Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Toll-Like Receptor- and Protein Kinase R-Induced Type I Interferon Sustains Infection of Leishmania donovani in Macrophages.

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-ß). Here, we show that the gene expression of IFN-ß by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2-/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2-/- macrophages completely abolished induction of IFN-ß gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2-/-) or from protein kinase R (PKR) knock-out mice (pkr-/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr-/- macrophages but was fully restored by the addition of exogenous IFN-ß, and parasite burdens were reduced in the spleen of pkr-/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.

PARP inhibitor niraparib as a radiosensitizer promotes antitumor immunity of radiotherapy in EGFR-mutated non-small cell lung cancer.

BACKGROUND: Poly-(ADP-Ribose)-Polymerase inhibitors (PARPi) were reported as radiosensitizers in non-small cell lung cancer (NSCLC) with wide-type epidermal growth factor receptor (EGFR), but the effects of radiation combined with PARPi were not investigated in EGFR-mutated NSCLC. Moreover, the underlying mechanisms were not well examined. This study aimed to study the efficacy of radiation combined with niraparib in EGFR-mutated NSCLC and explore their influence on the immune system. METHODS: Clone formation and apoptosis assay were conducted to explore the effects of niraparib and radiation. Immunofluorescence was conducted to detect the double-strand DNA breaks. Real-time PCR and immunoblotting were employed to evaluate the activation of STING/TBK1/TRF3 pathway and the expression levels of interferon ß, CCL5 and CXCL10. Immunocompetent mice model bearing with subcutaneous Lewis lung cancer was established to confirm the results in vivo. RESULTS: Niraparib and radiation were synergistic to inhibit tumor both in vitro and in vivo. Radiation plus niraparib could activate anti-tumor immunity, which appeared as increased CD8+ T lymphocytes and activated STING/TBK1/IRF3 pathway. CONCLUSION: PARPi not only as a radiosensitizer inhibited EGFR-mutated NSCLC tumor growth, but also cooperated with radiation to promote anti-tumor immune responses.

Intranasal delivery of interferon-ß-loaded nanoparticles induces control of neuroinflammation in a preclinical model of multiple sclerosis: A promising simple, effective, non-invasive, and low-cost therapy.

Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS). Interferon (IFN)-ß constitutes one of the first-line therapies to treat MS, but has limited efficacy due to the injectable systemic administration, short half-life, and limited CNS access. To address these limitations, we developed IFN-ß-loaded chitosan/sulfobutylether-ß-cyclodextrin nanoparticles (IFN-ß-NPs) for delivery of IFN-ß into the CNS via the intranasal (i.n.) route. The nanoparticles (NPs) (≈200 nm, polydispersity ≈0.1, and zeta potential ≈20 mV) were prepared by mixing two aqueous solutions and associated human or murine IFN-ß with high efficiency (90%). Functional in vitro assays showed that IFN-ß-NPs were safe and that IFN-ß was steadily released while retaining biological activity. Biodistribution analysis showed an early and high fluorescence in the brain after nasal administration of fluorescent probe-loaded NPs. Remarkably, mice developing experimental autoimmune encephalomyelitis (EAE), an experimental model of MS, exhibited a significant improvement of clinical symptoms in response to intranasal IFN-ß-NPs (inIFN-ß-NPs), whereas a similar dose of intranasal or systemic free IFN-ß had no effect. Importantly, inIFN-ß-NPs treatment was equally effective despite a reduction of 78% in the total amount of weekly administered IFN-ß. Spinal cords obtained from inIFN-ß-NPs-treated EAE mice showed fewer inflammatory foci and demyelination, lower expression of antigen-presenting and costimulatory proteins on CD11b+ cells, and lower astrocyte and microglia activation than control mice. Therefore, IFN-ß treatment at tested doses was effective in promoting clinical recovery and control of neuroinflammation in EAE only when associated with NPs. Overall, inIFN-ß-NPs represent a potential, effective, non-invasive, and low-cost therapy for MS.

In cardiac fibroblasts, interferon-beta attenuates differentiation, collagen synthesis, and TGF-ß1-induced collagen gel contraction.

