Down regulation of macrophage IFNGR1 exacerbates systemic L. monocytogenes infection.
ABSTRACT: Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFN?) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFN? and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFN? alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFN? in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFN? and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.
Project description:The ability of type I IFNs to increase susceptibility to certain bacterial infections correlates with downregulation of myeloid cell surface IFNGR, the receptor for the type II IFN (IFN-?), and reduced myeloid cell responsiveness to IFN-?. In this study, we show that the rapid reductions in mouse and human myeloid cell surface IFNGR1 expression that occur in response to type I IFN treatment reflect a rapid silencing of new ifngr1 transcription by repressive transcriptional regulators. Treatment of macrophages with IFN-? reduced cellular abundance of ifngr1 transcripts as rapidly and effectively as actinomycin D treatment. IFN-? treatment also significantly reduced the amounts of activated RNA polymerase II (pol II) and acetylated histones H3 and H4 at the ifngr1 promoter and the activity of an IFNGR1-luc reporter construct in macrophages. The suppression of IFNGR1-luc activity required an intact early growth response factor (Egr) binding site in the proximal ifngr1 promoter. Three Egr proteins and two Egr/NGFI-A binding (Nab) proteins were found to be expressed in bone macrophages, but only Egr3 and Nab1 were recruited to the ifngr1 promoter upon IFN-? stimulation. Knockdown of Nab1 in a macrophage cell line prevented downregulation of IFNGR1 and prevented the loss of acetylated histones from the ifngr1 promoter. These data suggest that type I IFN stimulation induces a rapid recruitment of a repressive Egr3/Nab1 complex that silences transcription from the ifngr1 promoter. This mechanism of gene silencing may contribute to the anti-inflammatory effects of type I IFNs.
Project description:The type II IFN (IFN?) enhances antimicrobial activity yet also drives expression of genes that amplify inflammatory responses. Hence, excessive IFN? stimulation can be pathogenic. Here, we describe a previously unappreciated mechanism whereby IFN? itself dampens myeloid cell activation. Staining of monocytes from <i>Listeria monocytogenes</i>-infected mice provided evidence of type I IFN-independent reductions in IFNGR1. IFN? was subsequently found to reduce surface IFNGR1 on cultured murine myeloid cells and human CD14<sup>+</sup> peripheral blood mononuclear cells. IFN?-driven reductions in IFNGR1 were not explained by ligand-induced receptor internalization. Rather, IFN? reduced macrophage <i>Ifngr1</i> transcription by altering chromatin structure at putative <i>Ifngr1</i> enhancer sites. This is a distinct mechanism from that used by type I IFNs. Ligand-induced reductions in IFNGR1 altered myeloid cell sensitivity to IFN?, blunting activation of STAT1 and 3. Our data, thus, reveal a mechanism by which IFNGR1 abundance and myeloid cell sensitivity to IFN? can be modulated in the absence of type I IFNs. Multiple mechanisms, thus, exist to calibrate macrophage IFNGR1 abundance, likely permitting the fine tuning of macrophage activation and inflammation.
Project description:Macrophages express a spectrum of proinflammatory and regulatory mediators during African trypanosomiasis. Microarray analyses revealed similar profiles of induced genes in macrophages stimulated with the trypanosome soluble variant surface glycoprotein in vitro and in macrophages taken from infected mice. Genes associated with the acute phase response and with type I IFN responses were prominent components of the macrophage activation profiles expressed within 72 h in vitro and in vivo. Thus, induction of proinflammatory gene expression is a characteristic of early trypanosome infection that is driven primarily by soluble variant surface glycoprotein exposure, and it may be that IFN-alpha/beta plays a central role in regulation of early resistance to trypanosomes. To test this hypothesis, we assessed parameters of infection in mouse strains with genetic alterations in the IFN-alpha/beta response pathway. We found that Ifnar1(-/-) mice, which lack the receptor for type I IFNs, exhibited delayed control of parasite burden during the first week of infection and died earlier than did wild-type controls. However, infection of Ubp43(-/-) mice, which are hyperresponsive to type I IFNs, did not exhibit enhanced resistance to trypanosomes. Instead, these animals also failed to control parasite burden and were more susceptible than wild-type animals. Additionally, the Ubp43(-/-) mice exhibited a significant defect in IFN-gamma production, which is definitively linked to host resistance in trypanosomiasis. These results show that type I IFNs play a role in early control of parasites in infected mice but may contribute to down-regulation of IFN-gamma production and subsequent loss of host resistance later in infection.
