Interferon Regulatory Factor-1 Knockout Mice Research Articles

The Modulation of Interferon Regulatory Factor-1 via Caspase-1-Mediated Alveolar Macrophage Pyroptosis in Ventilator-Induced Lung Injury.

Background To examine the role of interferon regulatory factor-1 (IRF-1) and to explore the potential molecular mechanism in ventilator-induced lung injury. Methods Wild-type C57BL/6 mice and IRF-1 gene knockout mice/caspase-1 knockout mice were mechanically ventilated with a high tidal volume to establish a ventilator-related lung injury model. The supernatant of the alveolar lavage solution and the lung tissues of these mice were collected. The degree of lung injury was examined by hematoxylin and eosin staining. The protein and mRNA expression levels of IRF-1, caspase-1 (p10), and interleukin (IL)-1β (p17) in lung tissues were measured by western blot and quantitative real-time polymerase chain reaction, respectively. Pyroptosis of alveolar macrophages was detected by flow cytometry and western blotting for active caspase-1 and cleaved GSDMD. An enzyme-linked immunosorbent assay was used to measure the levels of IL-1β, IL-18, IL-6, TNF-α, and high mobility group box protein 1 (HMGB-1) in alveolar lavage fluid. Results IRF-1 expression and caspase-1-dependent pyroptosis in lung tissues of wild-type mice were significantly upregulated after mechanical ventilation with a high tidal volume. The degree of ventilator-related lung injury in IRF-1 gene knockout mice and caspase-1 knockout mice was significantly improved compared to that in wild-type mice, and the levels of GSDMD, IL-1β, IL-18, IL-6, and HMGB-1 in alveolar lavage solution were significantly reduced (P < 0.05). The expression levels of caspase-1 (p10), cleaved GSDMD, and IL-1β (p17) proteins in lung tissues of IRF-1 knockout mice with ventilator-related lung injury were significantly lower than those of wild-type mice, and the level of pyroptosis of macrophages in alveolar lavage solution was significantly reduced. Conclusions IRF-1 may aggravate ventilator-induced lung injury by regulating the activation of caspase-1 and the focal death of alveolar macrophages.

Interferon Regulatory Factor-1 (IRF1) activates autophagy to promote liver ischemia/reperfusion injury by inhibiting β-catenin in mice.

Autophagy is an important factor in liver ischemia-reperfusion injury. In the current study we investigate the function of interferon regulatory factor-1 (IRF1) in regulating autophagy to promote hepatic ischemia reperfusion injury (IR). The high expression of IRF1 during hepatic IR exhibited increased liver damage and was associated with activation of autophagy shown by Western blot markers, as well as immunofluorescent staining for autophagosomes. These effects were diminished by IRF1 deficiency in IRF1 knock out (KO) mice. Moreover, the autophagy inhibitor 3-MA decreased IR-induced liver necrosis and markedly abrogated the rise in liver injury tests (AST/ALT). β-catenin expression decreased during liver IR and was increased in the IRF1 KO mice. Immunoprecipitation assay showed the binding between IRF1 and β-catenin. Overexpression of IRF1 induced autophagy and also inhibited the expression of β-catenin. β-catenin inhibitor increased autophagy while β-catenin agonist suppressed autophagy in primary mouse hepatocytes. These results indicate that IRF1 induced autophagy aggravates hepatic IR injury in part by inhibiting β-catenin and suggests that targeting IRF1 may be an effective strategy in reducing hepatic IR injury.

Role of interferon regulatory factor-1 in lipopolysaccharide-induced mitochondrial damage and oxidative stress responses in macrophages.

