs) are the most recently discovered IFNs in the human immune system and have important, but as yet poorly characterized, functions in innate and adaptive immunity that complement their antiviral functions. It is now becoming clear that these type-III IFNs have a functional niche where epithelial surfaces interact with the adaptive immune system, that their antiviral capability is not as highly developed as that of the type-I IFNs, and that they have their own profile of immunomodulatory functions; specifically, they are key modulators of the T-helper (Th)2 response.
Assuntos
Epitélio/imunologia , Interleucinas/farmacologia , Células Th2/imunologia , Viroses/imunologia , Imunidade Adaptativa , Antivirais/uso terapêutico , Epitélio/efeitos dos fármacos , Humanos , Imunomodulação , Interferons , Interleucinas/uso terapêutico , Células Th2/efeitos dos fármacos , Viroses/tratamento farmacológicoRESUMO
The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]), IFN-lambda2 (IL-28A), and IFN-lambda3 (IL-28B), each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently, it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here, we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However, pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments, we showed that IL-4-responsive monocytes are an intermediary cell, responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus, our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function, and suggests an expanded immunomodulatory role for Th2-associated cytokines.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Interleucina-4/farmacologia , Interleucinas/biossíntese , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Células Dendríticas/metabolismo , Herpesvirus Humano 1/imunologia , Humanos , Técnicas In Vitro , Interferons , Interleucinas/genética , Modelos Imunológicos , Monócitos/metabolismo , Proteínas Recombinantes/farmacologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismoRESUMO
The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1, -2, and -3) signals through the same heterodimeric receptor complex, which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution, the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells, IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition, IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13, IFN-gamma, and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential.
Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Interleucinas/imunologia , Plasmócitos/imunologia , Adjuvantes Imunológicos/farmacologia , Aminoquinolinas/farmacologia , Células Dendríticas/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imiquimode , Interferon gama/imunologia , Interferons , Interleucina-10/imunologia , Subunidade beta de Receptor de Interleucina-10/imunologia , Interleucina-13/imunologia , Interleucinas/farmacologia , Plasmócitos/citologia , RNA Mensageiro/imunologia , Receptores de Citocinas/imunologiaRESUMO
IFN-lambda1 (IL-29) plays a novel, emerging role in the inhibition of human Th2 responses. Here, we demonstrate that both naive and memory human CD4(+) T cells express mRNA for the IFN-lambda1-specific receptor, IL-28Ralpha, and are responsive to IFN-lambda1. Expression of Th2 cytokines (IL-4 and IL-13) was suppressed in naive and memory CD4(+) T cells by IFN-lambda1, without affecting their proliferation. Further, acquisition of IL-4Ralpha expression after stimulation was inhibited by IFN-lambda1, as was GATA3 expression. Finally, IFN-lambda1 diminished the change in cell-surface phenotype that accompanies differentiation of "central memory" T cells into "effector memory" T cells. Taken together, our data describe unique immunomodulatory effects of IFN-lambda1 and identify novel mechanisms for the reduction of existing Th2 responses and the regulation of new ones, in circulating naive and memory CD4(+) T cells.
Assuntos
Fator de Transcrição GATA3/metabolismo , Imunidade Inata/imunologia , Memória Imunológica/imunologia , Interleucinas/farmacologia , Células Th2/imunologia , Células Th2/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Humanos , Imunidade Inata/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Interleucinas/biossíntese , Receptores de Citocinas/imunologia , Células Th2/citologia , Células Th2/efeitos dos fármacosRESUMO
Interferon (IFN)-lambda1 [interleukin (IL)-29] is a member of the interferon lambda family (also known as type III interferons), whose members are distantly related to both the type I interferons and members of the IL-10 family. While IFN-lambda1 has significant antiviral activity, it is also becoming apparent that it has important immunoregulatory properties, especially with regard to the T helper type 2 (Th2) response. Previously, we have shown that IFN-lambda1 is capable of down-regulating IL-13 production in an IFN-gamma-independent manner and that this is mediated in part via monocyte-derived dendritic cells. Here, we have extended our knowledge of IFN-lambda1 regulation of the human in vitro Th2 response by examining the regulation of three major Th2 cytokines, IL-4, IL-5 and IL-13, by IFN-lambda1. Our results reveal that IFN-lambda1 preferentially inhibits IL-13 production, compared with IL-4 or IL-5. Levels of IL-13 mRNA, the amount of secreted IL-13 protein and numbers of IL-13-positive CD3(+) CD4(+) cells were all significantly diminished by IFN-lambda1. IFN-lambda1 significantly decreased some aspects of IL-4 and IL-5 production, but its effects were not as consistent as those seen on IL-13. IFN-lambda1 was also effective at decreasing IL-13 secretion under conditions designed to support the generation of Th2 cells. Irrespective of whether Concanavalin-A or T-cell-stimulatory microbeads were used, IFN-lambda1 markedly diminished IL-13 secretion in cultures where IL-4 had been added. Thus, IFN-lambda1 appears to be an inhibitor of human Th2 responses whose action is primarily directed towards IL-13 but which may also affect Th2 responses generally and does not invoke a complementary elevation of IFN-gamma secretion.