Project description:Production of type I interferon (IFN; IFN-alphabeta) increases host susceptibility to Listeria monocytogenes, whereas type II IFN (IFN-gamma) activates macrophages to resist infection. We show that these opposing immunological effects of IFN-alphabeta and IFN-gamma occur because of cross talk between the respective signaling pathways. We found that cultured macrophages infected with L. monocytogenes were refractory to IFN-gamma treatment as a result of down-regulation of the IFN-gamma receptor (IFNGR). The soluble factor responsible for these effects was identified as host IFN-alphabeta. Accordingly, macrophages and dendritic cells (DCs) showed reduced IFNGR1 expression and reduced responsiveness to IFN-gamma during systemic infection of IFN-alphabeta-responsive mice. Furthermore, the increased resistance of mice lacking the IFN-alphabeta receptor (IFNAR(-/-)) to L. monocytogenes correlated with increased expression of IFN-gamma-dependent activation markers by macrophages and DCs and was reversed by depletion of IFN-gamma. Thus, IFN-alphabeta produced in response to bacterial infection and other stimuli antagonizes the host response to IFN-gamma by down-regulating the IFNGR. Such cross talk permits prioritization of IFN-alphabeta-type immune responses and may contribute to the beneficial effects of IFN-beta in treatment of inflammatory diseases such as multiple sclerosis.
Project description:Type 2 diabetes (T2D) is associated with increased risk for atherosclerosis; however, the mechanisms underlying this relationship are poorly understood. Macrophages, which are activated in T2D and causatively linked to atherogenesis, are an attractive mechanistic link. Here, we use proteomics to show that diet-induced obesity and insulin resistance (obesity/IR) modulate a pro-atherogenic "macrophage-sterol-responsive-network" (MSRN), which, in turn, predisposes macrophages to cholesterol accumulation. We identify IFN? as the mediator of obesity/IR-induced MSRN dysregulation and increased macrophage cholesterol accumulation and show that obesity/IR primes T cells to increase IFN? production. Accordingly, myeloid cell-specific deletion of the IFN? receptor (Ifngr1-/-) restores MSRN proteins, attenuates macrophage cholesterol accumulation and atherogenesis, and uncouples the strong relationship between hyperinsulinemia and aortic root lesion size in hypercholesterolemic Ldlr-/- mice with obesity/IR, but does not affect these parameters in Ldlr-/- mice without obesity/IR. Collectively, our findings identify an IFN?-macrophage pathway as a mechanistic link between obesity/IR and accelerated atherogenesis.
Project description:Type I interferons (IFNs) induced by an endogenous <i>Leishmania</i> RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous <i>Leishmania</i> parasites, however, the impact of type I IFNs in visceral <i>Leishmania</i> infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with <i>L. infantum</i> and <i>L. donovani</i> and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-?) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-? particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-?. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent <i>L. infantum</i> promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral <i>Leishmania</i> promastigotes and that targeting of Sn may have some protective effects in early infection.
Project description:Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFN? in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated upregulation of IFN-stimulated genes and a cell-autonomous defence pathway acting on replicating and non-replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN-stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria.