Sepsis causes many early deaths; both macrophage mitochondrial damage and oxidative stress responses are key factors in its pathogenesis. Although the exact mechanisms responsible for sepsis-induced mitochondrial damage are unknown, the nuclear transcription factor, interferon regulatory factor-1 (IRF-1) has been reported to cause mitochondrial damage in several diseases. Previously, we reported that in addition to promoting systemic inflammation, IRF-1 promoted the apoptosis of and inhibited autophagy in macrophages. In the present study, we hypothesized that lipopolysaccharide (LPS)-induced IRF-1 activation in macrophages may promote mitochondrial damage and oxidative stress. In vitro, LPS was found to promote IRF-1 activation, reactive oxygen species (ROS) production, adenosine triphosphate (ATP) depletion, superoxide dismutase (SOD) consumption, malondialdehyde (MDA) accumulation and mitochondrial depolarization in macrophages in a time- and dose-dependent manner. These effects were abrogated in cells in which IRF-1 was knocked down. Furthermore, IRF-1 overexpression increased LPS-induced oxidative stress responses and mitochondrial damage. In vivo, peritoneal macrophages obtained from IRF-1 knockout (KO) mice produced less ROS and had less mitochondrial depolarization and damage following the administration of LPS, when compared to their wild-type (WT) counterparts. In addition, IRF-1 KO mice exhibited a decreased release of mitochondrial DNA (mtDNA) following the administration of LPS. Thus, IRF-1 may be a critical factor in augmenting LPS-induced oxidative stress and mitochondrial damage in macrophages.

Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

Interferon regulatory factor-1 mediates the release of high mobility group box-1 in endotoxemia in mice

Background The extracellular release of the danger signal high mobility group box-1 (HMGB1) has been implicated in the pathogenesis and outcomes of sepsis. Understanding the mechanisms responsible for HMGB1 release can lead to the identification of targets that may inhibit this process. The transcription factor interferon regulatory factor-1 (IRF-1) is an important mediator of innate immune responses and has been shown to participate in mortality associated with endotoxemia; however, its role in mediating the release of HMGB1 in these settings is unknown. Methods Male IRF-1 knockout (KO) and age matched C57BL/6 wild type (WT) mice were given intraperitoneal (IP) injections of lipopolysaccharide (LPS). In some experiments, 96 hours survival rates were observed. In other experiments, mice were sacrificed 12 hours after LPS administration and sera were harvested for future analysis. In in vitro study, RAW 264.7 murine monocyte/macrophage-like cells or primary peritoneal macrophage obtained from IRF-1 KO and WT mice were cultured for LPS mediated HMGB1 release analysis. And the mechanism for HMGB1 release was analyzed by immune-precipitation. Results IRF-1 KO mice experienced less mortality, and released less systemic HMGB1 compared to their WT counterparts. Exogenous administration of recombinant HMGB1 to IRF-1 KO mice returned the mortality rate to that seen originally in IRF-1 WT mice. Using cultures of peritoneal macrophages or RAW264.7 cells, in vitro LPS stimulation induced the release of HMGB1 in an IRF-1 dependent manner. And the janus associated kinase (JAK)-IRF-1 signal pathway appeared to participate in the signaling mechanisms of LPS-induced HMGB1 release by mediating acetylation of HMGB1. Conclusion IRF-1 plays a role in LPS induced release of HMGB1 and therefore may serve as a novel target in sepsis.

Splenocyte Apoptosis and Autophagy Is Mediated by Interferon Regulatory Factor 1 During Murine Endotoxemia

Sepsis-induced lymphocyte and dendritic cell apoptosis contributes to immunosuppression, which results in an inability to eradicate the primary infection as well as a propensity to acquire new, secondary infections. Another cellular process, autophagy, is also activated in immune cells and plays a protective role. In the present study, we demonstrate that interferon regulatory factor 1 (IRF-1) regulates both immune cell apoptosis and autophagy in a murine endotoxemia model. Interferon regulatory factor 1 is activated at an early phase through a Toll-like receptor 4-dependent, myeloid differentiation primary response gene 88-independent manner in splenocytes. Furthermore, IRF-1 knockout (KO) mice are protected from a lethal endotoxemia model. This protection is associated with decreased apoptosis and increased autophagy in splenocytes. Interferon regulatory factor 1 KO mice experience decreased apoptotic cell loss, especially in CD4⁺ T lymphocytes and myeloid antigen-presenting cells. Meanwhile, IRF-1 KO mice demonstrate increased autophagy and improved mitochondrial integrity. This increased autophagy in KO mice is attributable, at least in part, to deactivation of mammalian target of rapamycin/P70S6 signaling--a main negative regulator of autophagy. Therefore, we propose a novel role for IRF-1 in regulating both apoptosis and autophagy in splenocytes in the setting of endotoxemia with IRF-1 promoting apoptosis and inhibiting autophagy.

