Toll-like receptor agonists and alpha-galactosylceramide synergistically enhance the production of interferon-gamma in murine splenocytes.
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ABSTRACT:
SUBMITTER: Ando T
PROVIDER: S-EPMC3759050 | biostudies-other | 2013
REPOSITORIES: biostudies-other
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Project description:Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been used as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases or psychoactive compounds. In this study, we aimed to explore the potential of IFN-γ and TLR agonists being applied simultaneously to boost dendritic cell activation and the subsequent antigen presentation. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic-polycytidylic acid (poly I:C), or resiquimod (R848). Next, the dendritic cells were stained for an activation marker, a cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. From the cytometric analysis, IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of dendritic cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 µg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% CD86-positive cells obtained by 10 ng/mL IFN-γ. These results suggested that IFN-γ and TLR agonists could be applied as complementary systems to promote dendritic cell activation and antigen presentation. There might be a synergy between the two classes of molecules, but further investigation is warranted to ascertain the interaction of their promotive activities.
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Project description:IntroductionDuring the course of infection, natural killer (NK) cells contribute to innate immunity by producing cytokines, particularly interferon-gamma (IFN-γ). In addition to their beneficial effects against infection, NK cells may play a detrimental role during systemic inflammation, causing lethality during sepsis. Little is known on the immune status of NK cells in patients with systemic inflammatory response syndrome (SIRS) or sepsis in terms of cell surface markers expression and IFN-γ production.MethodsWe investigated 27 sepsis patients and 11 patients with non-infectious SIRS. CD56bright and CD56dim NK cell subsets were identified by flow cytometry and Toll-like receptor (TLR)2, TLR4, TLR9, CX3CR1, CD16 and CD69 expression were analyzed, as well as ex vivo IFN-γ production by NK cells in whole blood samples.ResultsWe first showed that in NK cells from healthy controls, TLR2 and TLR4 expression is mainly intracellular, similarly to TLR9. Intracellular levels of TLR2 and TLR4, in both CD56bright and CD56dim NK cell subsets from sepsis patients, were increased compared to healthy subjects. In addition, the percentage of CD69+ cells was higher among NK cells of sepsis patients. No difference was observed for TLR9, CX3CR1, and CD16 expression. The ex vivo stimulation by TLR4 or TLR9 agonists, or whole bacteria in synergy with accessory cytokines (IL-15+IL-18), resulted in significant production of IFN-γ by NK cells of healthy controls. In contrast, for SIRS and sepsis patients this response was dramatically reduced.ConclusionsThis study reports for the first time an intracellular expression of TLR2 and TLR4 in human NK cells. Surface TLR4 expression allows discriminating sepsis and SIRS. Furthermore, during these pathologies, NK cells undergo an alteration of their immune status characterized by a profound reduction of their capacity to release IFN-γ.
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Project description:Sézary syndrome (SS) carries a poor prognosis, and infections represent the most frequent cause of death in SS patients. Toll-like receptors (TLRs) are a family of innate immune receptors that induce protective immune responses against infections. We sought to evaluate the ability of TLR agonists to induce inflammatory cytokine, Th2 cytokine, and type I interferon (IFN-I) production by peripheral blood mononuclear cells (PBMC) of untreated SS patients. We detected impaired IL-6, IL-10 and IL-13 secretion by PBMC induced by the agonists for TLR5, TLR3, TLR7 and TLR9 in SS patients, while it was partially recovered by TLR2/TLR4 and TLR7/8 agonists TNF secretion was restored following stimulation with TLR2/TLR4 agonists. IFN-γ was scarcely produced upon TLR activation in SS cells, albeit TLR 7/8 (CL097) enhanced their secretion at lower levels than the control group. TLR9 agonist efficiently induced IFN-I in SS patients, although this positive regulation was not observed for other cytokines, in direct contrast to the broad activity of CL097. Among the TLR agonists, TLR4 was able to induce pro-inflammatory, IL-10 and Th2 secretion, while TLR7-8 agonist induced the inflammatory cytokines, IFN-I and IFN-γ. These findings reveal a dysfunctional cytokine response upon both extracellular and intracellular TLR activation in SS patients, which was partially restored by TLRs agonists.
