Showing papers on "Interferon published in 2012"

Mycobacterial Disease and Impaired IFN-γ Immunity in Humans with Inherited ISG15 Deficiency

Abstract: ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes-granulocyte, in particular-reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity.

DENV inhibits type I IFN production in infected cells by cleaving human STING.

Abstract: Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein

Abstract: Innate sensing of microbial components is well documented to occur at many cellular sites, including at the cell surface, in the cytosol, and in intracellular vesicles, but there is limited evidence of nuclear innate signaling. In this study we have defined the mechanisms of interferon regulatory factor-3 (IRF-3) signaling in primary human foreskin fibroblasts (HFF) infected with herpes simplex virus 1 (HSV-1) in the absence of viral gene expression. We found that the interferon inducible protein 16 (IFI16) DNA sensor, which is required for induction of IRF-3 signaling in these cells, is nuclear, and its localization does not change detectably upon HSV-1 d109 infection and induction of IRF-3 signaling. Consistent with the IFI16 sensor being nuclear, conditions that block viral DNA release from incoming capsids inhibit IRF-3 signaling. An unknown factor must be exported from the nucleus to activate IRF-3 through cytoplasmic STING, which is required for IRF-3 activation and signaling. However, when the viral ICP0 protein is expressed in the nucleus, it causes the nuclear relocalization and degradation of IFI16, inhibiting IRF-3 signaling. Therefore, HSV-1 infection is sensed in HFF by nuclear IFI16 upon release of encapsidated viral DNA into the nucleus, and the viral nuclear ICP0 protein can inhibit the process by targeting IFI16 for degradation. Together these results define a pathway for nuclear innate sensing of HSV DNA by IFI16 in infected HFF and document a mechanism by which a virus can block this nuclear innate response.

Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium

Abstract: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a virulent pathogen that induces rapid host death. Here we observed that host survival after infection with S. Typhimurium was enhanced in the absence of type I interferon signaling, with improved survival of mice deficient in the receptor for type I interferons (Ifnar1(-/-) mice) that was attributed to macrophages. Although there was no impairment in cytokine expression or inflammasome activation in Ifnar1(-/-) macrophages, they were highly resistant to S. Typhimurium-induced cell death. Specific inhibition of the kinase RIP1 or knockdown of the gene encoding the kinase RIP3 prevented the death of wild-type macrophages, which indicated that necroptosis was a mechanism of cell death. Finally, RIP3-deficient macrophages, which cannot undergo necroptosis, had similarly less death and enhanced control of S. Typhimurium in vivo. Thus, we propose that S. Typhimurium induces the production of type I interferon, which drives necroptosis of macrophages and allows them to evade the immune response.

NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Abstract: Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-κB (NF-κB), type I interferon and inflammasome signalling. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis, but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-κB pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens.

The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response

Abstract: The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.

Abstract: Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

Abstract: Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

Bat3 promotes T cell responses and autoimmunity by repressing Tim-3-mediated cell death and exhaustion

Abstract: T cell immunoglobulin and mucin domain–containing 3 (Tim-3) is an inhibitory receptor that is expressed on exhausted T cells during infection with HIV-1 and hepatitis C virus. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms modulating Tim-3 function are not well understood. Here we show that human leukocyte antigen B (HLA-B)-associated transcript 3 (Bat3) binds to, and represses the function of, Tim-3. Bat3 protects T helper type 1 (TH1) cells from galectin-9–mediated cell death and promotes both proliferation and proinflammatory cytokine production. Bat3-deficient T cells have elevated expression of exhaustion-associated molecules such as Tim-3, Lag3, Prdm1 and Pbx3, and Bat3 knockdown in myelin-antigen–specific CD4+ T cells markedly inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3hi, interferon-γ (IFN-γ)loCD4+ cell population. Furthermore, expression of Bat3 is reduced in exhausted Tim-3+ T cells from mouse tumors and HIV-1–infected individuals. These data indicate that Bat3 acts as an inhibitor of Tim-3–dependent exhaustion and cell death. Bat3 may thus represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers.

Interplay between human cytomegalovirus and intrinsic/innate host responses: a complex bidirectional relationship

Abstract: The interaction between human cytomegalovirus (HCMV) and its host is a complex process that begins with viral attachment and entry into host cells, culminating in the development of a specific adaptive response that clears the acute infection but fails to eradicate HCMV. We review the viral and cellular partners that mediate early host responses to HCMV with regard to the interaction between structural components of virions (viral glycoproteins) and cellular receptors (attachment/entry receptors, toll-like receptors, and other nucleic acid sensors) or intrinsic factors (PML, hDaxx, Sp100, viperin, interferon inducible protein 16), the reactions of innate immune cells (antigen presenting cells and natural killer cells), the numerous mechanisms of viral immunoevasion, and the potential exploitation of events that are associated with early phases of virus-host interplay as a therapeutic strategy.

Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling.

Abstract: Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKe, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKe complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.

