Identification of Host Cytosolic Sensors and Bacterial Factors Regulating the Type I Interferon Response to Legionella pneumophila

Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires'' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN) in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.     

Type I IFN as a natural adjuvant for a protective immune response: lessons from the influenza vaccine model

The identification of natural adjuvants capable of selectively promoting an efficient immune response against infectious agents would represent an important advance in immunology, with direct implications for vaccine development, whose progress is generally hampered by the difficulties in defining powerful synthetic adjuvants suitable for clinical use. Here, we demonstrate that endogenous type I IFN is necessary for the Th1 type of immune response induced by typical adjuvants in mice and that IFN itself is an unexpectedly powerful adjuvant when administered with the human influenza vaccine, for inducing IgG2a and IgA production and conferring protection from virus challenge. The finding that these cytokines, currently used in patients, are necessary for full expression of adjuvant activity and are sufficient for the generation of a protective immune response opens new perspectives in understanding the basis of immunity and in vaccine development.     

Phenotypic Characterization of Porcine IFNγ-Producing Lymphocytes in Porcine Reproductive and Respiratory Syndrome Virus Vaccinated and Challenged Pigs

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most important swine diseases worldwide. Interferon-γ (IFNγ)-mediated type Ⅰ cell-mediated immune response plays an important role in protection from, and clearance of, PRRS virus (PRRSV). Several lymphocyte subsets including T-helper, CTLs, Th/memory cells, and γδ T lymphocytes were previously reported to produce IFNγ during PRRSV infection. However, the proportion and phenotypic characterization of these IFNγ-secreting lymphocytes have not been explored. In this study, IFNγ producted by different lymphocyte subsets was assessed by multi-color flow cytometry after vaccination with PRRSV modified live vaccine (PRRSV-MLV) and challenge with homogeneous or heterogeneous PRRSV. The results showed that T-helper cells were the major IFNγ-secreting cell population after PRRSV-MLV vaccination and PRRSV challenge. Additionally, the proportion of IFNγ producing Th/memory cells and γδ T cells increased after PRRSV challenge. This difference was accounted for an enhanced ability to produce IFNγ in Th/memory cells and an enlarged quantity of γδ T cells. The results presented here could contribute to our understanding of the roles of IFNγ in protective immunity against PRRSV infection and may be useful for assessment of cell-mediated immunity in vaccine tests.     

Differential regulation of IFNα, IFNβ and IFNε gene expression in human cervical epithelial cells

Interferonε (IFNε) is a unique type I IFN that has distinct functions from IFNα/β. IFNε is constitutively expressed at mucosal tissues, including the female genital mucosa, and is reported to be modulated by estrogen and seminal plasma. However, its regulation by cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-17, IL-22 and IFNα, which are commonly present in the female genital mucosa, is not well documented in freshly isolated primary cervical cells from tissues. We determined the effect of these cytokines on gene expression of type I IFNs in an immortalized endocervical epithelial cell line (A2EN) and in primary cervical epithelial cells. Several pro-inflammatory cytokines were found to induce IFNε, and TNFα induced the strongest response in both cell types. Pretreatment of cells with the IκB inhibitor, which blocks the NF-κB pathway, suppressed TNFα-mediated IFNε gene induction and promoter activation. Expression of IFNα, IFNβ, and IFNε was differentially regulated in response to various cytokines. Taken together, our results show that regulation of these IFNs depends on cell type, cytokine concentration, and incubation time, highlighting the complexity of the cytokine network in the cervical epithelium.     

Cell type-dependent requirement of autophagy in HSV-1 antiviral defense

Type I interferons (IFNs) are induced during most viral infections and are considered to be the primary and universal means of innate viral control. However, several other innate mechanisms, including autophagy, have recently been shown to play an important role in antiviral defense. In our recent study, we utilized a herpes simplex virus 1 (HSV-1) infection model to investigate the relationship between cell type and innate antiviral immune mechanisms. Our study demonstrates that dorsal root ganglion (DRG) neurons undergo an innate antiviral response to HSV-1 that differs from the antiviral program induced in mitotic cells in three distinct ways. First, DRG neurons produce less type I IFN and undergo a less effective IFN antiviral program vs. mitotic cells in response to HSV-1 infection. Second, the type I IFN program initiated in DRG neurons induces less cell death than in mitotic cells. Third, in the absence of a robust type I IFN response, DRG neurons, but not mitotic cells, repy on autophagy in HSV-1 defense. Our findings reveal a cell type-specific requirement for autophagy in defense against HSV-1, and offer insight into the cell-appropriate antiviral defense mechanism employed by neurons.     

