Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes

To determine the potential contribution of innate immune responses to the early proinflammatory cytokine response to Plasmodium falciparum malaria, we have examined the kinetics and cellular sources of IFN-gamma production in response to human PBMC activation by intact, infected RBC (iRBC) or freeze-thaw lysates of P. falciparum schizonts. Infected erythrocytes induce a more rapid and intense IFN-gamma response from malaria-naive PBMC than do P. falciparum schizont lysates correlating with rapid iRBC activation of the CD3(-)CD56(+) NK cell population to produce IFN-gamma. IFN-gamma(+) NK cells are detectable within 6 h of coculture with iRBC, their numbers peaking at 24 h in most donors. There is marked heterogeneity between donors in magnitude of the NK-IFN-gamma response that does not correlate with mitogen- or cytokine-induced NK activation or prior malaria exposure. The NK cell-mediated IFN-gamma response is highly IL-12 dependent and appears to be partially IL-18 dependent. Exogenous rIL-12 or rIL-18 did not augment NK cell IFN-gamma responses, indicating that production of IL-12 and IL-18 is not the limiting factor explaining differences in NK cell reactivity between donors or between live and dead parasites. These data indicate that NK cells may represent an important early source of IFN-gamma, a cytokine that has been implicated in induction of various antiparasitic effector mechanisms. The heterogeneity of this early IFN-gamma response between donors suggests a variation in their ability to mount a rapid proinflammatory cytokine response to malaria infection that may, in turn, influence their innate susceptibility to malaria infection, malaria-related morbidity, or death from malaria.     

Direct action of type I IFN on NK cells is required for their activation in response to vaccinia viral infection in vivo

Type I IFN plays an important role in the activation of NK cells. However, the mechanism underlying type I IFN-dependent NK cell activation remains largely unknown. A recent report suggested that type I IFN acted on accessory dendritic cells, leading to IL-15 production, and that subsequent trans-presentation of IL-15 was required for NK cell activation upon stimulation with synthetic TLR ligands. It is not clear how type I IFN regulates NK cell activation in response to live pathogens. Using a murine model of infection with vaccinia virus (VV), we previously demonstrated a critical role for type I IFN in the innate immune control of VV infection. In this study, we first showed that type I IFN did not directly protect L929 cells from VV infection in vitro and that type I IFN-dependent innate immune control of VV infection in vivo was mediated by activated NK cells. We further demonstrated that direct action of type I IFN on NK cells, but not on dendritic cells, is required for the activation of NK cells in response to VV infection both in vitro and in vivo, leading to efficient VV clearance. Our findings may help design effective strategies for the control of poxviral infections in vivo.     

Activation Mechanisms of Natural Killer Cells during Influenza Virus Infection

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.     

Expansion of Foxp3+ regulatory T cells in mice infected with the filarial parasite Brugia malayi

Many helminths, including Brugia malayi, are able to establish long-lived infections in immunocompetent hosts. Growing evidence suggests that the immune system's failure to eliminate parasites is at least partially due to the effects of regulatory T cells (Tregs). To test whether parasites may directly stimulate host regulatory activity, we infected mice with two key stages of B. malayi. Both mosquito-borne infective larvae and mature adults i.p. introduced were found to preferentially expand the proportion of CD25(+)Foxp3(+) cells within the CD4(+) T cell population. The induction of Foxp3 was accompanied by raised CD25, CD103, and CTLA-4 expression, and was shown to be an active process, which accompanied the introduction of live, but not dead parasites. CTLA-4 expression was also markedly higher on Foxp3(-) cells, suggesting anergized effector populations. Peritoneal lavage CD4(+)CD25(+) cells from infected mice showed similar suppressive activity in vitro to normal splenic "natural" Tregs. Both B. malayi larvae and adults were also able to induce Foxp3 expression in adoptively transferred DO11.10 T cells, demonstrating that filarial infection can influence the development of T cells specific to a third party Ag. In addition, we showed that induction was intact in IL-4R-deficient animals, in the absence of a Th2 or alternatively activated macrophage response. We conclude that filarial infections significantly skew the balance of the host immune system toward Treg expansion and activation, in a manner dependent on live parasites but independent of a concomitant Th2 response.     

Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis

Glucocorticoid-induced tumor necrosis factor receptor (GITR), found constitutively expressed on human primary natural killer (NK) cells at low levels was up-regulated upon stimulation by either Toll-like receptor ligand or NK cell growth factor, interleukin (IL)-15. cDNA microarray analysis showed that engagement of GITR primarily suppressed the activation of NF-KB pathway of NK cells and up-regulated anti-inflammatory genes heme oxygenase-1 and IL-10. Further analysis revealed that GITR activation suppressed NK cell proliferation in response to IL-15. GITR activation also suppressed proinflammatory cytokine secretion and increased NK cell apoptosis. GITR activation resulted in blocked phosphorylation of Stat5 and Akt, which may have contributed to the observed antiproliferative effect of GITR on NK cells. Increased apoptosis was independent of the Fas-FasL pathway, but Bcl-XL and phospho-Bad protein expressions were diminished, suggesting involvement of the mitochondrial apoptosis pathway. The results suggest that although GITR is an activation marker for NK cells similar to that for T cells, GITR serves as a negative regulator for NK cell activation. Our studies demonstrate a novel physiological role of GITR on NK cells.     

Regulation of APO-2 ligand/trail expression in NK cells-involvement in NK cell-mediated cytotoxicity.

Apo-2L is a new member of the tumour necrosis factor (TNF) family shown to induce apoptosis in a number of tumour cell lines. Apo-2L mRNA is expressed by numerous human tissues. Here we report that Apo-2L is expressed and utilized by human Natural Killer (NK) cells. NK cells were shown to express surface Apo-2L in response to interleukin 2 (IL-2) activation, and this response was restricted to the CD3(-)population of the NK cells. Apo-2L mRNA and intracellular Apo-2L were present in both CD3(-)and CD3(+)NK cells; however, increased expression in response to IL-2 was only observed in CD3(-)CD56(+)cells. Also, IL-2-activated NK cells were shown to utilize membrane-bound Apo-2L in mediating lysis of Jurkat cells. Furthermore, Apo-2L-induced apoptosis of Jurkat cells was more rapid than FasL-induced apoptosis, indicating an important and distinct role for Apo-2L in apoptotic cell destruction. In conclusion, we report that NK cells express Apo-2L and that IL-2 activated CD3(-)NK cells utilize the Apo-2L pathway in mediating target cell lysis.     

Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells

Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the future.     

IL-10 restricts activation-induced death of NK cells during acute murine cytomegalovirus infection

IL-10 is an immunomodulatory cytokine that acts to antagonize T cell responses elicited during acute and chronic infections. Thus, the IL-10R signaling pathway provides a potential therapeutic target in strategies aimed at combating infectious diseases. In this study, we set out to investigate whether IL-10 expression had an effect on NK cells. Murine CMV infection provides the best characterized in vivo system to evaluate the NK cell response, with NK cells being critical in the early control of acute infection. Blockade of IL-10R during acute murine CMV infection markedly reduced the accumulation of cytotoxic NK cells in the spleen and lung, a phenotype associated with a transient elevation of virus DNA load. Impaired NK cell responsiveness after IL-10R blockade was attributed to elevated levels of apoptosis observed in NK cells exhibiting an activated phenotype. Therefore, we conclude that IL-10 contributes to antiviral innate immunity during acute infection by restricting activation-induced death in NK cells.     

Gamma-interferon induces apoptosis of the B lymphoma WEHI-279 cell line through a CD95/CD95L-independent mechanism.

Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and natural killer (NK) cells, plays a central role in the development of humoral and cell-mediated immunity. IFN-gamma participates in the maturation and differentiation of B cells, but it has been previously reported that IFN-gamma may inhibit the early stages of B cell activation. We report that the inhibition of the B lymphoma cell WEHI-279-proliferation induced by IFN-gamma, involves the induction of typical features of apoptosis (nuclear chromatin condensation and fragmentation, cell shrinkage, phosphatidyl-serine (PS) exposure and mitochondrial membrane potential (delta psim) loss). IFN-gamma-mediated B cell apoptosis was decreased by the addition of the T helper type 2 cytokine, IL-4. WEHI-279 cells express CD95 and undergo apoptosis after treatment with either an agonistic anti-CD95 Ab or with a soluble recombinant CD95L. However, incubation with CD95-Fc or TRAIL-R1-Fc fusion proteins, did not prevent IFN-gamma-mediated apoptosis, suggesting that IFN-gamma-mediated apoptosis occurs independently of CD95/CD95L and TRAIL-R/TRAIL interactions. IFN-gamma-mediated apoptosis is associated with caspase-3 activation that can be prevented by the addition of the broad caspase inhibitor zVAD-fmk. These data indicate that IFN-gamma may play a major role in the regulation of B cell apoptosis, and suggest the involvement of an alternative pathway which is independent of the death receptors.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Expansion of NK cells with reduction of their inhibitory Ly-49A, Ly-49C, and Ly-49G2 receptor-expressing subsets in a murine helminth infection: contribution to parasite control

