Andrea Kröger

Review: Activities of IRF-1

Interferon (IFN) regulatory factor-1 (IRF-1) was isolated by virtue of its affinity to specific DNA sequences in the IFN-β promoter that mediate virus responsiveness. IRF-1 was the first factor identified of the IRF family and was most extensively characterized at the molecular level. Also, its physiologic role in host defense against pathogens, tumor prevention, and development of the immune system was investigated in detail. Even though some of the functions first associated with IRF-1 were later found to be mediated in part or predominantly by other activators of the IRF family of transcription factors, IRF-1 has remained a central paradigm in the transcriptional regulation of the IFN response.

DNA Damage Primes the Type I Interferon System via the Cytosolic DNA Sensor STING to Promote Anti-Microbial Innate Immunity

Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.

Hepatic dendritic cell subsets in the mouse

The CD11c+ cell population in the non‐parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c+ DC population from immunocompetent or immunodeficient [recombinase‐activating gene‐1 (RAG1)–/–] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220+ CD11cint subset of ‘plasmacytoid’ DC, and a B220– CD11c+ DC subset. The latter DC population could be subdivided into a major, immature (CD40lo CD80lo CD86lo MHC class IIlo) CD11cint subset, and a minor, mature (CD40hi CD80hi CD86hi MHC class IIhi) CD11chi subset. Stimulated B220+ but not B220– DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220– DC three‐ to fourfold within 18 h post‐injection, and up‐regulated their surface expression of activation marker, while it contracted the B220+ DC population. Early in virus infection, the hepatic B220+ DC subset expanded, and both, the B220+ as well as B220– DC populations in the liver matured. In vitro, B220– but not B220+ DC primed CD4+ or CD8+T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220+ and B220– subsets in CD11c+ DC populations freshly isolated from the mouse liver.

GARP: a key receptor controlling FOXP3 in human regulatory T cells

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein‐A repetitions predominant), LGALS3 (lectin, galactoside‐binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T‐cell receptor stimulation. Retroviral overexpression of GARP in antigen‐specific Th cells leads to an efficient and stable re‐programming of an effector T cell towards a regulatory T cell, which involves up‐regulation of FOXP3, LGALS3, LGMN and other Treg‐associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down‐regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down‐regulation of FOXP3. Moreover, down‐regulation of FOXP3 resulted in similar phenotypic changes and down‐regulation of GARP. This provides compelling evidence for a GARP‐FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor‐β induced Treg cells as we show here that the latter do not up‐regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T‐cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response

The cellular recognition of viruses evokes the secretion of type‐I interferons (IFNs) that induce an antiviral protective state. By live‐cell imaging, we show that key steps of virus‐induced signal transduction, IFN‐β expression, and induction of IFN‐stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular—and not viral—origin, and temporal unpredictability of IFN‐β expression is largely due to cell‐intrinsic noise generated both upstream and downstream of the activation of nuclear factor‐κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all‐or‐nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi‐layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.

Type I IFN Negatively Regulates CD8+ T Cell Responses through IL-10-Producing CD4+ T Regulatory 1 Cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8+ T cell responses in IFN-β (IFN-β−/−)- or type I IFN receptor (IFNAR−/−)-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR−/− or IFN-β−/− mice have normal numbers of CD4+ and CD8+ T cells, and CD25+FoxP3+ T regulatory (TR) cells in liver and spleen. Twice as many CD8+ T cells specific for different class I-restricted epitopes develop in IFNAR−/− or IFN-β−/− mice than in normal animals after peptide- or DNA-based vaccination. IFN-γ and TNF-α production and clonal expansion of specific CD8+ T cells from normal and knockout mice are similar. CD25+FoxP3+ TR cells down-modulate vaccine-primed CD8+ T cell responses in normal, IFNAR−/−, or IFN-β−/− mice to a comparable extent. Low IFN-α or IFN-β doses (500–103 U/mouse) down-modulate CD8+ T cells priming in vivo. IFNAR- and IFN-β-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4+ T cells after polyclonal or specific stimulation in vitro or in vivo. CD8+ T cell responses are thus subjected to negative control by both CD25+FoxP3+ TR cells and CD4+IL-10+ TR1 cells, but only development of the latter TR cells depends on type I IFN.