Assuntos
Regulação para Baixo , Interleucina-13/imunologia , Interleucinas/imunologia , Células Th2/imunologia , Células Cultivadas , Concanavalina A , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Humanos , Terapia de Imunossupressão , Interferons , Interleucina-13/análise , Interleucina-13/genética , Interleucina-4/análise , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/análise , Interleucina-5/genética , Interleucina-5/imunologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Plasmacytoid dendritic cells (pDC) are well-known for their ability to produce large quantities of interferon-alpha (IFN-alpha) in response to viruses. In addition, pDC produce IFN-alpha in response to HSV-infected cells. We demonstrate that both tonsil and PBMC contain pDC that respond to stimulation with HSV either in suspension or in tonsil tissue-fragment culture. We hypothesized that other DC subsets acquire virus in the periphery and deliver the interferongenic signals to the pDC in the draining lymphoid tissue. As a model for pDC/myeloid DC interaction, we studied the interaction of pDC derived from blood with HSV-infected and uninfected monocyte derived dendritic cells (MDDC). Infected, but not uninfected, MDDC induced IFN-alpha in pDC. To further study pDC/infected MDDC interactions, we labeled MDDC with fluorescent cell trackers PKH67 or CFSE prior to infection with HSV and co-cultured with pDC. Cells were then analyzed using conventional and imaging flow cytometry. In addition, we infected MDDC with a GFP-expressing HSV prior to co-culture with pDC. Using traditional flow cytometry, we observed that pDC became fluorescent after co-incubation with uninfected or infected, fluorescently labeled MDDC, indicating that MDDC transferred fluorescent protein and membrane to pDC. By imaging flow cytometry, we observed formation of conjugates between pDC and MDDC as well as transfer and internalization of cellular components from the labeled MDDC by pDC, with preferential uptake from, and association with, infected vs. uninfected MDDC. These studies demonstrate that MDDC infected with HSV are able to stimulate IFN-alpha and chemokine production by pDC through the transfer of cellular materials from the HSV-infected MDDC to the pDC. Together, these observations indicate that heterogeneous populations of DC interact to generate an effective IFN-alpha response.
Assuntos
Células Dendríticas/imunologia , Citometria de Fluxo/métodos , Interferon Tipo I/biossíntese , Monócitos/metabolismo , Plasmócitos/metabolismo , Animais , Células Sanguíneas , Células Dendríticas/efeitos dos fármacos , Diagnóstico por Imagem , Citometria de Fluxo/instrumentação , Humanos , Interferon Tipo I/metabolismo , Monócitos/imunologia , Monócitos/virologia , Compostos Orgânicos/metabolismo , Plasmócitos/virologia , Simplexvirus/fisiologia , Técnicas de Cultura de TecidosRESUMO
Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.
Assuntos
Células Dendríticas/imunologia , Herpesvirus Humano 1/fisiologia , Interferon-alfa/biossíntese , Receptores Imunológicos/metabolismo , Anticorpos/farmacologia , Antígenos de Superfície/efeitos dos fármacos , Apoptose , Antígenos CD4/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Células Dendríticas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Fator Regulador 7 de Interferon/metabolismo , Interferon-alfa/genética , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-3/efeitos dos fármacos , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/efeitos dos fármacos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/efeitos dos fármacos , Microesferas , Fosforilação , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/efeitos dos fármacos , Receptores de Interleucina-3/antagonistas & inibidores , Receptores de Interleucina-3/efeitos dos fármacos , Tirosina/metabolismoRESUMO
Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.
