Targeted Transcriptomics of Frog Virus 3 in Infected Frog Tissues Reveal Non-Coding Regulatory Elements and microRNAs in the Ranaviral Genome and Their Potential Interaction with Host Immune Response.

Background: Frog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae. Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions. Results: Using a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis-regulatory elements (CREs) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3'-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs). Conclusions: Using the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response.

Kidney Subcapsular Allograft Transplants as a Model to Test Virus-Derived Chemokine-Modulating Proteins as Therapeutics.

Solid tissue transplant is a growing medical need that is further complicated by a limited donor organ supply. Acute and chronic rejection occurs in nearly all transplants and reduces long-term graft survival, thus increasing the need for repeat transplantation. Viruses have evolved highly adapted responses designed to evade the host's immune defenses. Immunomodulatory proteins derived from viruses represent a novel class of potential therapeutics that are under investigation as biologics to attenuate immune-mediated rejection and damage. These immune-modulating proteins have the potential to reduce the need for traditional posttransplant immune suppressants and improve graft survival. The myxoma virus-derived protein M-T7 is a promising biologic that targets chemokine and glycosaminoglycan pathways central to kidney transplant rejection. Orthotopic transplantations in mice are prohibitively difficult and costly and require a highly trained microsurgeon to successfully perform the procedure. Here we describe a kidney-to-kidney subcapsular transplant model as a practical and simple method for studying transplant rejection, a model that requires fewer mice. One kidney can be used as a donor for transplants into six or more recipient mice. Using this model there is lower morbidity, pain, and mortality for the mice. Subcapsular kidney transplantation provides a first step approach to testing virus-derived proteins as new potential immune-modulating therapeutics to reduce transplant rejection and inflammation.

IFN-γ regulates the transformation of microglia into dendritic-like cells via the ERK/c-myc signaling pathway during cerebral ischemia/reperfusion in mice.

Cerebral ischemia-reperfusion injury induces a secondary immune inflammatory reaction that exacerbates brain injury and clinical prognosis. Dendritic cells (DCs) and microglia are both important regulators of neuroinflammation. Studies have confirmed that a large number of cells express the DC surface marker CD11c in the ischemic area, and some of these cells also express microglial markers. However, the specific mechanism of transformation between microglia and DCs and their roles in the process of cerebral ischemia-reperfusion injury are still not clear. In this study, we established a mouse model and flow cytometry was used to detect the expression of mature DC surface molecules in activated microglia. IFN-γ knockout mice were used to determine the regulatory effect of IFN-γ on microglial transformation. We found that CD11c+ cells were derived from microglia after ischemia-reperfusion injury, and this group of cells highly expressed MHC-II molecules and other costimulatory molecules, such as CD80 and CD86, which were regulated by IFN-γ and its downstream signaling molecules ERK/c-myc. In summary, our results showed in cerebral ischemia-reperfusion injury, IFN-γ regulates the transformation of microglia to DC-like cells. Microglial-derived DC-like cells possess the ability to present antigens and activate naïve T cells which is regulated by the ERK/c-myc signaling pathway.

Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells.

Type III interferons (IFN-lambdas(λ)) are important cytokines that inhibit viruses and modulate immune responses by acting through a unique IFN-λR1/IL-10RB heterodimeric receptor. Until now, the primary antiviral function of IFN-λs has been proposed to be at anatomical barrier sites. Here, we examine the regulation of IFN-λR1 expression and measure the downstream effects of IFN-λ3 stimulation in primary human blood immune cells, compared with lung or liver epithelial cells. IFN-λ3 directly bound and upregulated IFN-stimulated gene (ISG) expression in freshly purified human B cells and CD8+ T cells, but not monocytes, neutrophils, natural killer cells, and CD4+ T cells. Despite similar IFNLR1 transcript levels in B cells and lung epithelial cells, lung epithelial cells bound more IFN-λ3, which resulted in a 50-fold greater ISG induction when compared to B cells. The reduced response of B cells could be explained by higher expression of the soluble variant of IFN-λR1 (sIFN-λR1), which significantly reduced ISG induction when added with IFN-λ3 to peripheral blood mononuclear cells or liver epithelial cells. T-cell receptor stimulation potently, and specifically, upregulated membrane-bound IFNLR1 expression in CD4+ T cells, leading to greater antiviral gene induction, and inhibition of human immunodeficiency virus type 1 infection. Collectively, our data demonstrate IFN-λ3 directly interacts with the human adaptive immune system, unlike what has been previously shown in published mouse models, and that type III IFNs could be potentially utilized to suppress both mucosal and blood-borne viral infections.