Interferon Regulatory Factor-1 Regulates the Autophagic Response in LPS-Stimulated Macrophages through Nitric Oxide

The pathogenesis of sepsis is complex and, unfortunately, poorly understood. The cellular process of autophagy is believed to play a protective role in sepsis; however, the mechanisms responsible for its regulation in this setting are ill defined. In the present study, interferon regulatory factor 1 (IRF-1) was found to regulate the autophagic response in lipopolysaccharide (LPS)-stimulated macrophages. In vivo, tissue macrophages obtained from LPS-stimulated IRF-1 knockout (KO) mice demonstrated increased autophagy and decreased apoptosis compared to those isolated from IRF-1 wild-type (WT) mice. In vitro, LPS-stimulated peritoneal macrophages obtained from IRF-1 KO mice experienced increased autophagy and decreased apoptosis. IRF-1 mediates the inhibition of autophagy by modulating the activation of the mammalian target of rapamycin (mTOR). LPS induced the activation of mTOR in WT peritoneal macrophages, but not in IRF-1 KO macrophages. In contrast, overexpression of IRF-1 alone increased the activation of mTOR and consequently decreased autophagic flux. Furthermore, the inhibitory effects of IRF-1 mTOR activity were mediated by nitric oxide (NO). Therefore, we propose a novel role for IRF-1 and NO in the regulation of macrophage autophagy during LPS stimulation in which IRF-1/NO inhibits autophagy through mTOR activation.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus ΔwbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-α when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Δ wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.

The role of the vacB gene in the pathogenesis of Brucella abortus

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus ΔvacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12–14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.

The transcription factor interferon regulatory factor-1 mediates liver damage during ischemia-reperfusion injury

Hepatic ischemia occurs in the settings of trauma, transplantation, and elective liver resections. The initiating events that account for local organ damage are only partially understood. Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that regulates the expression of a number of genes involved in both innate and acquired immunity; however, its function in liver injury is unknown. Therefore, the purpose of this study was to investigate the role of IRF-1 in hepatic ischemia-reperfusion (I/R) injury. In C57BL/6 mice undergoing 60 min of hepatic ischemia, IRF-1 protein expression increased as early as 1 h after reperfusion. IRF-1 knockout mice were significantly protected from hepatic I/R-induced damage compared with their wild-type controls. Hepatic I/R injury resulted in marked activation of the MAP kinase c-Jun NH(2)-terminal kinase (JNK) in wild-type mice but not IRF-1 knockout mice. IRF-1 knockout mice also exhibited significantly lower hepatic expression of TNF-alpha, IL-6, ICAM-1, and inducible nitric oxide synthase (iNOS) mRNA. Adenoviral delivery of IRF-1 into C57BL/6 mice resulted in increased liver damage even without an ischemic insult. This injury was associated with increased JNK activation and hepatic iNOS expression. Because IRF-1 contributed to liver injury, we also examined for inflammatory signals that regulated IRF-1 gene expression in cultured hepatocytes. Whereas IFN-gamma and IFN-beta were strong inducers of IRF-1 mRNA (>10-fold) in a time- and dose-dependent manner, TNF-alpha and IL-1beta also induced IRF-1 mRNA to a lesser extent (2- to 3-fold). IL-6 and lipopolysaccharide had no effect on IRF-1 expression. This study demonstrates that IRF-1 exerts a harmful role in hepatic I/R injury by modulating the expression of multiple inflammatory mediators. We further show that IRF-1-mediated injury involves the activation of JNK and that hepatocellular IRF-1 expression itself is regulated by specific cytokines.

Dual role of interferon-gamma signalling pathway in sensitivity of pancreatic beta cells to immune destruction.

