The role of CD11c(+) hepatic dendritic cells in the induction of innate immune responses

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May-Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo-activation potential. natural killer (NK)1.1(-)CD11c(+) liver DC subsets (conventional DCs, T cell receptor (TcR)beta(-)NK1.1(-)CD11c(+)B220(-) and plasmacytoid DCs, TcRbeta(-)NK1.1(-)CD11c(+)B220(+)) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40 in response to Toll-like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c(+) hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated-allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo-stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.     

Stimulation of hematopoiesis by the Fms-like tyrosine kinase 3 ligand restores bacterial induction of Th1 cytokines in thermally injured mice

Patients with large burn injuries are susceptible to opportunistic infections due to impaired functions of multiple effector cells of innate immunity and acquired immunity, including macrophages, dendritic cells (DC), natural killer (NK) cells, and T cells. The ability of a host to produce Th1 cytokines, such as gamma interferon (IFN-gamma) and interleukin-12 (IL-12), upon infectious challenge is also impaired after burn injury. Stimulation of hematopoiesis, to regenerate new immune cells, may be an effective strategy for improving resistance to infections after severe burn trauma. Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that stimulates the expansion and differentiation of NK cells and DC. Using a mouse model, we tested the hypothesis that Flt3L treatments after burn injury stimulate the production of functional effector cells of innate immunity and restore appropriate Th1 cytokine responses to Pseudomonas aeruginosa, a common source of pneumonia and wound infections in burn victims. Flt3L increased splenic cellularity in sham (uninjured) and burned mice and increased the numbers of NK cells (DX5(+)) and DC (CD11c(+)). In response to P. aeruginosa, significant increases in the serum IFN-gamma levels and the numbers of splenic IFN-gamma-producing DC, NK cells, and T cells were observed in Flt3L-treated burned mice compared to the values obtained for untreated burned mice. The splenic levels of IL-12 and IL-15 mRNAs and the IL-12 and IL-15 receptors were also increased. In addition, Flt3L treatment restored the ability of splenic cultures prepared from burned mice to produce IFN-gamma and IL-12 after in vitro challenge with P. aeruginosa. Flt3L may have potential for restoring NK cell and DC functions and improving immunity after burn injury.     

IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.     

NK cells and CD1d-restricted NKT cells respond in different ways with divergent kinetics to IL-2 treatment in primary HIV-1 infection

Cytokine immunotherapy is being evaluated as adjunct treatment in infectious diseases. The effects on innate and adaptive immunity in vivo are insufficiently known. Here, we investigate whether combination treatment with antiretroviral therapy (ART) and Interleukin-2 (IL-2) of patients with primary HIV-1 infection induces sustained increases in circulating NKT cell and NK cell numbers and effector functions and investigate how changes are coordinated in the two compartments. Patients with primary HIV-1 infection starting ART were analyzed for numbers, phenotype and function of NKT cells, NK cells and dendritic cells (DC) in peripheral blood before, during and after IL-2 treatment. NKT cells expanded during IL-2 treatment as expected from previous studies. However, their response to α-galactosyl ceramide antigen were retained but not boosted. Myeloid DC did not change their numbers or CD1d-expression during treatment. In contrast, the NK cell compartment responded with rapid expansion of the CD56(dim) effector subset and enhanced IFNγ production. Expansions of NKT cells and NK cells retracted back towards baseline values at 12 months after IL-2 treatment ended. In summary, NKT cells and NK cells respond to IL-2 treatment with different kinetics. Effects on cellular function are distinct between the cell types and the effects appear not to be sustained after IL-2 treatment ends. These results improve our understanding of the effects of cytokine immunotherapy on innate cellular immunity in early HIV-1 infection.     

TRAF6 is a critical factor for dendritic cell maturation and development

IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily members are critical for regulating multiple aspects of dendritic cell (DC) biology. Several signaling pathways associated with each family utilize the adapter molecule, TRAF6, but its role in DCs is unclear. By examining TRAF6-deficient mice and bone marrow (BM) chimeras reconstituted with TRAF6-deficient fetal liver cells, we show that proper DC maturation requires TRAF6. In response to either microbial components or CD40L, TRAF6-deficient DCs fail to upregulate surface expression of MHCII and B7.2, or produce inflammatory cytokines. Moreover, LPS-treated TRAF6-deficient DCs do not exhibit an enhanced capacity to stimulate naive T cells. Interestingly, a major population of splenic DCs, the CD4(+)CD8alpha(-) subset, is nearly absent in both TRAF6-deficient mice and BM chimeras. Together these results indicate that TRAF6 regulates the critical processes required for maturation, activation, and development of DCs, the primary cellular bridge between innate and adaptive immunity.     