Analysis of interferon-γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal-conceptus interface and the role of interferon-γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs.

It has long been known that pig conceptuses produce interferon-γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal-conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin-1ß did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy- and cell-type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.

Variability of interferon-λ induction and antiviral activity in Nipah virus infected differentiated human bronchial epithelial cells of two human donors.

Highly pathogenic Nipah virus (NiV) generally causes severe encephalitis in humans. Respiratory symptoms are infrequently observed, likely reflecting variations in infection kinetics in human airways. Supporting this idea, we recently identified individual differences in NiV replication kinetics in cultured airway epithelia from different human donors. As type III interferons (IFN-λ) represent major players in the defence mechanism against viral infection of the respiratory mucosa, we studied IFN-λ induction and antiviral activity in NiV-infected primary differentiated human bronchial epithelial cells (HBEpCs) cultured under air-liquid interface conditions. Our studies revealed that IFN-λ was upregulated in airway epithelia upon NiV infection. We also show that IFN-λ pretreatment efficiently inhibited NiV replication. Interestingly, the antiviral activity of IFN-λ varied in HBEpCs from two different donors. Increased sensitivity to IFN-λ was associated with higher expression levels of IFN-λ receptors, enhanced phosphorylation of STAT1, as well as enhanced induction of interferon-stimulated gene expression. These findings suggest that individual variations in IFN-λ receptor expression affecting IFN responsiveness can play a functional role for NiV replication kinetics in human respiratory epithelial cells of different donors.

Pulmonary C-fiber degeneration downregulates IFN-γ receptor 1 via IFN-α induction to attenuate RSV-induced airway hyperresponsiveness.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection in infants. Unfortunately, no effective vaccine or treatment against RSV is currently available. Pulmonary C-fibers (PCFs) are critical for regulating pulmonary inflammation and airway hyperresponsiveness (AHR). We previously reported that IFN-γ partially mediated RSV-induced airway disorders. In this study, we found that PCF degeneration alleviated RSV-induced airway inflammation, especially AHR by downregulating IFN-γ receptor 1 (IFNGR1), but had no effect on IFN-γ induction. In contrast, PCF degeneration actually increased IFN-α/ß levels, as were the levels of STAT1 and phosphorylated STAT1 (pSTAT1). Exogenous IFN-α treatment induced STAT1 activation and downregulated IFNGR1 expression. These results suggest that PCFs affect IFNGR1 expression by inducing IFN-α to regulate IFN-γ-mediated airway inflammation and AHR. Thus, targeting PCFs activation may help control RSV-induced airway disorders, especially AHR, even with the presence of inflammation.

White adipose tissue IFN-γ expression and signalling along the progression of rodent cancer cachexia.

Cachexia is associated with increased morbidity and mortality in cancer. The White adipose tissue (WAT) synthesizes and releases several pro-inflammatory cytokines that play a role in cancer cachexia-related systemic inflammation. IFN-γ is a pleiotropic cytokine that regulates several immune and metabolic functions. To assess whether IFN-γ signalling in different WAT pads is modified along cancer-cachexia progression, we evaluated IFN-γ receptors expression (IFNGR1 and IFNGR2) and IFN-γ protein expression in a rodent model of cachexia (7, 10, and 14days after tumour implantation). IFN-γ protein expression was heterogeneously modulated in WAT, with increases in the mesenteric pad and decreased levels in the retroperitoneal depot along cachexia progression. Ifngr1 was up-regulated 7days after tumour cell injection in mesenteric and epididymal WAT, but the retroperitoneal depot showed reduced Ifngr1 gene expression. Ifngr2 gene expression was increased 7 and 14days after tumour inoculation in mesenteric WAT. The results provide evidence that changes in IFN-γ expression and signalling may be perceived at stages preceding refractory cachexia, and therefore, might be employed as a means to assess the early stage of the syndrome.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells.

CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma.

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8+ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8+ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8+ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8+ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8+ T cells was observed in female recipient mice. Male CD8+ T cells produced higher levels of IFN-γ than female CD8+ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4+ T cells. Interestingly, IFN-γ receptor expression on CD4+ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8+ T cells and the lower expression of IFN-γ receptor on CD4+ T cells in females compared with males.

Defective NK Cells in Acute Myeloid Leukemia Patients at Diagnosis Are Associated with Blast Transcriptional Signatures of Immune Evasion.

Acute myeloid leukemia (AML) is a heterogeneous group of malignancies that may be sensitive to the NK cell antitumor response. However, NK cells are frequently defective in AML. In this study, we found in an exploratory cohort (n = 46) that NK cell status at diagnosis of AML separated patients in two groups with a different clinical outcome. Patients with a deficient NK cell profile, including reduced expression of some activating NK receptors (e.g., DNAX accessory molecule-1, NKp46, and NKG2D) and decreased IFN-γ production, had a significantly higher risk of relapse (p = 0.03) independently of cytogenetic classification in multivariate analysis. Patients with defective NK cells showed a profound gene expression decrease in AML blasts for cytokine and chemokine signaling (e.g., IL15, IFNGR1, IFNGR2, and CXCR4), Ag processing (e.g., HLA-DRA, HLA-DRB1, and CD74) and adhesion molecule pathways (e.g., PVR and ICAM1). A set of 388 leukemic classifier genes defined in the exploratory cohort was independently validated in a multicentric cohort of 194 AML patients. In total, these data evidenced the interplay between NK cells and AML blasts at diagnosis allowing an immune-based stratification of AML patients independently of clinical classifications.

IFN-λ is able to augment TLR-mediated activation and subsequent function of primary human B cells.

During the past decade, increased emphasis has been placed on finding alternatives to IFN-α-based therapies. One such alternative, IFN-λ, has shown therapeutic promise in a variety of diseases, but research of this family of cytokines has been primarily focused on their antiviral activities. The goal of the present study was to investigate the role of IFN-λ in the regulation and modulation of B cell function. We show that, similar to IFN-α, IFN-λ1 is able to augment TLR-mediated B cell activation, partially attributed to an upregulation of TLR7 expression, and that both naïve and memory B cells express the limiting type III IFN receptor component, IFN-λR1. Furthermore, this IFN-λ-enhanced B cell activation resulted in increased cytokine and Ig production during TLR7 challenge, most prominently after the addition of helper T cell signals. Ultimately, these elevated cytokine and Ig levels could be partially attributed to the increase in proliferation of TLR7-challenged B cells by both type I and type III IFNs. These findings demonstrate the ability of IFN-λ to boost humoral immunity, an important attribute to consider for further studies on immunity to pathogens, vaccine development, and ongoing advancement of therapeutic strategies aimed at replacing IFN-α-based treatments with IFN-λ.

Identification, Characterization, and Developmental Expression Pattern of Type III Interferon Receptor Gene in the Chinese Goose.

Interferons, as the first line of defense against the viral infection, play an important role in innate immune responses. Type III interferon (IFN-λ) was a newly identified member of IFN family, which plays IFN-like antiviral activity. Towards a better understanding of the type III interferon system in birds, type III interferon lambda receptor (IFNLR1) was first identified in the Chinese goose. In this paper, we had cloned 1952 bp for goose IFNLR1 (goIFNLR1), including an ORF of 1539 bp, encoding a 512-amino acid protein with a 20 aa predict signal peptide at its N terminal and a 23 aa transmembrane region. The predicted amino acid sequence of goIFNLR1 has 90%, 73%, and 34% identity with duck IFNLR1 (predicted sequence), chicken IFNLR1, and human IFNLR1, respectively. And the age-related tissue distribution of goIFNLR1 was identified by Real Time quantitative PCR (RT-qPCR), we found that the goIFNLR1 has a mainly expression in epithelium-rich tissues similar to other species', such as small intestinal, lung, liver, and stomach. Moreover, a relatively high expression of goIFNLR1 was also observed in the secondary immune tissues (harderian gland and cecal tonsil). The identification and tissue distribution of goIFNLR1 will facilitate further study of the role of IFN-λ in goose antiviral defense.

