Innate IFN-γ Research Articles

P265 Essential role of Card9 in innate IFN-γ production and Th17 differentiation in the host defense against cryptococcal infection

P265 Essential role of Card9 in innate IFN-γ production and Th17 differentiation in the host defense against cryptococcal infection

IL-18 provided in dying bacterial-infected macrophages induces IFN-γ production in functional T-cell hybridoma B6HO3 through cell conjugates

We have previously reported that the co-culture of functional T-cell hybridoma B6HO3 with dying J774 macrophage cells infected with Listeria monocytogenes (LM) results in the production of IFN-γ by B6HO3 cells. Here, we explore the mechanism underlying this phenomenon. We found that IFN-γ production was dependent on IL-18, but that the dying LM-infected macrophages produced no more than 100 pg/ml of IL-18, much less than the amount of IL-18 required for stimulating B6HO3 cells to produce IFN-γ. Furthermore, IL-18 binding protein added to the co-culture was unable to easily gain access to IL-18 for neutralisation. B6HO3 cells formed cell conjugates with J774 macrophages, and IFN-γ-producing B6HO3 cells were spatially and temporally associated with LM-infected macrophage cell death that exhibited neither pyroptosis nor pyronecrosis. These results suggest that the IL-18 produced by dying LM-infected macrophages is released to the interface of the cell conjugates, thereby inducing B6HO3 cells to produce IFN-γ. Based on the present and also previous findings, we propose that IL-18 released from macrophages because of cell death caused by bacteria may be the primary cytokine that triggers the innate IFN-γ production that is required for activating the bactericidal functions of macrophages at early stages of bacterial infection.

Reciprocal interaction between NK cells and DCs regulates the Th17 response by controlling the innate IFN-γ/IL-27 axis (P4067)

Abstract Experimental autoimmune uveitis (EAU) is a model for human uveitis. IFN-γ deficient mice develop enhanced EAU with elevated Th17 responses. This protective effect of IFN-γ appears at odds with the ability of IFN-γ-producing Th1 cells to induce EAU. We previously showed that it is innate IFN-γ that is protective, whereas adaptive IFN-γ is pathogenic. We now demonstrate that NK cells, a major cellular source of innate IFN-γ, interact with DCs in draining lymph nodes of EAU-challenged mice to ameliorate disease by controlling the innate IFN-γ/IL-27 axis. After immunization, both NK cells and DCs were recruited to the draining lymph nodes, with recruitment of NK cells being at least partially dependent on DCs and CXCR3. Infusion of IFN-γ-sufficient WT NK cells into IFN-γ-/- recipients reduced their EAU scores and enhanced production of IL 27, a suppressor of the Th17 response, from DCs of these mice. In vivo neutralization of IL-27 abolished the protective effect of WT NK cells on EAU. In vitro experiments revealed that mature DCs induced NK cells to produce IFN-γ, which in turn caused DCs to produce IL-27. IL-27 then stimulated NK cells to produce more IFN-γ, suggesting the presence of a positive feedback loop. IL-27 also promoted generation of IL-10-producing Tr1-like cells. Our results suggest that the magnitude of the Th17 response, and consequently the severity of disease, is controlled by the interaction between NK cells and DCs through the IFN-γ/IL-27 axis.

Solute Carrier 11A1 Is Expressed by Innate Lymphocytes and Augments Their Activation

