ABSTRACT: 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 Listeria monocytogenes-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+ peripheral blood mononuclear cells. IFN?-driven reductions in IFNGR1 were not explained by ligand-induced receptor internalization. Rather, IFN? reduced macrophage Ifngr1 transcription by altering chromatin structure at putative Ifngr1 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: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:Although IFN-? is required for resolution of Listeria monocytogenes infection, the identities of the IFN-?-responsive cells that initiate the process remain unclear. We addressed this question using novel mice with conditional loss of IFN-?R (IFNGR1). Itgax-cre(+)Ifngr1(f/f) mice with selective IFN-? unresponsiveness in CD8?(+) dendritic cells displayed increased susceptibility to infection. This phenotype was due to the inability of IFN-?-unresponsive CD8?(+) dendritic cells to produce the initial burst of IL-12 induced by IFN-? from TNF-?-activated NK/NKT cells. The defect in early IL-12 production resulted in increased IL-4 production that established a myeloid cell environment favoring Listeria growth. Neutralization of IL-4 restored Listeria resistance in Itgax-cre(+)Ifngr1(f/f) mice. We also found that Itgax-cre(+)Ifngr1(f/f) mice survived infection with low-dose Listeria as the result of a second wave of IL-12 produced by Ly6C(hi) monocytes. Thus, an IFN-?-driven cascade involving CD8?(+) dendritic cells and NK/NKT cells induces the rapid production of IL-12 that initiates the anti-Listeria response.
Project description:Brain expression of AAV-Ifn-? leads to reactive gliosis, nigrostriatal degeneration and midbrain calcification in wild type mice. This mouse model phenocopies idiopathic basal ganglia calcification which is associated with Parkinsonian symptoms. To understand how the nigro-striatal pathway is selectively vulnerable to Ifn-?, we determined if the phenotype is driven by canonical signaling intermediates, Ifngr1 and Stat1. Using focused bioinformatic analysis and rotarod testing, we show that neuroinflammation and motor abnormalities precede the appearance of midbrain neuropathologies in the brains of Ifn-? mouse model. To test whether canonical Ifn-? signaling is a key driver of progressive nigrostriatal degeneration, we overexpressed Ifn-? in the brains of Ifngr1-/- and Stat1-/- mice. Expression of Ifn-? in Ifngr1-/- mice did not result in any neuroinflammation, midbrain calcinosis or nigrostriatal degenerative pathology. Interestingly, in Stat1-/- mice, Ifn-? expression resulted in gliosis without recapitulating the neurodegenerative phenotype. Overall, our data shows that canonical Ifn-? signaling triggers midbrain calcinosis and nigrostriatal neurodegeneration, providing mechanistic insights into cytokine-driven selective neuronal vulnerability. Our study establishes the broader relevance of inflammatory signaling in neurodegenerative diseases and can potentially identify novel immunological targets for Parkinsonian syndromes.
Project description:Complicated/severe cases of placental pathology due to Plasmodium falciparum and P. vivax, especially adverse pregnancy outcomes during P. vivax infection, have been increasing in recent years. However, the pathogenesis of placental pathology during severe malaria is poorly understood, while responses against IFN-? are thought to be associated with adverse pregnancy outcomes. In the present study, we explored the role of IFN-? receptor 1 (IFNGR1) signaling in placental pathology during severe malaria using luciferase-expressing rodent malaria parasites, P. berghei NK65 (PbNK65L). We detected luciferase activities in the lung, spleen, adipose tissue, and placenta in pregnant mice, suggesting that infected erythrocytes could accumulate in various organs during infection. Importantly, we found that fetal mortality in IFNGR1-deficient mice infected with PbNK65L parasites was much less than in infected wild type (WT) mice. Placental pathology was also improved in IFNGR1-deficient mice. In contrast, bioluminescence imaging showed that parasite accumulation in the placentas of IFNGR1-deficient pregnant mice was comparable to that in WT mice infected with PbNK65L. These findings suggest that IFNGR1 signaling plays a pivotal role in placental pathology and subsequent adverse pregnancy outcomes during severe malaria. Our findings may increase our understanding of how disease aggravation occurs during malaria during pregnancy.
Project description:The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.
Project description:Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003%) was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity.
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:Post-kala-azar dermal leishmanaisis (PKDL) in Sudan is associated with elevated interferon-gamma (IFN-gamma). To study interferon-gamma pathways in PKDL, we genotyped 80 trios from the Masalit ethnic group for polymorphisms at -470 ins/delTT, -270T/C, -56T/C and +95T/C in IFNGR1 and at -179G/A and +874T/A in IFNG. No associations occurred at IFNG. Global association with haplotypes comprising all four markers at IFNGR1 (chi(2)(10df)=21.97, P=0.015) was observed, associated with a significant (chi(2)(1df)=4.54, P=0.033) bias in transmission of the haplotype insTT T T T and less (chi(2)(1df)=5.59, P=0.018) than expected transmission of insTT C C C. When compared with data on malaria associations from Gambia, the results suggest a complex pattern of haplotypic variation at the IFNGR1 promoter locus associated with different infectious disease in African populations that reflect the complex roles of IFN-gamma in parasite killing versus inflammation and pathogenesis.
Project description:Little is known about the parasite/host factors that lead to Post Kala-azar Dermal Leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after drug-cure. Studies in Sudan provide evidence for association between polymorphisms in the gene (IFNGR1) encoding the alpha chain of interferon-? receptor type I and risk of PKDL. This study aimed to identify putative functional polymorphisms in the IFNGR1 gene, and to determine whether differences in expression of interferon-? (IFNG) and IFNGR1 at the RNA level are associated with pathogenesis of VL and/or PKDL in Sudan.Sanger sequencing was used to re-sequence 841 bp of upstream, exon1 and intron1 of the IFNGR1 gene in DNA from 30 PKDL patients. LAGAN and SYNPLOT bioinformatics tools were used to compare human, chimpanzee and dog sequences to identify conserved noncoding sequences carrying putative regulatory elements. The relative expression of IFNG and IFNGR1 in paired pre- and post-treatment RNA samples from the lymph nodes of 24 VL patients, and in RNA samples from skin biopsies of 19 PKDL patients, was measured using real time PCR. Pre- versus post-treatment expression was evaluated statistically using the nonparametric Wilcoxon matched pairs signed-rank test.Ten variants were identified in the 841 bp of sequence, four of which are novel polymorphisms at -77A/G, +10 C/T, +18C/T and +91G/T relative to the IFNGR1 initiation site. A cluster of conserved non-coding sequences with putative regulatory variants was identified in the distal promoter of IFNGR1. Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P?=?0.006) in expression following treatment. IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment. IFNG expression was undetectable in the skin biopsies of PKDL cases, while IFNGR1 expression was also uniformly low.Uniformly low expression of IFN and IFNGR1 in PKDL skin biopsies could explain parasite persistence and is consistent with prior demonstration of genetic association with IFNGR1 polymorphisms. Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.