Cardiac fibroblasts (CF) play a key role in the homeostasis of the extracellular matrix in cardiac tissue and are newly recognized as inflammatory supporter cells. Besides, CF-to-Cardiac myofibroblast differentiation is commanded by TGF-b, through SMAD signaling pathways, and these last cells are strongly implicated in cardiac fibrosis. In the heart IFN-ß is produced by CF; however, the role of IFN-ß, STAT proteins, and STAT-homo or heterodimers in the regulation of CF function with or without a fibrotic environment is unknown. CF were isolated from hearts of adult rats, and by western blot analysis we studied STAT1, STAT2, and STAT3 phosphorylation and through specific siRNA against these proteins we analyzed their role in CF functions such as differentiation (α-SMA expression); and pro-collagen type-I synthesis and secretion expression levels; collagen gels contraction and CF migration. In cultured adult rats CF, IFN-ß increases phosphorylation of STAT1, STAT2, and STAT3. Both STAT1 and STAT2 were involved in decreasing α-SMA and CF migration induced by TGF-ß1. Also, IFN-ß through STAT1 regulated pro-collagen type-I protein expression levels, and collagen gels contraction induced by TGF-ß1. STAT3 was not involved in any effects of IFN-ß studied. In conclusion, IFN-ß through STAT1 and STAT2 shows antifibrotic effects on CF TGF-ß1-treated, whereas STAT3 did not participate in such effect.

p19Arf sensitizes B16 melanoma cells to interferon-ß delivered via mesenchymal stem cells in vitro.

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-ß (IFNß) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNß was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNß production. MSCs used to produce IFNß inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNß produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNß and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

p19Arf sensitizes B16 melanoma cells to interferon-β delivered via mesenchymal stem cells in vitro

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-ß production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela-/-), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased NO and decreased TGF-ß production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN-ß mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-ß. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-ß necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.

Amazonian Phlebovirus (Bunyaviridae) potentiates the infection of Leishmania (Leishmania) amazonensis: Role of the PKR/IFN1/IL-10 axis.

BACKGROUND: Leishmania parasites are transmitted to vertebrate hosts by phlebotomine sandflies and, in humans, may cause tegumentary or visceral leishmaniasis. The role of PKR (dsRNA activated kinase) and Toll-like receptor 3 (TLR3) activation in the control of Leishmania infection highlights the importance of the engagement of RNA sensors, which are usually involved in the antiviral cell response, in the fate of parasitism by Leishmania. We tested the hypothesis that Phlebovirus, a subgroup of the Bunyaviridae, transmitted by sandflies, would interfere with Leishmania infection. METHODOLOGY/PRINCIPAL FINDINGS: We tested two Phlebovirus isolates, Icoaraci and Pacui, from the rodents Nectomys sp. and Oryzomys sp., respectively, both natural sylvatic reservoir of Leishmania (Leishmania) amazonensis from the Amazon region. Phlebovirus coinfection with L. (L.) amazonensis in murine macrophages led to increased intracellular growth of L. (L.) amazonensis. Further studies with Icoaraci coinfection revealed the requirement of the PKR/IFN1 axis on the exacerbation of the parasite infection. L. (L.) amazonensis and Phlebovirus coinfection potentiated PKR activation and synergistically induced the expression of IFNß and IL-10. Importantly, in vivo coinfection of C57BL/6 mice corroborated the in vitro data. The exacerbation effect of RNA virus on parasite infection may be specific because coinfection with dengue virus (DENV2) exerted the opposite effect on parasite load. CONCLUSIONS: Altogether, our data suggest that coinfections with specific RNA viruses shared by vectors or reservoirs of Leishmania may enhance and sustain the activation of host cellular RNA sensors, resulting in aggravation of the parasite infection. The present work highlights new perspectives for the investigation of antiviral pathways as important modulators of protozoan infections.

The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide dampens lipopolysaccharide-induced transcriptomic changes in macrophages.