Disruption of the interferon-gamma (IFN-gamma) signalling pathway at the level of interferon regulatory factor-1 (IRF-1) protects islets against cytokine-induced nitric oxide production and cell death in vitro. The aim of this study was to investigate the effects of a global disruption of IFN-gamma signalling, or a selective disruption of IRF-1, on beta-cell sensitivity to in vivo immune destruction. In a first set of experiments, IFN-gamma receptor knockout mice (IFN-gammaR-/-) and interferon regulatory factor-1 knockout mice (IRF-1-/-) were rendered diabetic by injections of 50 mg streptozotocin i. p. on 5 consecutive days (MLDSTZ). Whereas no difference in sensitivity to MLDSTZ-induced diabetes could be observed between IFN-gammaR-/- mice and their 129/Sv/Ev controls (50% vs 55%, NS), there was an increased incidence of diabetes in IRF-1-/- mice (100% vs 67% in C57B1/6 mice, p < 0.05). A similar increased sensitivity to immune destruction of IRF-1-/- islets was observed when these islets were used as allografts. Islet graft survival rate of IFN-gammaR-/- and 129/Sv/Ev islets, when transplanted in alloxan-diabetic BALB/c recipients, was comparable (12.0 +/- 1.9 days vs 12.9 +/- 2.3 days, NS). Allograft rejection, however, of IRF-1-/- islets by BALB/c recipients occurred more rapidly than following transplantation to their C57B1/6 controls (9.1 +/- 2.0 days vs 13.1 +/- 1.5 days, p < 0.003). These data indicate that IFN-gamma signal transduction at the beta-cell level is not essential for immune beta-cell destruction in vivo. Moreover, disruption of the IRF-1 gene in pancreatic islets increases susceptibility to beta-cell killing, suggesting that IRF-1 might be necessary for the expression of putative beta-cell "defence and/or repair" genes.

Role of interferon regulatory factor-1 in the regulation of IL-18 production and activity

Interferon regulatory factor-1 (IRF-1) is a transcription factor regulating the expression of several cytokines. Using IRF-1 knockout (KO) mice, the role of IRF-1 in the production and activity of IL-18 was evaluated. Administration of IL-12 or concanavalin A significantly increased levels of circulating IL-18 in wild-type (WT), but not in IRF-1 KO mice. However, despite these differences in circulating IL-18 levels, no or only minor differences in constitutive or inducible IL-18 mRNA and tissue-associated protein levels were observed between WT and IRF-1 KO mice. On the other hand, we observed that constitutive and inducible levels of the IL-18-processing enzyme caspase-1 were markedly reduced in the spleen and the liver of IRF-1 KO compared to WT mice. In addition, both constitutive and inducible liver mRNA levels for the IL-18 binding protein (IL-18BP), a specific IL-18 antagonist, were significantly lower in IRF-1 KO than in WT mice. Compared to IL-12, IL-18 only weakly induced IRF-1 mRNA in cultured splenocytes. However, IL-18-induced IFN-gamma production was strongly reduced in splenocytes from IRF-1 KO compared to WT cells. In conclusion, IRF-1 regulates IL-18 production and activity mostly by modulating expression of caspase-1 and IL-18BP. In addition, IRF-1 participates in the induction of IFN-gamma by IL-18.

Interferon-gamma induces interleukin-1 converting enzyme expression in pancreatic islets by an interferon regulatory factor-1-dependent mechanism.

Whereas nitric oxide (NO) production is associated with the toxic effect of cytokines on rodent pancreatic beta-cells, cytokine-induced apoptosis in human islets may occur independently of NO. The cysteine protease interleukin (IL)-1 converting enzyme (ICE) is a key proapoptotic caspase. Our aim was therefore to analyze the effect of cytokines on ICE expression in human, rat, and mouse islets and rat insulinoma cells. ICE messenger RNA (mRNA) expression was highly up-regulated after 6-, 24-, and 72-h exposure of human islets to interferon (IFN)gamma, tumor necrosis factor (TNF)alpha + IFNgamma or IL-1beta + TNFalpha + IFNgamma, paralleled by increased iNOS (the inducible form of NO synthase) expression and NO production after exposure to the combined cytokines but not to IFNgamma or TNFalpha + IFNgamma. Cytokine-induced NO-independent ICE transcription was confirmed using iNOS inhibitors. Exposure of rat and mouse islets, or rat insulinoma cells, for 24 h to IFNgamma alone or in combination with the two other cytokines also resulted in a highly significant ICE mRNA expression. ICE transcription was not inducible in islets from IFN regulatory factor-1 knock-out mice, suggesting a key-role of this transcription-factor in cytokine-mediated ICE expression in pancreatic islets. In conclusion, cytokines and IFNgamma in particular increase ICE mRNA expression in pancreatic islet cells and beta-cell lines, independently of NO synthesis, suggesting that ICE up-regulation may be involved in cytokine-induced NO-independent apoptosis of human islets.