Expression signature of IFN/STAT1 signaling genes predicts poor survival outcome in glioblastoma multiforme in a subtype-specific manner

Previous reports have implicated an induction of genes in IFN/STAT1 (Interferon/STAT1) signaling in radiation resistant and prosurvival tumor phenotypes in a number of cancer cell lines, and we have hypothesized that upregulation of these genes may be predictive of poor survival outcome and/or treatment response in Glioblastoma Multiforme (GBM) patients. We have developed a list of 8 genes related to IFN/STAT1 that we hypothesize to be predictive of poor survival in GBM patients. Our working hypothesis that over-expression of this gene signature predicts poor survival outcome in GBM patients was confirmed, and in addition, it was demonstrated that the survival model was highly subtype-dependent, with strong dependence in the Proneural subtype and no detected dependence in the Classical and Mesenchymal subtypes. We developed a specific multi-gene survival model for the Proneural subtype in the TCGA (the Cancer Genome Atlas) discovery set which we have validated in the TCGA validation set. In addition, we have performed network analysis in the form of Bayesian Network discovery and Ingenuity Pathway Analysis to further dissect the underlying biology of this gene signature in the etiology of GBM. We theorize that the strong predictive value of the IFN/STAT1 gene signature in the Proneural subtype may be due to chemotherapy and/or radiation resistance induced through prolonged constitutive signaling of these genes during the course of the illness. The results of this study have implications both for better prediction models for survival outcome in GBM and for improved understanding of the underlying subtype-specific molecular mechanisms for GBM tumor progression and treatment response.     

Stimulation of immature lung macrophages with intranasal interferon gamma in a novel neonatal mouse model of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral death in infants. Reduced CD8 T-cells and negligible interferon gamma (IFNγ) in the airway are associated with severe infant RSV disease, yet there is an abundance of alveolar macrophages (AM) and neutrophils. However, it is unclear, based on our current understanding of macrophage functional heterogeneity, if immature AM improve viral clearance or contribute to inflammation and airway obstruction in the IFNγ-deficient neonatal lung environment. The aim of the current study was to define the age-dependent AM phenotype during neonatal RSV infection and investigate their differentiation to classically activated macrophages (CAM) using i.n. IFNγ in the context of improving viral clearance. Neonatal and adult BALB/cJ mice were infected with 1×10(6) plaque forming units (PFU)/gram (g) RSV line 19 and their AM responses compared. Adult mice showed a rapid and robust CAM response, indicated by increases in major histocompatibility complex class II (MHC II), CD86, CCR7, and a reduction in mannose receptor (MR). Neonatal mice showed a delayed and reduced CAM response, likely due to undetectable IFNγ production. Intranasal (i.n.) treatment with recombinant mouse IFNγ (rIFNγ) increased the expression of CAM markers on neonatal AM, reduced viral lung titers, and improved weight gain compared to untreated controls with no detectable increase in CD4 or CD8 T-cell infiltration. In vitro infection of J774A.1 macrophages with RSV induced an alternatively activated macrophage (AAM) phenotype however, when macrophages were first primed with IFNγ, a CAM phenotype was induced and RSV spread to adjacent Hep-2 cells was reduced. These studies demonstrate that the neonatal AM response to RSV infection is abundant and immature, but can be exogenously stimulated to express the antimicrobial phenotype, CAM, with i.n. rIFNγ.     

Interferon gamma-induced EGF receptor transactivation depends on the level of expression of EGF receptor in epithelial cells

Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to interferon γ (IFNγ) in epidermoid carcinoma A431 cells. It was shown that IFNγ-induced EGFR transactivation is impossible in the some cancer epithelial cells. Here, we hypothesize that IFNγ-dependent EGFR transactivation in these cells correlates to the amount of EGFR on the surface of the cell. To test this suggestion, a line of stably transfected HEK293 cells (HEK293Δ99 cells) expressing a high level of mutant EGFR that lacked 99 C-terminal residues was obtained. Unlike the parent HEK293 cells, which lacked transactivation, HEK293Δ99 cells demonstrated EGFR transactivation in response to IFNγ. In HEK293Δ99 and A431 cells, the time courses of EGFR activation induced by IFNγ have the same pattern. In HEK293Δ99, as in A431 cells, IFNγ-induced EGFR transactivation requires EGFR kinase activity and occurs via an autophosphorylation mechanism. Taken together, these data provide direct evidence for the dependence of IFNγ-induced EGFR transactivation upon EGFR expression level in epithelial cells.     

Putting the brakes on the anti-viral response: negative regulators of type I interferon (IFN) production

Type I IFNs (IFNα/β) are essential anti-viral cytokines produced in response to the detection of viral components by host pattern recognition receptors. IFNα/β production is transient, and aberrant activation can be hazardous to the host. In this article, we review our current understanding of host negative regulatory mechanisms that control IFNα/β production.     