Solute carrier 11A1 (SLC11A1) is a divalent ion transporter formerly known as the natural resistance-associated macrophage protein (NRAMP1) and the Bcg/Lsh/Ity locus. SLC11A1 was thought to be exclusively expressed in monocyte/macrophages and to have roles in phagosome maturation and cell activation. We characterized the expression of SLC11A1 in the majority of human and bovine γδ T cells and NK cells and in human CD3(+)CD45RO(+) T cells. Consistent with a role for iron-dependent inhibition of protein tyrosine phosphatases, SLC11A1(+) lymphocytes were more prone to activation and retained tyrosine phosphorylation. Transfection of SLC11A1 into a human γδ T cell-like line rendered the cells more prone to activation. Nonadherent splenocytes from wild-type mice expressed significantly greater IFN-γ compared with cells from Sv/129 (SLC11A1(-/-)) mice. Our data suggest that SLC11A1 has a heretofore unknown role in activation of a large subset of innate lymphocytes that are critical sources of IFN-γ. SLC11A1(+) animals have enhanced innate IFN-γ expression in response to Salmonella infection compared with SLC11A1(-) mice, which include commonly used inbred laboratory mice. Expression of SLC11A1 in innate lymphocytes and its role in augmenting their activation may account for inconsistencies in studies of innate lymphocytes in different animal models.

Blockade of IL-10 Signaling during Bacillus Calmette-Guérin Vaccination Enhances and Sustains Th1, Th17, and Innate Lymphoid IFN-γ and IL-17 Responses and Increases Protection to Mycobacterium tuberculosis Infection

Vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the only prophylactic vaccine against tuberculosis, caused by Mycobacterium tuberculosis, but gives variable protection against pulmonary disease. The generation of host Th1 responses following BCG vaccination is accepted as the major mechanism of protection against M. tuberculosis infection. Early production of IL-17 in the lungs following M. tuberculosis challenge of mice previously vaccinated with M. tuberculosis peptides in adjuvant has been shown to be required for efficient Th1 cell recruitment. IL-10 regulates various processes involved in generation of Th1 and Th17 responses. Previous studies have shown IL-10 as a negative regulator of the immune response to primary M. tuberculosis infection, with Il10(-/-) mice having reduced lung bacterial loads. In this study we show that inhibition of IL-10 signaling during BCG vaccination enhances host-generated Ag-specific IFN-γ and IL-17A responses, and that this regimen gives significantly greater protection against aerogenic M. tuberculosis challenge in both susceptible and relatively resistant strains of mice. In M. tuberculosis-susceptible CBA/J mice, Ab blockade of IL-10R specifically during BCG vaccination resulted in additional protection against M. tuberculosis challenge of >1-log(10) compared with equivalent isotype-treated controls. The protection observed following BCG vaccination concurrent with anti-IL-10R mAb treatment was sustained through chronic M. tuberculosis infection and correlated with enhanced lung Th1 and Th17 responses and increased IFN-γ and IL-17A production by γδ T cells and an innate-like Thy1.2(+)CD3(-) lymphoid population. We show that IL-10 inhibits optimal BCG-elicited protection, therefore suggesting that antagonists of IL-10 may be of great benefit as adjuvants in preventive vaccination against tuberculosis.

Innate IFN-γ Is Essential for Programmed Death Ligand-1–Mediated T Cell Stimulation following Listeria monocytogenes Infection

Although best characterized for sustaining T cell exhaustion during persistent viral infection, programmed death ligand-1 (PDL-1) also stimulates the expansion of protective T cells after infection with intracellular bacterial pathogens. Therefore, establishing the molecular signals that control whether PDL-1 stimulates immune suppression or activation is important as immune modulation therapies based on manipulating PDL-1 are being developed. In this study, the requirement for PDL-1 blockade initiated before infection with the intracellular bacterium Listeria monocytogenes in reducing pathogen-specific T cell expansion is demonstrated. In turn, the role of proinflammatory cytokines triggered early after L. monocytogenes infection in controlling PDL-1-mediated T cell stimulation was investigated using mice with targeted defects in specific cytokines or cytokine receptors. These experiments illustrate an essential role for IL-12 or type I IFNs in PDL-1-mediated expansion of pathogen-specific CD8(+) T cells. Unexpectedly, direct stimulation by neither IL-12 nor type I IFNs on pathogen-specific CD8(+) cells was essential for PDL-1-mediated expansion. Instead, the absence of early innate IFN-γ production in mice with combined defects in both IL-12 and type I IFNR negated the impacts of PDL-1 blockade. In turn, IFN-γ ablation using neutralizing Abs or in mice with targeted defects in IFN-γR each eliminated the PDL-1-mediated stimulatory impacts on pathogen-specific T cell expansion. Thus, innate IFN-γ is essential for PDL-1-mediated T cell stimulation.