Natural killer cell-associated direct cytotoxicity and cytokine production are crucial mechanisms for early innate host resistance against viruses, bacteria, or protozoa. The engagement of inhibitory NK cell receptors can influence host responses to viruses. However, these receptors have not been investigated to date in parasitic infections, and little is known about the role of NK cells in the defense against helminths. Therefore, we have correlated the frequencies of cells expressing the pan-NK marker DX5 and subsets bearing inhibitory Ly-49 receptors with worm survival and cytokine production during infection with Litomosoides sigmodontis in BALB/c mice (H2(d)), the only fully permissive model of filariasis. A marked influx of DX5(+)/CD3(-) NK cells and DX5(+)/CD3(+) T cells into the pleural cavity, where the parasites were located, was observed. The frequency of pleural NK cells expressing the H2(d)-reactive inhibitory receptors Ly-49A, Ly-49C, or Ly-49G2 declined most strongly compared with spleen and blood. In the peripheral blood, longitudinal analysis revealed an early and stable reduction of Ly-49C(+) and Ly-49G2(+) NK cells, a subsequent significant increase of the entire NK cell and DX5(+)/CD3(+) T cell populations, and a reduction in the Ly-49A(+) subset. The in vivo depletion of NK cells strongly enhanced the worm load and influenced IL-4 and IL-5 plasma levels. These data demonstrate a new role for NK cells in the host defense against filariae and, for the first time, alterations of Ly-49 receptor-expressing NK cell subsets in a parasitic infection.     

Familial NK cell deficiency associated with impaired IL-2- and IL-15-dependent survival of lymphocytes

We previously reported the clinical phenotype of two siblings with a novel inherited developmental and immunodeficiency syndrome consisting of severe intrauterine growth retardation and the impaired development of specific lymphoid lineages, including transient CD8 alphabeta T lymphopenia and a persistent lack of blood NK cells. We describe here the elucidation of a plausible underlying pathogenic mechanism, with a cellular phenotype of impaired survival of both fresh and herpesvirus saimiri-transformed T cells, in the surviving child. Clearly, NK cells could not be studied. However, peripheral blood T lymphocytes displayed excessive apoptosis ex vivo. Moreover, the survival rates of CD4 and CD8 alphabeta T cell blasts generated in vitro, and herpesvirus saimiri-transformed T cells cultured in vitro, were low, but not nil, following treatment with IL-2 and IL-15. In contrast, Fas-mediated activation-induced cell death was not enhanced, indicating a selective excess of cytokine deprivation-mediated apoptosis. In keeping with the known roles of IL-2 and IL-15 in the development of NK and CD8 T cells in the mouse model, these data suggest that an impaired, but not abolished, survival response to IL-2 and IL-15 accounts for the persistent lack of NK cells and the transient CD8 alphabeta T lymphopenia documented in vivo. Impaired cytokine-mediated lymphocyte survival is likely to be the pathogenic mechanism underlying this novel form of inherited and selective NK deficiency in humans.     

Compartmental differences in NK cell responsiveness to IL-12 during lymphocytic choriomeningitis virus infection

Some, but not all, viral infections induce endogenous IL-12 to drive NK cell IFN-gamma production and downstream antiviral defenses during innate immune responses. Even though lymphocytic choriomeningitis virus (LCMV) can be sensitive to IFN-gamma-mediated antiviral effects, infections with this agent do not elicit IL-12 or early IFN-gamma in immunocompetent hosts. Studies presented here demonstrate that LCMV infections of mice not only fail to induce IL-12, but also modify responsiveness to exogenous IL-12 for IFN-gamma production. IFN-gamma responses induced by IL-12 administration were greatly diminished in splenic populations, but significantly increased in serum and hepatic leukocytes, during the early course of LCMV infections. The IFN-gamma production was NK cell dependent, and the compartmental dichotomy between spleen and liver was also demonstrated in response to in vitro IL-12 stimulation. Although infections did increase proportions and numbers of liver NK cells, changes in responsiveness for IFN-gamma expression could not be explained by cell redistribution. Corroborating changes in proportions of NK cells induced to express intracellular IFN-gamma protein within the compartments were observed. The reduction in ability of splenic populations to produce IL-12-induced IFN-gamma after infection by LCMV was associated with decreased efficacy of administered IL-12 for promoting IFN-gamma-dependent antiviral effects in the spleen. Concomitantly, the maintenance of hepatic population IFN-gamma production was associated with preserved efficacy of administered IL-12 to elicit IFN-gamma-dependent antiviral effects in the liver. Taken together, these results demonstrate modifications of compartmental responses to IL-12 by viral infections and the consequences of these changes for efficacy of cytokine therapy.     

