RESUMO
Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1ß/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1ß processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1ß expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores Corda/imunologia , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium/imunologia , Peptidoglicano/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Adjuvantes Imunológicos , Animais , Proteínas Adaptadoras de Sinalização CARD , Diferenciação Celular/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Mycobacterium/química , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-18/metabolismo , Transdução de Sinais , Células Th17/citologia , Receptores Toll-Like/metabolismoRESUMO
Dendritic cells (DCs), monocytes, and/or macrophages initiate host-protective immune responses to intracellular pathogens in part through interleukin-12 (IL-12) production, although the relative contribution of tissue resident versus recruited cells has been unclear. Here, we showed that after intraperitoneal infection with Toxoplasma gondii cysts, resident mononuclear phagocytes are replaced by circulating monocytes that differentiate in situ into inflammatory DCs (moDCs) and F4/80(+) macrophages. Importantly, NK cell-derived interferon-γ (IFN-γ) was required for both the loss of resident mononuclear phagocytes and the local differentiation of monocytes into macrophages and moDCs. This newly generated moDC population and not the resident DCs (or macrophages) served as the major source of IL-12 at the site of infection. Thus, NK cell-derived IFN-γ is important in both regulating inflammatory cell dynamics and in driving the local differentiation of monocytes into the cells required for initiating the immune response to an important intracellular pathogen.
Assuntos
Células Dendríticas/imunologia , Interferon gama/fisiologia , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Transferência Adotiva , Animais , Antígenos Ly/análise , Diferenciação Celular , Quimiotaxia de Leucócito , Células Dendríticas/patologia , Células Dendríticas/transplante , Genes Reporter , Subunidade p40 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/genética , Células Matadoras Naturais/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/transplante , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/química , Monócitos/patologia , Monócitos/transplante , Fator 88 de Diferenciação Mieloide/fisiologia , Neutrófilos/imunologia , Peritonite/imunologia , Peritonite/parasitologia , Fagócitos/classificação , Fagócitos/imunologia , Fagócitos/patologia , Receptores de Interferon/deficiência , Receptores de Interferon/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologia , Toxoplasmose Animal/imunologia , Receptor de Interferon gamaRESUMO
Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition mouse dendritic cells (DCs) to promote Th2 lymphocyte differentiation. Using an in vitro bystander polarization assay as the readout, we purified and identified the major Th2-inducing component from soluble egg extract (SEA) as the secreted T2 ribonuclease, omega-1. The Th2-promoting activity of omega-1 was found to be sensitive to ribonuclease inhibition and did not require MyD88/TRIF signaling in DCs. In common with unfractioned SEA, the purified native protein suppresses lipopolysaccharide-induced DC activation, but unlike SEA, it fails to trigger interleukin 4 production from basophils. Importantly, omega-1-exposed DCs displayed pronounced cytoskeletal changes and exhibited decreased antigen-dependent conjugate formation with CD4(+) T cells. Based on this evidence, we hypothesize that S. mansoni omega-1 acts by limiting the interaction of DCs with CD4(+) T lymphocytes, thereby lowering the strength of the activation signal delivered.
Assuntos
Antígenos de Helmintos/imunologia , Células Dendríticas/imunologia , Proteínas de Helminto/imunologia , Ribonucleases/imunologia , Schistosoma mansoni/imunologia , Células Th2/imunologia , Animais , Adesão Celular , Diferenciação Celular , Meios de Cultivo Condicionados , Células Dendríticas/citologia , Feminino , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óvulo/imunologia , Ribonucleases/antagonistas & inibidores , Células Th2/citologiaRESUMO
Toxoplasma gondii tachyzoites infect host cells by an active invasion process leading to the formation of a specialized compartment, the parasitophorous vacuole (PV). PVs resist fusion with host cell endosomes and lysosomes and are thus distinct from phagosomes. Because the parasite remains sequestered within the PV, it is unclear how T. gondii-derived antigens (Ag's) access the major histocompatibility complex (MHC) class I pathway for presentation to CD8(+) T cells. We demonstrate that recruitment of host endoplasmic reticulum (hER) to the PV in T. gondii-infected dendritic cells (DCs) directly correlates with cross-priming of CD8(+) T cells. Furthermore, we document by immunoelectron microscopy the transfer of hER components into the PV, a process indicative of direct fusion between the two compartments. In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these findings demonstrate that pathogen-driven hER-PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8(+) T cell cross-priming.
Assuntos
Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Retículo Endoplasmático/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Vacúolos/imunologia , Transferência Adotiva , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/ultraestrutura , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Vacúolos/parasitologiaRESUMO
To investigate if transporter associated with antigen processing (TAP)-1 is required for CD8(+) T cell-mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1(-/-), CD8(-/-), and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1(-/-) mice displayed greater susceptibility than CD8(-/-), beta(2)-microglobulin(-/-) (beta(2)m(-/-)), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1(-/-) mice correlated with a reduction in the frequency of activated (CD62L(low) CD44(hi)) and interferon (IFN)-gamma-producing CD4(+) T cells. Interestingly, infected TAP-1(-/-) mice also showed reduced numbers of IFN-gamma-producing natural killer (NK) cells relative to WT, CD8(-/-), or beta(2)m(-/-) mice, and after NK cell depletion both CD8(-/-) and WT mice succumbed to infection with the same kinetics as TAP-1(-/-) animals and displayed impaired CD4(+) T cell IFN-gamma responses. Moreover, adoptive transfer of NK cells obtained from IFN-gamma(+/+), but not IFN-gamma(-/-), animals restored the CD4(+) T cell response of infected TAP-1(-/-) mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-gamma-producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.