Human invariant natural killer T cells acquire transient innate responsiveness via histone H4 acetylation induced by weak TCR stimulation

Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent T(H1) differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.

Innate IFN-γ-Producing Cells in the Spleen of Mice Early after Listeria monocytogenes Infection: Importance of Microenvironment of the Cells Involved in the Production of Innate IFN-γ

Production of innate interferon-γ (IFN-γ) is a crucial step in immunological defense against bacteria. However, there is little information regarding cellular mechanisms underlying IFN-γ production in vivo early after bacterial infection. Here we analyze innate IFN-γ production in the spleen of mice early after Listeria monocytogenes (LM) infection ex vivo by flow-cytometry and in situ by immunohistochemistry, and compare them with the IFN-γ-producing cells reported previously in our in vitro coculture system in which cell-cell interaction between lymphocytes and dying bacterial-infected macrophages is required for the production of IFN-γ. In the spleen at 20 h after LM infection, natural killer (NK) cells, a subset of αβ T cells, and subsets of NKT and γδ T cells produced IFN-γ with features similar to the IFN-γ-producing cells in our in vitro coculture system. Immunohistochemistry revealed that LM bacteria were first phagocytosed mainly by ER-TR9+ marginal zone macrophages (MZMs), then forming infectious foci in close vicinity of the marginal zone (MZ) at 20-h postinfection. At this time point, the IFN-γ-producing cells were accumulating at the same site of infectious foci, around which ER-TR9+ MZMs were clustered but most of bacteria were no longer associated with ER-TR9+ MZMs. These results indicate that innate IFN-γ production by innate lymphocytes takes place at infectious foci formed in close vicinity of the MZ, and they also suggest an important role for the microenvironment of the cells accumulated at infectious foci in inducing the production of innate IFN-γ.

CD8 T Cells in Old Mice Contribute to the Innate Immune Response toMycobacterium tuberculosisvia Interleukin-12p70-Dependent and Antigen-Independent Production of Gamma Interferon

Elderly individuals have increased morbidity and mortality associated with infectious diseases due in part to the progressive age-associated decline in immune function. Despite this, the old mouse model of Mycobacterium tuberculosis infection has revealed a CD8- and gamma interferon (IFN-gamma)-dependent early resistance to infection. In this study, we investigated the mechanism by which CD8 T cells from old mice contributed to the early immune response to M. tuberculosis. Following a low-dose aerosol infection with M. tuberculosis, CD8 T cells were identified as being a dominant source of IFN-gamma expression in the lungs of old mice early after infection, before the typical onset of antigen-specific immunity. In addition, M. tuberculosis-induced IFN-gamma production by CD8 T cells isolated from naïve old mice was major histocompatibility complex class I independent but was dependent on interleukin-12p70, confirming an innate role of CD8 T cells during M. tuberculosis infection. Moreover, the ability of CD8 T cells from old mice to produce increased innate IFN-gamma levels in response to M. tuberculosis infection was defined as a unique function of CD8 T cells from old mice and not the aged lung environment. Finally, we have identified increased expression of SET as being one possible mechanism by which CD8 T cells from old mice produce enhanced levels of IFN-gamma. Additional characterizations of the signaling events that lead to enhanced innate IFN-gamma production by CD8 T cells in old mice may lead to novel strategies to further enhance or perpetuate beneficial immune responses in the elderly.