IL-1R8: A molecular brake of anti-tumor and anti-viral activity of NK cells and ILC

Interleukin-1 receptor family members (ILRs) and Toll-Like Receptors (TLRs) play pivotal role in immunity and inflammation and are expressed by most cell types including cells of both the innate and adaptive immune system. In this context, IL-1 superfamily members are also important players in regulating function and differentiation of adaptive and innate lymphoid cells. This system is tightly regulated in order to avoid uncontrolled activation, which may lead to detrimental inflammation contributing to autoimmune or allergic responses. IL-1R8 (also known as TIR8 or SIGIRR) is a member of the IL-1R family that acts as a negative regulator dampening ILR and TLR signaling and as a co-receptor for human IL-37. Human and mouse NK cells, that are key players in immune surveillance of tumors and infections, express high level of IL-1R8. In this review, we will summarize our current understanding on the structure, expression and function of IL-1R8 and we will also discuss the emerging role of IL-1R8 as an important checkpoint regulating NK cells function in pathological conditions including cancer and viral infections.     

IL-2 mediates NK cell proliferation but not hyperactivity

Natural killer cells play a major role in innate immunity against tumor and virus-infected cells. NK cells express activating and inhibitory receptors to regulate their function. It has been established that modulation in the NK cell receptor profile results in altered function of NK cell against target cells. Here, we study the effect of IL-2 stimulation on NK cell inhibitory receptors Ly49A, Ly49C, and activating receptor Ly49D in C57BL/6 mice. It was observed that there was significant increase in expression of Ly49A but no change in expression of Ly49C and Ly49D on IL-2 stimulation. We further noticed that although IL-2 stimulation increased the NK cell population and expression of activation marker NK1.1 but IL-2 stimulation does not cause hyper-responsiveness in NK cells, as there was no increase in MIP-1α and IFN-γ production in IL-2 stimulated NK cells as compared to unstimulated controls. These findings provide a framework to understand the effect of IL-2 stimulation on cognate and non-cognate receptor ligand interactions and suggest stratagies for immunotherapies in conjunction with IL-2 combinatorial therapies.     

Natural killer cells activated by MHC class I(low) targets prime dendritic cells to induce protective CD8 T cell responses

Conserved molecular patterns derived from pathogenic microorganisms prime antigen-presenting dendritic cells (DC) to induce adaptive T cell responses. In contrast, virus-infected or tumor cells that express low levels of major histocompatibility complex (MHC) class I activate natural killer (NK) cells for direct killing. It is unknown whether NK cell recognition of MHC class I(low) targets can also induce adaptive T cell responses. Here, we show that MHC class I(low) targets initiate a cascade of immune responses, starting with the immediate activation of NK cells. The activated NK cells then prime DC to produce IL-12 and to induce highly protective CD8 T cell memory responses. Therefore, sensing of MHC class I(low) targets by NK cells can link innate and adaptive immunity to induce protective T cell responses and may alarm the immune system during early infection with noncytopathic viruses.     

Cytokine-activated natural killer cells exert direct killing of hepatoma cells harboring hepatitis C virus replicons.

Hepatitis C virus (HCV)-specific impairments in host immunity have been described at multiple levels of the innate and adaptive response, which may lead to viral persistence in the majority of infections. Understanding of HCV-associated immune defects could lead to novel therapeutic advances. Natural killer (NK) cells, the major effector cells of the innate immune system, are functionally impaired in chronic HCV infection. It has been suggested that this phenotype is a result of virus-specific defects in antigen-presenting cells (APCs) that regulate NK cell activity, as normal NK function is restored when they are stimulated ex vivo. In this study, we used human NK cell cytotoxicity assays to evaluate the activation-induced effects of NK cells on the HCV replicon-containing hepatic cells. We found that cytokine-activated NK cells were capable of inducing an HCV-associated, perforin/granzyme-dependent lysis of human hepatoma cells and that this required direct cellular contact and was independent of MHC class I expression levels. In contrast, on removal of cytokine stimulation, NK cells failed to exert any direct cytolytic effect on replicon targets. These findings suggest an important underlying mechanism by which NK cells control HCV infection and, with appropriate understanding of HCV-associated immune defects, could lead to novel therapeutic advances.     