T-bet is critical for the development of acute graft-versus-host disease through controlling T cell differentiation and function.

T-bet is a master regulator for IFN-γ production and Th1 differentiation. We evaluated the roles of T-bet and IFN-γ in T cell responses in acute graft-versus-host disease (GVHD) and found that T-bet(-/-) T cells induced significantly less GVHD compared with wild-type or IFN-γ(-/-) counterparts in both MHC-mismatched and MHC-matched but minor histocompatibility Ag-mismatched models driven by CD4 T cells. T-bet(-/-), but not IFN-γ(-/-), CD4 T cells had a markedly reduced ability to cause tissue damage in liver and gut. This distinct outcome is reflected by the differential gene expression on donor CD4 T cells deficient for T-bet or IFN-γ. At mRNA and protein levels, we defined several T-bet-dependent molecules that may account for the impaired ability of T-bet(-/-) T cells to migrate into target organs and to produce Th1-related cytokines. Moreover, these molecules were independent of either endogenous IFN-γ, such as CXCR3 and programmed death-1, or systematic IFN-γ, such as NKG2D, I-A(b), and granzyme B. Although both T-bet(-/-) and IFN-γ(-/-) CD4 T cells are prone to differentiate into Th17 cells, polarized Th17 cells deficient for T-bet but not for IFN-γ had a significantly reduced ability to cause GVHD. Finally, T-bet(-/-) T cells had a compromised graft-versus-leukemia effect, which could be essentially reversed by neutralization of IL-17 in the recipients. We conclude that T-bet is required for Th1 differentiation and migration, as well as for optimal function of Th17 cells. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic.

Human monocytes have increased IFN-γ-mediated IL-15 production with age alongside altered IFN-γ receptor signaling.

IL-15 is involved in regulating host defense and inflammation. Monocytes produce the biologically active cell surface IL-15 in response to IFN-γ. Although aging can alter the immune system, little is known about whether and how aging affects IFN-γ-mediated IL-15 production in human monocytes. We showed that monocytes of healthy older adults (age ≥ 65) had increased cell surface IL-15 expression in response to IFN-γ compared to those of healthy young adults (age ≤ 40). This finding stems in part from increased IFN-γ receptor (R)1/2 expression on monocytes in older adults, leading to enhanced STAT1 activation and interferon regulatory factor 1 synthesis with increased IL15 gene expression. Our study suggests that with aging the IFN-γ-mediated IL-15 production pathway in human monocytes is uncompromised, but rather augmented, and could be considered as a therapeutic target point to modulate host defense and inflammation in older adults.

Sodium butyrate inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via STAT1 in nasopharyngeal carcinoma cells.

AIMS: Indoleamine 2,3-dioxygenase (IDO) inhibits T-cell proliferation by catalyzing the conversion of l-tryptophan to l-kynurenine. IDO-induced immune tolerance weakens the clinical outcomes of immunotherapies. Sodium butyrate (NaB), one of the histone deacetylase inhibitors (HDACIs), has potential anti-tumor effects. Our previous studies revealed that NaB could inhibit IFN-γ induced IDO expression in nasopharyngeal carcinoma cells, CNE2. In the present study, we aim to investigate to the mechanism of NaB interfering with the interferon-gamma (IFN-γ)-mediated IDO expression signaling transduction. MAIN METHODS: IDO expression and STAT1 phosphorylation in CNE2 cells were analyzed by western blotting and STAT1 acetylation was evaluated by immunoprecipitation. STAT1 nuclear translocation and NF-κB activity were detected by transient transfection and reporter gene assay. KEY FINDINGS: We found that NaB inhibited IFN-γ-induced IDO expression in CNE2 cells via decreasing phosphorylation and nuclear translocation of STAT1, but not via down-regulation of IFN-γ-receptor (IFNGR). Immunoprecipitation assays revealed that NaB increased STAT1 acetylation. Furthermore, NaB elevated the activity of NF-κB in CNE2 cells, and blocking the NF-κB activity had no effect on the IFN-γ-induced IDO expression. SIGNIFICANCE: These results suggest that NaB inhibited IFN-γ-induced IDO expression via STAT1 increased acetylation, decreased phosphorylation, and reduced nuclear translocation. These provided new evidence for the anti-tumor action of NaB and potential drug targets to reduce the IDO-induced immune tolerance.