Psychological stress suppresses innate IFN-γ production via glucocorticoid receptor activation: Reversal by the anxiolytic chlordiazepoxide

Studies in humans and in animals indicate that psychological stress can modulate immune responses. Here we demonstrate that exposure to psychological stress (restraint stress) suppresses innate interferon (IFN)-γ production in mice following an in vivo lipopolysaccharide (LPS) challenge. IFN-γ signaling was also impaired by stress, as indicated by reduced STAT1 phosphorylation and reduced expression of the IFN-γ-inducible genes, inducible nitric oxide synthase (iNOS) and IFN-γ-inducible protein 10 (IP-10/CXCL10). Furthermore, restraint stress suppressed production of the IFN-γ inducing cytokine interleukin (IL)-12 and increased production of the anti-inflammatory cytokine IL-10, which can inhibit both IL-12 and IFN-γ production. However, using IL-10 knockout mice, we demonstrate that IL-10 does not mediate the suppressive effect of restraint stress on innate IFN-γ production. Restraint stress increased corticosterone concentrations in serum and spleen, and consistent with a role for glucocorticoids in the immunosuppressive actions of stress, pre-treatment with the glucocorticoid receptor antagonist mifepristone completely blocked the stress-related suppression of innate IFN-γ production. Addition of exogenous IL-12 to LPS-stimulated spleen cells reversed the suppressive effect of both restraint stress and corticosterone on IFN-γ production. These data suggest that reduced IL-12 production is a key event in stress-induced suppression of innate IFN-γ production. Finally, we demonstrate that pre-treatment with the anxiolytic drug chlordiazepoxide prevents the suppressive effect of stress on innate IFN-γ production, and also attenuates the stress-induced increase in circulating corticosterone concentrations.

Innate Immune Response Profiles and Their Relationship to the Frequency of Respiratory Illnesses in the First Three Years of Life

RATIONALE: Previously, mitogen-induced interferon (IFN) responses at birth were shown to be inversely correlated with illness frequency. Here we more comprehensively evaluate whether variations in immunologic responses at birth to multiple innate and mitogen stimuli are associated with the risk of acquiring respiratory illnesses during early childhood. METHODS: Cord blood mononuclear cells were obtained at birth from 285 children enrolled in the Childhood Origins of ASThma (COAST) study. After stimulating them with multiple mitogen and innate stimuli (including PHA, LPS, PMA, RV, RSV, and SAC), IFN-γ, IL-6, IL-10, IL-12, IFN-α, and TNF-α were measured. Cytokine levels were correlated to the frequency of moderate to severe respiratory illnesses (MSIs) during the first 3 years of life. A Spearman correlation was utilized for cytokine/stimuli pairs for which ≥75% of patient samples had detectable results; a Wilcoxon rank-sum test was used below this threshold. RESULTS: Of all stimuli/cytokine pairs analyzed, the most consistent finding was with IFNs. IFN-γ levels in cord blood were lower in children who developed more respiratory illnesses for the following stimulants: PMA (r = -0.16, p = 0.008), PHA (r = -0.12, p = 0.05), RSV (detectable vs. undetectable: 5.03 vs. 5.93 MSIs, p = 0.07), and LPS (detectable vs. undetectable: 3.83 vs. 6.2 MSIs, p = 0.1). Similarly, LPS-induced IFN-α levels were lower in children who developed more respiratory illnesses (r = -0.23, p = 0.02). CONCLUSIONS: Greater interferon responses to multiple innate and mitogen stimuli at birth appear to be protective against the development of frequent respiratory illnesses during early childhood.

Activation of Invariant NKT Cells Ameliorates Experimental Ocular Autoimmunity by A Mechanism Involving Innate IFN-γ Production and Dampening of the Adaptive Th1 and Th17 Responses

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.

Transgenic Parasites: Improving Our Understanding of Innate Immunity to Malaria

Clinical immunity to Plasmodium falciparum malaria takes years to develop and is never complete. One explanation for these observations is that antigenic variation enables malaria parasites to evade humoral immunity; another is that P. falciparum induces immune dysregulation, which inhibits the development of protective cellular immunity. Research described by D'Ombrain et al. in this Cell Host & Microbe issue probes how the parasite's main virulence factor PfEMP-1 might significantly alter human innate immune responses.