OBJECTIVE: M1-like inflammatory phenotype of macrophages plays a critical role in tissue damage in chronic inflammatory diseases. Previously, we found that the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) dampens lipopolysaccharide (LPS)-triggered inflammatory priming of RAW 264.7 cells. Herein, we tested whether DMPO by itself can induce changes in macrophage transcriptome, and that these effects may prevent LPS-induced activation of macrophages. MATERIALS AND METHODS: To test our hypothesis, we performed a transcriptomic and bioinformatics analysis in RAW 264.7 cells incubated with or without LPS, in the presence or in the absence of DMPO. RESULTS: Functional data analysis showed 79 differentially expressed genes (DEGs) when comparing DMPO vs Control. We used DAVID databases for identifying enriched gene ontology terms and Ingenuity Pathway Analysis for functional analysis. Our data showed that DMPO vs Control comparison of DEGs is related to downregulation immune-system processes among others. Functional analysis indicated that interferon-response factor 7 and toll-like receptor were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses of the DMPO + LPS vs LPS DEGs were consistent with DMPO-dampening LPS-induced inflammatory transcriptomic profile in RAW 264.7. These changes were confirmed using Nanostring technology. CONCLUSIONS: Taking together our data, surprisingly, indicate that DMPO by itself affects gene expression related to regulation of immune system and that DMPO dampens LPS-triggered MyD88- and TRIF-dependent signaling pathways. Our research provides critical data for further studies on the possible use of DMPO as a structural platform for the design of novel mechanism-based anti-inflammatory drugs.

Protective Effects of Moringa oleifera on HBV Genotypes C and H Transiently Transfected Huh7 Cells.

Chronic hepatitis B infection treatment implicates a long-lasting treatment. M. oleifera extracts contain compounds with antiviral, antioxidant, and antifibrotic properties. In this study, the effect of M. oleifera was evaluated in Huh7 cells expressing either HBV genotypes C or H for the antiviral, antifibrotic, anti-inflammatory, and antioxidative responses. Huh7 cells were treated with an aqueous extract of M. oleifera (leaves) at doses of 0, 30, 45, or 60 µg/mL. The replicative virus and TGF-ß1, CTGF, CAT, IFN-ß1, and pgRNA expressions were measured by real time. HBsAg and IL-6 titers were determined by ELISA. CTGF, TGF-ß1, IFN-ß1, and pgRNA expressions decreased with M. oleifera treatment irrespective of the HBV genotype. HBsAg secretion in the supernatant of transfected Huh7 cells with both HBV genotypes was decreased regardless of the dose of M. oleifera. Similar effect was observed in proinflammatory cytokine IL-6, which had a tendency to decrease at 24 hours of treatment. Transfection with both HBV genotypes strongly decreased CAT expression, which is retrieved with M. oleifera treatment. M. oleifera treatment reduced fibrosis markers, IL-6, and HBsAg secretion in HBV genotypes C and H. However, at the level of replication, only HBV-DNA genotype C was slightly reduced with this treatment.

Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-α/ß signaling.

Mosquito-transmitted flavivirus Rocio (ROCV) was responsible for an outbreak of encephalitis in the Ribeira Valley, located in the south coast of Sao Paulo State, Brazil, in 1975-1976. ROCV also causes fatal encephalitis in adult mice. Seroprevalence studies in humans, horses and water buffaloes in different regions of Brazil have suggested that ROCV is still circulating in the country, indicating the risk of re-emergence of this virus. West Nile virus (WNV) is also a mosquito-transmitted encephalitic flavivirus, however, WNV strains circulating in Australia have not been associated with outbreaks of disease in humans and exhibit low virulence in adult mice. To identify viral determinants of ROCV virulence, we have generated reciprocal chimeric viruses between ROCV and the Australian strain of WNV by swapping structural prM and E genes. Chimeric WNV containing ROCV prM-E genes replicated more efficiently than WNV or chimeric ROCV containing WNV prM-E genes in mammalian cells, was as virulent as ROCV in adult mice, and inhibited type I IFN signaling as efficiently as ROCV. The results show that ROCV prM and E proteins are major virulence determinants and identify unexpected function of these proteins in inhibition of type I interferon response.