CD137-deficient mice have reduced NK/NKT cell numbers and function, are resistant to lipopolysaccharide-induced shock syndromes, and have lower IL-4 responses

CD137, a member of the TNF superfamily, is involved in T cell and NK cell activation and cytokine production. To establish its in vivo role in systems dependent on NK and NKT cells, we studied the response of CD137-/- mice to LPS-induced shock, tumor killing, and their IL-4-controlled Th2 responses. In both high and low dose shock models, all the CD137-deficient mice, but none of the wild-type BALB/c mice, survived. After injection of LPS/2-amino-2-deoxy-D-galactose (D-gal), CD137-/- mice had reduced serum cytokine levels and substantially impaired liver IFN-gamma and TNF-alpha mRNA levels. Phenotypic analysis of mononuclear cells revealed fewer NK and NKT cells in the CD137-/- mice. The knockout mice did not generate a rapid IL-4 response after systemic T cell activation, or effective Ag-specific Th2 responses. In addition, both in vitro and in vivo NK-specific cytolytic activities were reduced. These findings suggest that CD137-directed NK/NKT cells play an important role in the inflammatory response leading to the production of proinflammatory cytokines, LPS-induced septic shock, and tumor killing, as well as IL-4-dependent Th2 responses.     

Natural Killer Cell Cytokine Response to M. bovis BCG Is Associated with Inhibited Proliferation,Increased Apoptosis and Ultimate Depletion of NKp44+CD56bright Cells

Mycobacterium bovis BCG, a live attenuated strain of M. bovis initially developed as a vaccine against tuberculosis, is also used as an adjuvant for immunotherapy of cancers and for treatment of parasitic infections. The underlying mechanisms are thought to rely on its immunomodulatory properties including the recruitment of natural killer (NK) cells. In that context, we aimed to study the impact of M. bovis BCG on NK cell functions. We looked at cytotoxicity, cytokine production, proliferation and cell survival of purified human NK cells following exposure to single live particles of mycobacteria. We found that M. bovis BCG mediates apoptosis of NK cells only in the context of IL-2 stimulation during which CD56^bright NK cells are releasing IFN-γ in response to mycobacteria. We found that the presence of mycobacteria prevented the IL-2 induced proliferation and surface expression of NKp44 receptor by the CD56^bright population. In summary, we observed that M. bovis BCG is modulating the functions of CD56^bright NK cells to drive this subset to produce IFN-γ before subsequent programmed cell death. Therefore, IFN-γ production by CD56^bright cells constitutes the main effector mechanism of NK cells that would contribute to the benefits observed for M. bovis BCG as an immunotherapeutic agent.     

T cell immunity induced by live, necrotic, and apoptotic tumor cells

The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor's immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-gamma. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.     

Induction of natural killer cell responses by ectromelia virus controls infection

Natural killer (NK) cells play a pivotal role in the innate immune response to viral infections, particularly murine cytomegalovirus (MCMV) and human herpesviruses. In poxvirus infections, the role of NK cells is less clear. We examined disease progression in C57BL/6 mice after the removal of NK cells by both antibody depletion and genetic means. We found that NK cells were crucial for survival and the early control of virus replication in spleen and to a lesser extent in liver in C57BL/6 mice. Studies of various knockout mice suggested that gammadelta T cells and NKT cells are not important in the C57BL/6 mousepox model and CD4+ and CD8+ T cells do not exhibit antiviral activity at 6 days postinfection, when the absence of NK cells has a profound effect on virus titers in spleen and liver. NK cell cytotoxicity and/or gamma interferon (IFN-gamma) secretion likely mediated the antiviral effect needed to control virus infectivity in target organs. Studies of the effects of ectromelia virus (ECTV) infection on NK cells demonstrated that NK cells proliferate within target tissues (spleen and liver) and become activated following a low-dose footpad infection, although the mechanism of activation appears distinct from the ligand-dependent activation observed with MCMV. NK cell IFN-gamma secretion was detected by intracellular cytokine staining transiently at 32 to 72 h postinfection in the lymph node, suggesting a role in establishing a Th1 response. These results confirm a crucial role for NK cells in controlling an ECTV infection.     

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

The kinetics of CD8 T cell IFN-gamma responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-alphabeta and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-gamma response detectable in serum samples and spleen and liver homogenates. Production of IFN-gamma was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-gammadelta T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-gamma induction, and viral peptide stimulation enhanced CD8 T cell IFN-gamma expression in culture. The IFN-gamma response was associated with IL-18 and IFN-alphabeta expression. Furthermore, both factors augmented peptide-driven IFN-gamma production in culture, and mice lacking IL-18 or IFN-alphabeta functions had reduced day 4 IFN-gamma. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.