Cord-blood-derived mesenchymal stromal cells downmodulate CD4+ T-cell activation by inducing IL-10-producing Th1 cells.

The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response.

Type III interferons, IL-28 and IL-29, are increased in chronic HCV infection and induce myeloid dendritic cell-mediated FoxP3+ regulatory T cells.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is difficult to eradicate and type III interferons (IFN-λ, composed of IL-28A, IL-28B and IL-29) are novel therapeutic candidates. We hypothesized that IFN-λ have immunomodulatory effects in HCV- infected individuals. MATERIALS AND METHODS: We analyzed the expression of IFN-λ and its receptor (composed of IL-10R2 and IFN-λR subunits) in the blood and livers of patients with chronic (c)HCV infection compared to controls (those who cleared HCV by sustained virological response, SVR, and those with liver inflammation of non-viral origin, non-alcoholic steatohepatitis, NASH). We also compared the proliferative capacity of dendritic cells (DCs) obtained from healthy individuals and those with chronic HCV using a mixed leukocyte reaction combined with 3H-Td incorporation. In addition, the composition of the IFN-λ receptor (IFN-λR) on myeloid DCs, plasmacytoid DCs, PBMCs, and T cells was determined by FACS analysis. RESULTS: We report that the expression of IFN-λ protein in serum and mRNA in liver is increased in cHCV patients, but not in those with HCV SVR or NASH, compared to controls. Liver level of IFN-λR mirrored the expression of serum IFN-λ and was higher in cHCV, compared to controls and HCV-SVR patients, suggesting that elevation of IFN-λ and IFN-λR are HCV-dependent. We further identified that innate immune cell populations expressed complete IFN-λ receptor. In vitro, recombinant IFN-λ promoted differentiation of monocyte-derived dendritic cells (DCs) into a phenotype with low T cell stimulatory capacity and high PD-L1 expression, which further promoted expansion of existing regulatory T cells. IFN-λ-DCs failed to induce de novo generation of regulatory T cells. The inhibitory capacity of IFN-λ-DCs was counteracted by recombinant IL-12 and by neutralization of the PD-1/PD-L1 system. CONCLUSIONS: Our novel findings of the immunomodulatory effect of IFN-λ contribute to the understanding of the anti-inflammatory and/or anti-viral potential of IFN-λ in cHCV.

Role of caspases in cytokine-induced barrier breakdown in human brain endothelial cells.

During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood-brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.

Reduced IFN-γ receptor expression and attenuated IFN-γ response by dendritic cells in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is characterized by a predominance of T(H)2 immune reactions but weaker T(H)1 immune responses in acute skin lesions. OBJECTIVES: To evaluate whether enhanced T(H)2 immunity in patients with AD might impair T(H)1 immune responses by affecting the IFN-γ responsiveness of antigen-presenting cells, we investigated IFN-γ receptor and IL-4 receptor α chain expression, IFN-γ signaling, and the expression of IFN-γ-responsive mediators in dendritic cells (DCs) and their precursors from patients with AD compared with those from healthy subjects. METHODS: Skin biopsy specimens were obtained and both monocytes and monocyte-derived dendritic cells (MoDCs) from patients with AD (n = 86) and control subjects (n = 84) were analyzed by means of flow cytometry, real-time PCR, ELISA, and HPLC. RESULTS: We observed lower IFN-γ receptor II expression combined with higher IL-4 receptor α chain expression on epidermal DCs, monocytes, and MoDCs from patients with AD. Induction of IFN-γ-inducible factors, such as interferon regulatory factor 1, interferon-inducible protein 10, and indoleamine 2,3-dioxygenase, was attenuated in IFN-γ-pulsed MoDCs from patients with AD. Weaker signal transducer and activator of transcription 1 activation mirrored by lower phosphorylated signal transducer and activator of transcription 1 levels in response to IFN-γ stimulation could be observed in epidermal DCs, monocytes, and MoDCs from patients with AD. CONCLUSION: Impaired IFN-γ signaling together with attenuated IFN-γ responses in DCs and their precursor cells might contribute to the T(H)2 bias in patients with AD.