Elevated frequency of gamma interferon-producing NK cells in healthy adults vaccinated against influenza virus

Recent studies indicate that innate immunity in influenza virus infection is an area of substantial importance for our understanding of influenza virus pathogenesis, yet our knowledge of the mechanisms controlling innate immunity remains limited. Further delineation of the roles of NK cells and innate immunity in viral infection may have important implications for the development of improved influenza virus vaccines. In this study, we evaluated the phenotype and function of NK and T lymphocytes, as well as influenza virus-specific immunoglobulin G production, prior to and following vaccination with the routinely administered trivalent influenza virus vaccine. We demonstrate influenza virus antigen-specific innate and adaptive cellular responses and evaluate changes in NK cell receptor expression over time. Our results demonstrate increased innate and adaptive cellular immune responses and show that NK cells are a significant source of gamma interferon (IFN-γ) following influenza virus vaccination. An increase in the frequency of IFN-γ-producing NK cells was observed in many subjects postvaccination. The subset distribution with respect to CD56^dim and CD56^bright NK cell subsets remained stable, as did the NK cell phenotype with respect to expression of cell surface activating and inhibitory receptors. These results may form the basis for further investigations of the role of NK cells in immunity to influenza.     

Novel molecular mechanism for generating NK‐cell fitness and memory

The mammalian immune system has been traditionally subdivided into two compartments known as the innate and the adaptive. T cells and B cells, which rearrange their antigen‐receptor genes using the RAG recombinase, comprise the adaptive arm of immunity. Meanwhile, every other white blood cell has been grouped together under the broad umbrella of innate immunity, including NK cells. NK cells are considered innate lymphocytes because of their rapid responses to stressed cells and their ability to develop without receptor gene rearrangement (i.e. in RAG‐deficient mice). However, new findings implicate a critical function for RAG proteins during NK‐cell ontogeny, and suggest a novel mechanism by which controlled DNA breaks during NK‐cell development dictate the fitness, function, and longevity of these cells. This review highlights recent work describing how DNA break events can impact cellular differentiation and fitness in a variety of cell types and settings.     

The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells

NK and DC reciprocal interactions have only recently been investigated. In this study, we focused on the interplay between NK cells and DC in two models of bacterial infection. Immature monocyte-derived DC were cultured in the presence of live Escherichia coli or bacillus Calmette-Guérin. Upon exposure to either extracellular or intracellular bacteria, DC underwent maturation as assessed by the increased levels of expression of CD80,CD86, and HLA molecules and the de novo expression of CD83 and CCR7. Significant amounts of TNF-alpha and IL-12 were released by DC upon infection, whereas IL-2 and IL-15 were barely detectable in culture supernatants. Both infected and uninfected DC were capable of inducing in fresh autologous NK cells the expression of CD69 and HLA-DR and of inducing cell proliferation. Remarkably, however, infected DC were much stronger inducers of NK cell activation and proliferation than uninfected DC. Thus, after just 24 h of NK/DC coculture, only those NK cells that had been exposed to bacteria-infected DC had acquired the ability to lyse autologous immature DC. In addition, infected DC were more resistant to NK-mediated lysis as a consequence of the up-regulation of HLA class I molecule expression on their surface. This study suggests a regulatory circuit involving NK cells and DC in which DC-induced NK cell activation is effectively enhanced by the presence of pathogens. Activated NK cells, by limiting the supply of immature DC, may then exert a control on subsequent innate and adaptive immune responses.     