RIG-I, MDA5 and TLR3 synergistically play an important role in restriction of dengue virus infection

Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus.     

Proteomic analysis of the autoantibody response following immunization with a single autoantigen

In most autoimmune diseases, the autoantibody response is directed against several antigens of the target organ whose identification is crucial for understanding the physiopathological process. Thus, technologies allowing a characterization of the whole autoantibody pattern of both human and experimental autoimmune diseases are required. Here we have used immunoproteomic analysis of human epidermal extracts to characterize the diversity of the anti-desmosome antibody response induced in normal mice immunized with desmoglein 1, the major autoantigen of pemphigus foliaceus, an autoimmune blistering skin disease. In particular, this analysis enables us to characterize the binding properties of anti-desmosome mAbs derived from these mice and to show that the autoantibody response induced upon immunization with a single autoantigen targets different epidermal autoantigens with a pattern similar to that observed in certain variety of human pemphigus.     

The effect of febrile temperatures on biologic actions of interferons: abrogation of suppression of delayed-type hypersensitivity and antibody production

Interferons (IFN) have a complex immunoregulatory effect on all cells of the immune system. In most cases in which IFN had an enhancing effect, the suggested mechanism was inhibition of the generation or activity of suppressor cells. In the present study, we examined the effect of IFN on suppression of the delayed-type hypersensitivity (DTH) response. Suppression was induced with a low antigen dose of sheep erythrocytes (SRBC), and IFN was found to abrogate both the suppressed state and the transferability of this state. Cyclophosphamide had the same effect. However, the in vitro generation of suppressor cells was not altered by the addition of IFN to the culture medium at a normal temperature (37 degrees C). To reconcile the disparity between the successful anti-suppressive action of IFN in vivo compared with its failure in vitro, we considered the possibility that the pyrogenic action of IFN in vivo might create the optimal thermal environment for its anti-suppressive action. Indeed, when IFN was then tested in vitro at a febrile temperature (39.3 degrees C), it completely blocked the generation of suppressor cells. On the other hand, once suppressor cells were generated at 37 degrees C, IFN had no effect on their ability to suppress a fresh culture either at 37 degrees C or at 39.3 degrees C. IFN also had no effect on the generation of helper cells at either temperature, but help was greatly enhanced by high temperature alone. In vivo, we found our IFN preparation to be pyrogenic and observed that an anti-pyretic drug given before and during antigen stimulation abrogated the anti-suppressive effect of IFN. We suggest, therefore, that the febrile state induced by IFN promotes its action on suppressor cells.     

Protection by polyI:polyC against infection with herpes simplex virus in mice pretreated with Corynebacterium parvum

The effect of Corynebacterium parvum on the protection by polyinosinic-polycytidylic acid (polyI:polyC) against lethal infection with Herpes simplex virus type 1 (HSV) was studied in mice. Pretreatment with C. parvum resulted in prolonged survival times in all experiments. One third of the mice survived an infection with 100 LD50, whereas all mice died when treated with polyI:polyC alone. Increased protection was observed up to 6 weeks after pretreatment and only seen when both C. parvum and polyI:polyC were given at the same site of injection (intraperitoneally). Protection against HSV correlated with increased interferon (IFN) activities induced by polyI:polyC in the peritoneal cavity of C. parvum-pretreated mice. In these mice, natural killer cell activity of peritoneal exudate cells (PEC) was also augmented in response to polyI:polyC. Protection was markedly decreased by intraperitoneal injection of silica or of an antiserum against murine IFN. It appears that increased local levels of IFN presumably produced by macrophages in response to polyI:polyC in C. parvum-pretreated mice play the major role in the antiviral defence in our model and that activation of NK cells may be a secondary effect of IFN.     

Sphingosine analogue AAL-R increases TLR7-mediated dendritic cell responses via p38 and type I IFN signaling pathways

Sphingosine analogues display immunosuppressive activities and thus have therapeutic potential in the treatment of autoimmune diseases. In this study, we investigated the effects of the sphingosine analogue AAL-R (FTY720 derivative) on dendritic cell (DC) response upon TLR stimulation. Unlike its known immunosuppressive activity, AAL-R increased TLR7-mediated DC responses by elevating the levels of MHC class I and costimulatory molecules and type I IFN expression and by enhancing the capacity of DCs to induce CD8(+) T cell proliferation. Importantly, the stimulatory activity of AAL-R was dependent on type I IFN signaling, as type I IFN receptor-deficient DCs failed to respond to AAL-R. Also, AAL-R activated p38 MAPK to increase type I IFN synthesis and TLR7-mediated DC maturation. These findings enhance our understanding of sphingosine regulation of the host immune system, in particular upon pathogenic infections.