MK-5172, a Selective Inhibitor of Hepatitis C Virus NS3/4a Protease with Broad Activity across Genotypes and Resistant Variants

Abstract: HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

Molecular regulation of interferon antiviral response in fish

Abstract: Interferon (IFN) response is the first line of host defense against virus infection The recent years have witnessed tremendous progress in understanding of fish IFN antiviral response Varied number of IFN genes has been identified in different fish species but obviously, they do not show a one-to-one orthologous relationship with mammalian IFN homologs These genes are divided into two groups with different abilities to induce downstream gene expression through binding to different receptor complexes Consistently, some fish IFN-stimulated genes such as Mx and PKR have been confirmed for their antiviral effects In this review, we focus on how fish cells respond to IFNs and how fish IFNs are triggered through TLR pathway and RLR pathway We highlight the roles of IRF3 and IRF7 in activation of fish IFN response In addition, the unique mechanisms underlying IRF3/7-dependent fish IFN response and auto-regulation of fish IFN gene expression are discussed

Dengue virus targets the adaptor protein MITA to subvert host innate immunity.

Abstract: Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

NLRP4 negatively regulates type I interferon signaling by targeting the kinase TBK1 for degradation via the ubiquitin ligase DTX4

Abstract: Stringent control of the type I interferon signaling pathway is important for maintaining host immune responses and homeostasis, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. Here we report that the pattern-recognition receptor NLRP4 regulated the activation of type I interferon mediated by double-stranded RNA or DNA by targeting the kinase TBK1 for degradation. NLRP4 recruited the E3 ubiquitin ligase DTX4 to TBK1 for Lys48 (K48)-linked polyubiquitination at Lys670, which led to degradation of TBK1. Knockdown of either DTX4 or NLRP4 abrogated K48-linked ubiquitination and degradation of TBK1 and enhanced the phosphorylation of TBK1 and the transcription factor IRF3. Our results identify a previously unrecognized role for NLRP4 in the regulation of type I interferon signaling and provide molecular insight into the mechanisms by which NLRP4-DTX4 targets TBK1 for degradation.

Beneficial effect of novel proteasome inhibitors in murine lupus via dual inhibition of type I interferon and autoantibody-secreting cells.

Abstract: Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by dysregulation in multiple arms of the immune system and the production of hallmark autoantibodies and α interferon. Because this disease continues to be associated with significant morbidity and mortality, there is great interest in the development of new and targeted treatment approaches and correspondingly a better understanding of disease pathogenesis (1). Although several open-label studies of B cell depletion (BCD) as a targeted treatment have demonstrated clinical benefit (2), only a minority of SLE patients have lasting clinical responses (3, 4). Moreover, the recent failure of two large randomized trials of BCD in SLE (5) highlights the need for other therapeutic strategies. In particular, anti-CD20 has variable impact on autoantibodies that are produced by CD20 negative plasma cells. Decreasing autoantibodies may be particularly critical in SLE where they are often directly pathogenic, e.g. antibody induction of cytopenias and antibodies to DNA or DNA- and RNA-binding proteins forming immune complexes that stimulate α interferon. Bortezomib is a proteasome inhibitor (PI) that effectively kills plasma cells and has demonstrated success in the treatment of multiple myeloma (6-8). Recent promising results suggest that PIs are an effective therapy in a murine model of lupus (9), with relatively selective induction of the unfolded protein response in plasma cells, killing of both long lived and short lived plasma cells, and elimination of autoantibodies. Despite the success of bortezomib in the clinic and in this pre-clinical lupus model, its use in human autoimmune disease is limited because of the development of painful neuropathy in >30% of patients (10). Thus, there is great interest in developing alternative proteasome inhibitors that are less toxic. Carfilzomib is an irreversible proteasome inhibitor that, like bortezomib, primarily inhibits the chymotrypsin-like active sites of the ubiquitously expressed constitutive proteasome and the immunoproteasome, which is found predominantly in immune effector cells (11, 12). In contrast to bortezomib, clinical trials of carfilzomib have shown a low rate of peripheral neuropathy (13). Recently, it has also been shown that the immunoproteasome and not the constitutive proteasome regulates cytokine production in human peripheral blood mononuclear cells (PBMCs) and that selective inhibition of the immunoproteasome with the irreversible inhibitor ONX 0914 (formerly PR-957) is efficacious in both anti-collagen antibody-induced arthritis and collagen-induced arthritis models in mice (14). Given that bortezomib and other PIs have been shown to block activation of myeloid dendritic cells (mDCs) by Toll-like receptor 4 (TLR) in vitro (15) we postulated that pDC activation via TLR signaling, a pathway that is known to be triggered via immune complex (IC) binding and lead to the production of large quantities of IFN-α, may be similarly blocked by the proteasome inhibitors (16-19). Here, we demonstrate for the first time that proteasome inhibitors target both plasmacytoid dendritic cells producing α interferon and plasma cells producing pathogenic antibodies, creating a powerful synergistic effect in the treatment of SLE.

Genome-Wide Expression Profiling Identifies Type 1 Interferon Response Pathways in Active Tuberculosis

Abstract: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/β) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient's blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.