Innate immunosenescence: Effect of aging on cells and receptors of the innate immune system in humans

Components of the innate immune response, including neutrophils and macrophages, are the first line of defense against infections. Their role is to initiate an inflammatory response, phagocyte and kill pathogens, recruit natural killer cells (NK), and facilitate the maturation and migration of dendritic cells that will initiate the adaptive immune response. Extraordinary advances have been made in the last decade on the knowledge of the receptors and mechanisms used by cells of the innate immunity not only to sense and eliminate the pathogen but also to communicate each other and collaborate with cells of adaptive immunity to mount an effective immune response. The analysis of innate immunity in elderly humans has evidenced that aging has a profound impact on the phenotype and functions of these cells. Thus altered expression and/or function of innate immunity receptors and signal transduction leading to defective activation and decreased chemotaxis, phagocytosis and intracellular killing of pathogens have been described. The phenotype and function of NK cells from elderly individuals show significant changes that are compatible with remodeling of the different NK subsets, with a decrease in the CD56bright subpopulation and accumulation of the CD56dim cells, in particular those differentiated NK cells that co-express CD57, as well as a decreased expression of activating natural cytotoxicity receptors. These alterations can be responsible of the decreased production of cytokines and the lower per-cell cytotoxicity observed in the elderly. Considering the relevance of these cells in the initiation of the immune response, the possibility to reactivate the function of innate immune cells should be considered in order to improve the response to pathogens and to vaccination in the elderly.     

Regulation of dendritic cell function by microbial stimuli

Dendritic cells (DC) initiate T cell responses and produce cytokines and other molecules that can regulate the class adaptive immunity. It is increasingly clear that DC in vivo are in a "resting" state and require exogenous signals to transit into an "effector" state in which they can prime T cells. Much of this DC activation process appears to be regulated by infection. Exposure of murine DC to certain pathogens or their products triggers DC migration to T cell areas of secondary lymphoid tissues, improves MHC presentation and increases DC co-stimulatory potential. Pathogen recognition can also initiate cytokine production and/or condition DC to produce cytokines in response to subsequent T cell feedback signals delivered via CD40 and similar receptors. Recognition of pathogens by DC is largely dependent on Toll-like receptors (TLRs). Interestingly, mouse splenic CD8alpha+ and CDalpha-CD4- DC have the ability to produce either IL-12 p70 or IL-10 depending on the nature of the pathogen encountered. In contrast, CD4+ DC seem incapable of producing IL-12 p70. Thus, the nature of the pathogen can dictate the type of cytokine that is made by some DC subsets, allowing them to prime distinct types of immune responses. Overall, DC display significant plasticity in their ability to respond to infection and direct adaptive immunity.     

The plague virulence protein YopM targets the innate immune response by causing a global depletion of NK cells

Yersinia pestis, the etiologic agent of plague, delivers six Yersinia outer proteins (Yops) into host cells upon direct bacterial contact. One of these, YopM, is necessary for virulence in a mouse model of septicemic plague, but its pathogenic function is unknown. We report here the immune processes affected by YopM during infection. To test whether the innate or adaptive immune system is targeted by YopM, C57BL/6 (B6) and B6 SCID mice were infected with either the conditionally virulent Y. pestis KIM5 or a yopM deletion mutant and evaluated for bacterial growth in spleen and liver. Both B6 and SCID mice succumbed to infection with Y. pestis KIM5, whereas both mouse strains survived infection by the YopM(-) mutant. These data showed that YopM counteracts innate defenses present in SCID mice. The YopM(-) strain grew more slowly than the parent Y. pestis during the first 4 days of infection in both mouse strains, indicating an early pathogenic role for YopM. In B6 mice, populations of cells of the immune system were not differentially affected by the two Y. pestis strains, with one major exception: the parent Y. pestis KIM5 but not the YopM(-) mutant caused a significant global decrease in NK cell numbers (blood, spleen, and liver), beginning early in infection. NK cells and macrophages isolated early (day 2) from livers and spleens of mice infected with either Y. pestis strain contained comparable levels of cytokine mRNA: interleukin (IL)-1 beta, IL-12, IL-15, IL-18, and tumor necrosis factor alpha in macrophages and gamma interferon in NK cells. However, by day 4 postinfection, cells from mice infected with the parent Y. pestis expressed lower levels of these messages, while those from mice infected with the mutant retained strong expression. Significantly, mRNA for the IL-15 receptor alpha chain was not expressed in NK cells from Y. pestis KIM5-infected mice as early as day 2 postinfection. These findings suggest that YopM interferes with innate immunity by causing depletion of NK cells, possibly by affecting the expression of IL-15 receptor alpha and IL-15.     