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Project description:ObjectiveFamilial hemophagocytic lymphohistiocytosis (FHLH) is a complex cytokine storm syndrome caused by genetic abnormalities rendering CD8+ T cells and natural killer cells incapable of cytolytic killing. In murine models of FHLH, interferon-γ (IFNγ) produced by CD8+ T cells has been identified as a critical mediator of disease, and an IFNγ-blocking antibody (emapalumab) has recently been approved by the Food and Drug Administration. However, development of hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) in patients who are genetically unresponsive to IFNγ questions the absolute necessity of IFNγ in driving disease. This study was undertaken to determine the necessity of IFNγ in driving HLH.MethodsIFNγ-/- Prf1-/- mice were infected with lymphocytic choriomeningitis virus (LCMV), and HLH immunopathologic features, including survival, weight loss, cytopenias, cytokine profiles, and immune cell phenotypes, were assessed. Mixed bone marrow chimeras were created to determine the immune cell-intrinsic role of IFNγ receptor signaling. CD8+ T cell depletion and interleukin-33 (IL-33)/ST2 blockade were performed using monoclonal antibodies.ResultsLCMV infection of IFNγ-/- Prf1-/- mice resulted in severe HLH-like disease. CD8+ T cells and the IL-33/ST2 axis remained essential mediators of disease; however, IFNγ-independent HLH immunopathology correlated with a 10-15-fold increase in neutrophilia (P < 0.001) and an altered cytokine milieu dominated by IL-6, IL-1β, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05). Furthermore, IFNγ regulated CD8+ T cell expression of GM-CSF and neutrophil survival.ConclusionIFNγ is not necessary for the development of fulminant HLH, requiring physicians to consider case-by-case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL-33/ST2 signaling, may be more universally applicable treatment options that ameliorate both IFNγ-dependent and -independent manifestations of HLH/MAS.
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Project description:ObjectiveMacrophage activation syndrome (MAS) is a life-threatening cytokine storm syndrome that occurs in patients with underlying rheumatic diseases. Preclinical and clinical data suggest that interferon-γ (IFNγ) is pathogenic in MAS, but how IFNγ may be linked to disease pathogenesis remains unknown. This study was undertaken to determine whether IFNγ signals synergize with systemic innate immune responses to drive the cytokine storm in a murine model of MAS.MethodsIFNγ-deficient mice were treated with 5 doses of the Toll-like receptor 9 (TLR-9) agonist CpG 1826, IFNγ, or a combination of the 2 stimuli over the course of 10 days. Immunopathologic features of MAS, including cytopenias, hepatitis, hepatosplenomegaly, and induction of inflammatory myelopoiesis, were assessed. Mixed bone marrow chimeras were created to determine whether TLR-9- and IFNγ receptor 1 (IFNγR1)-dependent signals induce enhanced myelopoiesis in a cell-intrinsic or cell-extrinsic manner.ResultsIFNγ-deficient mice did not develop features of MAS when treated with repeated doses of either the TLR-9 agonist or IFNγ alone. In contrast, IFNγ-deficient mice treated with both the TLR-9 agonist and IFNγ developed cytopenias, hepatitis, and hepatosplenomegaly, reproducing major clinical features of MAS. TLR-9- and IFNγR1-dependent signals synergized to enhance myeloid progenitor cell function and induce myelopoiesis in vivo, which occurred through cell-extrinsic mechanisms and correlated with the induction of disease.ConclusionThese findings demonstrate that TLR-9-driven signals potentiate the effects of IFNγ to initiate murine MAS, and provide evidence that induction of inflammatory myelopoiesis is a common TLR-9- and IFNγ-dependent pathway that may contribute to the pathogenesis of MAS.
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