Assuntos
Antituberculosos/farmacologia , Etambutol/farmacologia , Genes Bacterianos , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Adaptação Fisiológica , Transporte Biológico Ativo/genética , Clonagem Molecular , Tolerância a Medicamentos/genética , Inibidores Enzimáticos/farmacologia , Etídio/farmacologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Isoniazida/análise , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estrutura Quaternária de Proteína , Reserpina/farmacologia , Transformação GenéticaRESUMO
Human plasmacytoid dendritic cells (PDC) are a major source of IFN-alpha upon exposure to enveloped viruses and TLR-7 and TLR-9 ligands. Although IFN regulatory factor-7 (IRF-7) is known to play an essential role in virus-activated transcription of IFN-alpha genes, the molecular mechanisms of IFN-alpha production in human PDC remain poorly understood. We and others have recently reported high constitutive levels of IRF-7 expression in PDC as compared with other PBMC. In this study, we demonstrate that both LPS and HSV up-regulate the expression of IRF-7 in PDC, and that this enhancement of IRF-7 is dependent on NF-kappa B activation. The NF-kappa B inhibitors MG132 and pyrrolidinedithiocarbamate efficiently inhibited the induction of IRF-7 by HSV or LPS, and also down-regulated the constitutive expression of IRF-7 in PDC and blocked the HSV-induced production of IFN-alpha. In addition, we found that nuclear translocation of IRF-7 occurred rapidly in response to HSV stimulation, but not in response to LPS, which is consistent with the stimulation of IFN-alpha production by virus and not by LPS. Although LPS by itself was not able to induce IFN-alpha production, it led to rapid up-regulation of TLR-4 on PDC and increased the magnitude and accelerated the kinetics of HSV-induced IFN-alpha production in PDC, providing a mechanism that might be operative in a scenario of mixed infection. In contrast to the current concept of IFN-alpha regulation established in cell lines, this study strongly supports the immediate availability of high constitutive levels of IRF-7 expression in PDC, and suggests an activation required for IRF-7 that contributes to IFN-alpha production in virus-stimulated PDC.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Células Dendríticas/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Interferon-alfa/biossíntese , Lipopolissacarídeos/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/virologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Cães , Humanos , Vírus da Influenza A/fisiologia , Fator Regulador 7 de Interferon , Interferon-alfa/genética , Rim , Leupeptinas/farmacologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Polimixina B/farmacologia , Pirrolidinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Vírus Sendai/fisiologia , Tiocarbamatos/farmacologia , Receptor 4 Toll-Like , Receptor 7 Toll-Like , Receptor Toll-Like 9 , Receptores Toll-Like , Transcrição GênicaRESUMO
The natural interferon (IFN)-producing cell is now known to be identical to the plasmacytoid dendritic cell (PDC). These are Lin-, CD123+, CD11c-, and human leukocyte antigen-DR+ cells that secrete large amounts of IFN-alpha (1-2 IU/cell) when stimulated by enveloped viruses such as herpes simplex virus. In the current study, we have evaluated chemokine expression by virally stimulated PDC. Up-regulation of mRNA for CCL4, CCL3, CCL5, CCL2, and CXC chemokine ligand (CXCL)10 in herpes simplex virus-stimulated PDC was detected by RNAse protection assays. In contrast, PDC-depleted peripheral blood mononuclear cells did not up-regulate these mRNA species upon viral stimulation. Enzyme-linked immunosorbent assay and/or intracellular flow cytometry confirmed production of these proteins, and studies indicated overlapping production of IFN-alpha and the other cytokines/chemokines by PDC. Endocytosis plays a critical role in chemokine induction, as disruption of the pathway inhibits the response. However, transcription of viral genes is not required for chemokine induction. Autocrine IFN-alpha signaling in the PDC could account for a portion of the CXCL10 and CCL2 production in virally stimulated PDC but was not responsible for the induction of the other chemokines. To evaluate the functional role of the chemokines, chemotaxis assays were performed using supernatants from virally stimulated PDC. Activated T cells and natural killer cells, but not naïve T cells, were preferentially recruited by these PDC supernatants. Migration was subsequently inhibited by addition of neutralizing antibody to CCL4 and CXCL10. We hypothesize that virally induced chemokine production plays a pivotal role in the homing of leukocytes to PDC.