G45R mutation in the nonstructural protein 1 of A/Puerto Rico/8/1934 (H1N1) enhances viral replication independent of dsRNA-binding activity and type I interferon biology.

BACKGROUND: The nonstructural protein 1 (NS1) of influenza A viruses can act as a viral replication enhancer by antagonizing type I interferon (IFN) induction and response in infected cells. We previously reported that A/Puerto Rico/8/1934 (H1N1) (PR8) containing the NS1 gene derived from A/swine/IA/15/1930 (H1N1) (IA30) replicated more efficiently than the wild type virus. Here, we identified amino acids in NS1 critical for enhancing viral replication. METHODS: To identify a key amino acid in NS1 which can increase the virus replication, growth kinetics of PR8 viruses encoding single mutation in NS1 were compared in A549 cells. NS1 mutant functions were studied using dsRNA-protein pull down, RIG-I mediated IFNß-promoter activity assays and growth curve analysis in murine lung epithelial type I (Let1) cells. RESULTS: The G45R mutation in the NS1 of PR8 (G45R/NS1) virus is critical for the enhanced viral replication in A549 cells. G45R/NS1 slightly decreased NS1 binding to dsRNA but did not interfere with its suppression of RIG-I-mediated type I IFN production. Likewise, replication of G45R/NS1 virus was increased in comparison to wild type virus in both wild type and type I interferon receptor null Let1 cells. CONCLUSIONS: The non-conserved amino acid, R45, enhances viral replication which is apparently independent of dsRNA binding and suppression of type I IFN, suggesting a non-characterized function of NS1 for the enhanced viral replication. As G45R/NS1 virus induced the type I IFN induction and response in infected A549 cells, it is also interesting to investigate virus virulence for further studies.

Antiviral factors and type I/III interferon expression associated with regulatory factors in the oral epithelial cells from HIV-1-serodiscordant couples.

Individuals who remain HIV-seronegative despite repeated unprotected exposure to the virus are defined as exposed seronegative (ESN) individuals. Innate and adaptive immunity, as well as genetic factors, provide ESNs with important advantages that allow for low infection susceptibility. The majority of HIV-1-infected individuals undergo antiretroviral therapy, which can decrease the level of HIV-1 exposure in ESNs. We analyzed type I interferon (IFN)-related antiviral and regulatory factors in peripheral blood mononuclear cells (PBMCs) and oral epithelial cells from serodiscordant couples. Our findings revealed that ESNs did not induce the expression of antiviral factors (APOBEC-3G, TRIM5-α, SAMDH1, STING, TBk1) or regulatory factors (Trex, Foxo3, Socs3, IL-10) in PBMCs, unlike their HIV-1-infected partners. In contrast, ESNs upregulated APOBEC-3G and type I/III IFNs (IFNs-α,-ß/-λ) in oral mucosal epithelial cells similar to their HIV-infected partners. The serodiscordant groups exhibited an increased expression of type I IFN-induced regulators, such as Trex and Foxo3, in oral epithelial cells. TLR7, TLR8 and TLR9 were expressed in oral epithelial cells of both ESNs and HIV-1-infected subjects. These findings revealed evidence of antiviral factors, type I/III interferon and regulatory factor expression only in the oral mucosal compartment of ESNs, while HIV-1-infected partners systemically and oral mucosal expressed the antiviral profile.

Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder.

Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage "Protein and nucleic acid structure and sequence analysis".

RNA helicase DDX3: at the crossroad of viral replication and antiviral immunity.

Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3, or DDX3, belongs to the DEAD-box family of ATP-dependent RNA helicases and is known to play different roles in RNA metabolism ranging from transcription to nuclear export, translation, and assembly of stress granules. In addition, there is growing evidence that DDX3 is a component of the innate immune response against viral infections. As such, DDX3 has been shown to play roles both upstream and downstream of I-kappa beta kinase ε (IKKε)/TANK-binding kinase 1, leading to IFN-ß production. Interestingly, several RNA viruses, including human threats such as HIV-1 and hepatitis C virus, hijack DDX3 to accomplish various steps of their replication cycles. Thus, it seems that viruses have evolved to exploit DDX3's functions while threatening the innate immune response. Understanding this interesting dichotomy in DDX3 function will help us not only to improve our knowledge of virus-host interactions but also to develop novel antiviral drugs targeting the multifaceted roles of DDX3 in viral replication.