Human monocyte-derived and CD83(+) blood dendritic cells enhance NK cell-mediated cytotoxicity

Dendritic cells (DC) are known to be the most potent APC and to stimulate antigen-specific T cell responses. Recently it was reported that murine DC were also capable of modulating the innate immunity by stimulating NK cells through cell-to-cell contact. In the present study, we examined whether human DC could affect NK activity. Both monocyte-derived and CD83(+) blood DC were tested. The addition of DC to cultures of CD56(+) cells resulted in the significant dose-dependent enhancement of the killing activity against various NK-sensitive targets. The resultant activity was comparable to that induced by optimal concentrations of various cytokines, including IL-2, IL-12, IL-15 and IFN-gamma. Interestingly, DC enhanced the cytotoxicity of CD3(-)CD56(+) NK cells, but not that of CD3(+)CD56(+) T cells. Experiments using transwells clearly demonstrated that the enhancement of NK activity by DC was mediated by soluble factors produced by DC. The culture supernatants of DC also stimulated NK activity. The treatment of both DC and their supernatants with anti-human IL-12 or IL-18 antibodies did not block the enhancement of NK cell-mediated cytolysis by DC, indicating that other factor(s) produced by DC were responsible for the enhancement of NK activity. These results suggest that human myeloid DC can modulate innate immunity by enhancing NK activity.     

Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patients

Hepatitis C virus (HCV) readily establishes high-level lifelong persistent infection in the majority of immunocompetent adults with failure of HCV-specific CD8+ CTL to clear viral replication. Virus-induced conditioning of innate immune responses is a possible mechanism that may contribute to the impairment of virus-specific CD8+ CTL responses. Here, we analyzed whether triggering of NK cell receptor expression and function is affected during chronic viremic HCV infection. Flow cytometric analysis of purified resting peripheral NK cells showed no evidence of NK cell activation, while analysis of natural cytotoxicity receptors (NCR) showed that NK cells from HCV-infected patients had selective increased expression of NKp30 and NKp46. NK cells had corresponding conserved cytotoxic activity against all targets with the exception of HepG2 hepatoma cells. Freshly separated NK cells from HCV patients showed significant production of IL-10 and normal concentrations of IFN-gamma upon cell-mediated triggering. Thus, increased expression of NKp30 during HCV infection with increased IL-10 production could contribute, once NK cells localize in the liver, to a NK-DC crosstalk leading to skewing of subsequent adaptive immune responses and lack of virus control.     

IL-21的免疫调节作用

IL-21是近几年来被发现和关注的细胞因子,由活化的T细胞合成和分泌,主要通过激活NK细胞来发挥功能。已有的研究表明IL-21还可对T、B细胞和DC发挥重要的调节作用,并可能在促进天然免疫向获得性免疫转化中扮演了重要角色。     

The chemokine receptor CXCR3 limits injury after acute toxic liver damage

Although acute liver failure is a rare disease, its presence is associated with high morbidity and mortality in affected patients. While a contribution of the immune system to the outcome of toxic liver failure is anticipated, functionally relevant immune cell receptors for liver cell damage need to be better defined. We here investigate the relevance of the chemokine receptor CXCR3, which is important for hepatic immune cell infiltration, in a model of experimental acute liver failure. Liver injury was induced by a single intraperitoneal injection of carbon tetrachloride (CCl(4)) in CXCR3(-/-), CCR1(-/-), CCR5(-/-) and wild-type mice. In this model, CXCR3(-/-) mice displayed augmented liver damage compared with all other mouse strains as assessed by liver histology and serum transaminases 24 and 72?h after injury. Phenotypically, CXCR3(-/-) mice had significantly reduced intrahepatic NK and NKT cells after injury at all investigated time points (all P<0.05), but strongly elevated expression levels of IL1-β, TNF-α and IFN-γ. In line with a functional role of innate immune cells, wild-type mice depleted for NK cells with an anti-ASIALO GM1 antibody before liver injury also displayed increased liver injury after CCl(4) challenge. CXCR3(-/-) and NK cell-depleted mice show reduced apoptotic liver cells (TUNEL assay), but more necrotic hepatocytes. Functionally, the augmented liver cell necrosis in CXCR3(-/-) and NK cell-depleted mice was associated with increased expression of high mobility group 1 (HMGB1) protein and a consecutive enhanced infiltration of neutrophils into the liver. In conclusion, the results demonstrate a primarily unexpected beneficial role of CXCR3 in acute toxic liver injury. These findings should be taken into account when planning trials with CXCR3 antagonists.