Expression of the p60 Autolysin Enhances NK Cell Activation and Is Required forListeria monocytogenesExpansion in IFN-γ-Responsive Mice

Both peptidoglycan and muropeptides potently modulate inflammatory and innate immune responses. The secreted Listeria monocytogenes p60 autolysin digests peptidoglycan and promotes bacterial infection in vivo. Here, we report that p60 contributes to bacterial subversion of NK cell activation and innate IFN-gamma production. L. monocytogenes deficient for p60 (Deltap60) competed well for expansion in mice doubly deficient for IFNAR1 and IFN-gammaR1 or singly deficient for IFN-gammaR1, but not in wild-type, IFNAR1(-/-), or TLR2(-/-) mice. The restored competitiveness of p60-deficient bacteria suggested a specific role for p60 in bacterial subversion of IFN-gamma-mediated immune responses, since in vivo expansion of three other mutant L. monocytogenes strains (DeltaActA, DeltaNamA, and DeltaPlcB) was not complemented in IFN-gammaR1(-/-) mice. Bacterial expression of p60 was not required to induce socs1, socs3, and il10 expression in infected mouse bone marrow macrophages but did correlate with enhanced production of IL-6, IL-12p70, and most strikingly IFN-gamma. The primary source of p60-dependent innate IFN-gamma was NK cells, whereas bacterial p60 expression did not significantly alter innate IFN-gamma production by T cells. The mechanism for p60-dependent NK cell stimulation was also indirect, given that treatment with purified p60 protein failed to directly activate NK cells for IFN-gamma production. These data suggest that p60 may act on infected cells to indirectly enhance NK cell activation and increase innate IFN-gamma production, which presumably promotes early bacterial expansion through its immunoregulatory effects on bystander cells. Thus, the simultaneous induction of IFN-gamma production and factors that inhibit IFN-gamma signaling may be a common strategy for misdirection of early antibacterial immunity.

Pro-inflammatory actions of β-adrenoceptor antagonism in vivo: Enhancement of innate IFN-γ production and MHC class II and co-stimulatory molecule expression

Pro-inflammatory actions of β-adrenoceptor antagonism in vivo: Enhancement of innate IFN-γ production and MHC class II and co-stimulatory molecule expression

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis.

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.

A role for IFN-alpha beta in virus infection-induced sensitization to endotoxin.

Underlying viral infections can heighten sensitivity and worsen cytokine-mediated disease following secondary inflammatory challenges. Mechanisms for this are poorly understood. The impact of the innate response to lymphocytic choriomeningitis virus (LCMV) infection on sensitivity to endotoxin (LPS) was investigated. Compared with uninfected mice, infection with LCMV for 2-days-sensitized mice to LPS by approximately 2-fold for lethality and by 2- to 6-fold for serum TNF-alpha levels. Priming for LPS-induced TNF-alpha was also seen with splenic and peritoneal leukocytes isolated from infected mice and challenged with LPS ex vivo. The effect on TNF-alpha production was present in the absence of IFN-gamma, its major producers NK and T cells, and the major pathways for its induction through IL-12 and the signal transducer and activator of transcription 4 (STAT4), and therefore was IFN-gamma independent. Early LCMV infection induces high concentrations of the type 1 IFNs, IFN-alphabeta. Administration of recombinant IFN-alpha alone heightened the TNF-alpha response to LPS. Innate IFN-alphabeta and IFN-gamma responses to LCMV exist in a delicate balance. To reduce priming for LPS-induced TNF-alpha during LCMV, deficiencies in both the IFN-alphabeta and IFN-gamma receptors or STAT1, a transcription factor downstream to both IFNs, were required. These data demonstrate that early viral infection can enhance sensitivity to bacterial products, and that this sensitization can occur in part as a result of endogenously expressed IFN-alphabeta. This work also raises issues about potential complications associated with IFN-alphabeta therapies.

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo.

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.