IFN Regulatory Factor-1 Bypasses IFN-Mediated Antiviral Effects through Viperin Gene Induction

Viperin is an antiviral protein whose expression is highly upregulated during viral infections via IFN-dependent and/or IFN-independent pathways. We examined the molecular alterations induced by the transcriptional activator IFN regulatory factor (IRF)-1 and found viperin to be among the group of IRF-1 regulated genes. From these data, it was not possible to distinguish genes that are primary targets of IRF-1 and those that are targets of IRF-1–induced proteins, like IFN-β. In this study, we show that IRF-1 directly binds to the murine viperin promoter to the two proximal IRF elements and thereby induces viperin expression. Infection studies with embryonal fibroblasts from different gene knock-out mice demonstrate that IRF-1 is essential, whereas the type I IFN system is dispensable for vesicular stomatitis virus induced viperin gene transcription. Further, IRF-1, but not IFN type I, mediates the induction of viperin transcription after IFN-γ treatment. In contrast, IRF-1 is not required for IFN-independent viperin induction by Newcastle disease virus infection and by infection with a vesicular stomatitis virus mutant that is unable to block IFN expression and secretion. We conclude that the IRF-1 mediated type I IFN independent mechanism of enhanced viperin expression provides a redundant mechanism to protect cells from viral infections. This mechanism becomes important when viruses evade innate immunity by antagonizing the induction and function of the IFN system.

Activating Immunity in the Liver. II. IFN-β Attenuates NK Cell-Dependent Liver Injury Triggered by Liver NKT Cell Activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10–100 ng/mouse) of α-galactosyl ceramide (αGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following αGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to αGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-γ response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG−/− or HBs-tg/RAG−/−) hosts via the portal vein elicited IFN-γ responses of liver NK cells in situ. IFN-β down-regulates the pathogenic IL-12/IFN-γ cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-β suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (αGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-γ response of the specifically activated liver NKT cells. In vivo, IFN-β attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.

IRF-1 reverts the transformed phenotype of oncogenically transformed cells in vitro and in vivo

The expression of the transcriptional activator and tumor suppressor IRF-1 induces multiple effects that counteract the growth of tumor cells in vitro and in vivo. These include the inhibition of cell proliferation, the secretion of interferon-β (IFN-β), the induction of apoptosis specifically in certain cell types and the induction of a strong T-cell response. Here, we show that apart from its immune-activating properties, IRF-1 expression leads to a reversion of the tumorigenic phenotype of NIH3T3 cells transformed by different oncogenes. This was analysed in detail in a cell line in which the expression of c-Ha-ras and c-myc is under the control of a doxycycline-regulated promoter allowing to switch between the normal and oncogenic cell status. In the same cells, a β-estradiol activatable IRF-1 fusion protein is expressed. After IRF-1 activation the oncogene-mediated acceleration of the cell cycle is reverted. Further, a complete IRF-1-mediated reversion of the oncogenic phenotype is observed in soft-agar growth assays. IRF-1 activation induces IFN-β secretion; however, the observed effects are not mediated by IFN-β. Inhibition of tumor growth is observed in nude mice as long as IRF-1 is active, indicating that neither B- nor T-cells must become activated for tumor growth suppression.

Mast cells elicit proinflammatory but not type I interferon responses upon activation of TLRs by bacteria

Balanced induction of proinflammatory and type I IFN responses upon activation of Toll-like receptors (TLRs) determines the outcome of microbial infections and the pathogenesis of autoimmune and other inflammatory diseases. Mast cells, key components of the innate immune system, are known for their debilitating role in allergy and autoimmunity. However, their role in antimicrobial host defenses is being acknowledged increasingly. How mast cells interact with microbes and the nature of responses triggered thereby is not well characterized. Here we show that in response to TLR activation by Gram-positive and -negative bacteria or their components, mast cells elicit proinflammatory but not type I IFN responses. We demonstrate that in mast cells, bound bacteria and TLR ligands remain trapped at the cell surface and do not undergo internalization, a prerequisite for type I IFN induction. Such cells, however, can elicit type I IFNs in response to vesicular stomatitis virus which accesses the cytosolic retinoic acid-inducible gene I receptor. Although important for antiviral immunity, a strong I IFN response is known to contribute to pathogenesis of several bacterial pathogens such as Listeria monocytogenes. Interestingly, we observed that the mast cell-dependent neutrophil mobilization upon L. monocytogenes infection is highly impaired by IFN-β. Thus, the fact that mast cells, although endowed with the capacity to elicit type I IFNs in response to viral infection, elicit only proinflammatory responses upon bacterial infection shows that mast cells, key effector cells of the innate immune system, are well adjusted for optimal antibacterial and antiviral responses.