Project description:Microbial pattern recognition critically contributes to innate response, both at extracellular and intracellular cytosolic surveillance pathway (CSP) interface. However, the role of pattern recognition by host innate receptors in CSP is poorly understood in Leishmania donovani infection. Here, we have demonstrated that cytosolic targeting of L.donovani DNA (Ld-DNA) inhibits macrophage responsiveness to IFN?, through decreased MHC-II expression and lowered pSTAT1 (Y701) levels, involving host three-prime repair exonuclease-1 (TREX-1). The Ld-DNA potently induced type-1 IFNs, i.e. significant over-production of IFN? through activation of the IRF pathway. Interestingly, knockdown of TRIF or MyD88 expression in macrophages had no effect on cytosolic Ld-DNA transfection-mediated IFN-? production, indicating involvement of a TLR independent pathway. Contrastingly, Ld-DNA failed to induce IFN? in both TBK-1 and IRF3KO knockout macrophages. Although IFN? was not induced by Ld-DNA in STING- knockout macrophages, STING alone was not enough for the induction. Evidently, besides STING, Ld-DNA recognition for induction of IFN? critically required cytosolic cyclic GMP-AMP synthase (cGAS). Furthermore, the cGAS dependent targeting of Ld-DNA induced IFN? over-production that contributed to antimony resistance in L.donovani infection. We provide the first evidence that enhanced cytosolic sensing of Ld-DNA in infection by antimony resistant (SBR-LD), but not antimony sensitive L.donovani strains (SBS-LD), was critically regulated by host MDRs, multi drug resistant associated protein 1 (MRP 1) and permeability glycoprotein (P-gp) in macrophages. Collectively, our results disclose Ld-DNA as a vital pathogen associated molecular pattern (PAMP) driving host Type-I IFN responses and antimony resistance. The findings may help in future development of policies for novel anti-leishmanial therapeutics.
Project description:Triggering or enhancing antitumor activity of tumor-associated macrophages is an attractive strategy for cancer treatment. We have previously shown that the cytokine interferon-? (IFN-?), a type II IFN, could synergize with toll-like receptor (TLR) agonists for induction of antitumor M1 macrophages. However, the toxicity of IFN-? limits its clinical use. Here, we investigated whether the less toxic type I IFNs, IFN-?, and IFN-?, could potentially replace IFN-? for induction of antitumor M1 macrophages. We measured in vitro the ability of type I and II IFNs to synergize with TLR agonists for transcription of inducible nitric oxide synthase (iNOS) mRNA and secretion of nitric oxide (NO) by mouse bone marrow-derived macrophages (BMDMs). An in vitro growth inhibition assay was used to measure both cytotoxic and cytostatic activity of activated macrophages against Lewis lung carcinoma (LLC) cancer cells. We found that both type I and II IFNs could synergize with TLR agonists in inducing macrophage-mediated inhibition of cancer cell growth, which was dependent on NO. The ability of high dose lipopolysaccharide (LPS) to induce tumoricidal activity in macrophages in the absence of IFN-? was shown to depend on induction of autocrine type I IFNs. Antitumor M1 macrophages could also be generated in the absence of IFN-? by a combination of two TLR ligands when using the TLR3 agonist poly(I:C) which induces autocrine type I IFNs. Finally, we show that encapsulation of poly(I:C) into nanoparticles improved its potency to induce M1 macrophages up to 100-fold. This study reveals the potential of type I IFNs for activation of antitumor macrophages and indicates new avenues for cancer immunotherapy based on type I IFN signaling, including combination of TLR agonists.
Project description:Recognition of intracellular bacteria by macrophages leads to secretion of type I IFNs. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. In this study, we investigated the role of type I IFNs in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1(-/-)) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A(+) gammadelta T cell population, indicating that type I IFNs negatively regulate the number of IL-17A(+) gammadelta T cells during infection. Furthermore, IL-17A-deficient mice contained fewer neutrophils compared with wild-type mice during infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1(-/-) mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent F. tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A(+) gammadelta T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1(-/-) mice to infection with F. novicida and other intracellular bacteria.