Modulating APOBEC expression enhances DNA vaccine immunogenicity.

DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA (dsDNA) sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC (apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide)-mediated plasmid degradation. We showed that plasmid sensing via STING (stimulator of interferon (IFN) genes) and TBK-1 (TANK-binding kinase 1) leads to IFN-ß induction, which results in APOBEC3A mRNA upregulation through a mechanism involving protein kinase C signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to a 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and a 2.6-fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in dsDNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.

Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses.

To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity of rPR8-NS(0028) might be associated with the lower virus titer and IFN-ß level in the lungs of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028. In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous monitoring of their mammalian pathogenicity.

Human endogenous retrovirus expression is inversely related with the up-regulation of interferon-inducible genes in the skin of patients with lichen planus.

Lichen planus (LP) is a common inflammatory skin disease of unknown etiology. Reports of a common transactivation of quiescent human endogenous retroviruses (HERVs) support the connection of viruses to the disease. HERVs are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancer and autoimmune diseases. We explored the transcriptional activity of HERV sequences as well as the antiviral restriction factor and interferon-inducible genes in the skin from LP patients and healthy control (HC) donors. The study included 13 skin biopsies from patients with LP and 12 controls. Real-time PCR assay identified significant decrease in the HERV-K gag and env mRNA expression levels in LP subjects, when compared to control group. The expressions of HERV-K18 and HERV-W env were also inhibited in the skin of LP patients. We observed a strong correlation between HERV-K gag with other HERV sequences, regardless the down-modulation of transcripts levels in LP group. In contrast, a significant up-regulation of the cytidine deaminase APOBEC 3G (apolipoprotein B mRNA-editing), and the GTPase MxA (Myxovirus resistance A) mRNA expression level was identified in the LP skin specimens. Other transcript expressions, such as the master regulator of type I interferon-dependent immune responses, STING (stimulator of interferon genes) and IRF-7 (interferon regulatory factor 7), IFN-ß and the inflammassome NALP3, had increased levels in LP, when compared to HC group. Our study suggests that interferon-inducible factors, in addition to their role in innate immunity against exogenous pathogens, contribute to the immune control of HERVs. Evaluation of the balance between HERV and interferon-inducible factor expression could possibly contribute to surveillance of inflammatory/malignant status of skin diseases.

Role of CXCR3/CXCL10 axis in immune cell recruitment into the small intestine in celiac disease.

Lymphocytic infiltration in the lamina propria (LP), which is primarily composed of CD4(+) Th1 cells and plasma cells, and increased numbers of intraepithelial lymphocytes (IELs), is a characteristic finding in active celiac disease (CD). Signals for this selective cell recruitment have not been fully established. CXCR3 and its ligands, particularly CXCL10, have been suggested to be one of the most relevant pathways in the attraction of cells into inflamed tissues. In addition, CXCR3 is characteristically expressed by Th1 cells. The aim of this work was to investigate the participation of the chemokine CXCL10/CXCR3 axis in CD pathogenesis. A higher concentration of CXCL10 was found in the serum of untreated CD patients. The mRNA levels of CXCL10 and CXCL11 but not CXCL9 were significantly higher in duodenal biopsies from untreated CD patients compared with non-CD controls or treated patients. The results demonstrate that CXCL10 is abundantly produced in untreated CD and reduced in treated patients, and the expression of CXCL10 was found to be correlated with the IFNγ levels in the tissue. Plasma cells and enterocytes were identified as CXCL10-producing cells. Moreover, the CXCL10 expression in intestinal tissues was upregulated by poly I:C and IL-15. IELs, LP T lymphocytes, and plasma cells, which infiltrate the intestinal mucosa in untreated CD, express CXCR3. The CXCR3/CXCL10 signalling axis is overactivated in the small intestinal mucosa in untreated patients, and this finding explains the specific recruitment of the major cell populations that infiltrate the epithelium and the LP in CD.