Presence Of IFN-α Research Articles

Adapting SHIVs In Vivo Selects for Envelope-Mediated Interferon-α Resistance.

Lentiviruses are able to establish persistent infection in their respective hosts despite a potent type-I interferon (IFN-I) response following transmission. A number of IFN-I-induced host factors that are able to inhibit lentiviral replication in vitro have been identified, and these studies suggest a role for IFN-induced factors as barriers to cross-species transmission. However, the ability of these factors to inhibit viral replication in vivo has not been well characterized, nor have the viral determinants that contribute to evasion or antagonism of the host IFN-I response. In this study, we hypothesized that the host IFN-I response serves as a strong selective pressure in the context of SIV/HIV chimeric virus (SHIV) infection of macaques and sought to identify the viral determinants that contribute to IFN-I resistance. We assessed the ability of SHIVs encoding HIV-1 sequences adapted by serial passage in macaques versus SHIVs encoding HIV sequences isolated directly from infected individuals to replicate in the presence of IFNα in macaque lymphocytes. We demonstrate that passage in macaques selects for IFNα resistant viruses that have higher replication kinetics and increased envelope content. SHIVs that encode HIV-1 sequences derived directly from infected humans were sensitive to IFNα –induced inhibition whereas SHIVs obtained after passage in macaques were not. This evolutionary process was directly observed in viruses that were serially passaged during the first few months of infection–a time when the IFNα response is high. Differences in IFNα sensitivity mapped to HIV-1 envelope and were associated with increased envelope levels despite similar mRNA expression, suggesting a post-transcriptional mechanism. These studies highlight critical differences in IFNα sensitivity between HIV-1 sequences in infected people and those used in SHIV models.

E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation.

The CD34+ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8+ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8+ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.

Preferential Expansion of Th1 type-CD56+Vγ9γδT Cells by CD56high+IFNa-induced Dendritic Cells Derived from Cancer Patients in vitro and in vivo

The CD56+ cells freshly isolated from human peripheral blood contain a substantial subset of CD14+CD86+HLA-DR+ cells which spontaneously differentiate into enlarged DC (dendritic cell)-like cells. We show here that interferon-α (IFN-α)-induced DC-like cells expressing high levels of CD56 and CD14 (hereafter, CD56high+IFNDCs) can be differentiated from monocytes obtained as adherent cells from patients with various cancers, in the presence of IFN-α and granulocyte-macrophage colony stimulating factor (GM-CSF) in vitro. These cells expressed high levels of CD14, CD86, and HLA-DR and may be an in vitro counterpart of the circulating CD14+CD86+HLA-DR+ cells in blood. In contrast to conventional mature IL-4DCs which are differentiated from monocytes as adherent cells in the presence of GM-CSF, IL-4, and TNF-α (hereafter, mIL-4DCs), the CD56 high+ IFN-DCs exhibited a stronger capacity in stimulating autologous CD56+Vγ9γδT cells expressing high levels of CD16 which produced high amounts of IFNγ and TNF-α, in comparison with the mIL-4DCs. Intravenous administration of a large number of Vγ9γδT cells activated by CD56high+IFN-DCs pulsed with zoledronate (hereafter, CD56 high+ IFN-DC-activated Vγ9γδT cells) to patients with a non-small cell lung cancer resulted in the increase of PB Vγ9γδT cells among which CD56+Vγ9γδT cells and CD16+Vγ9γδT cells predominantly increased in vivo. Among γδT cell-based immunotherapy, vaccination with CD56high+IFN-DCactivated Vγ9γδT cells may lead to improved clinical outcomes of patients with cancer.

Phosphorylation of STAT1 and NF-κB by human plasmacytoid dendritic cells during HIV-infection (VIR9P.1154)

Abstract Plasmacytoid dendritic cells (pDC) are critical for anti-viral immunity. TLR7 and TLR9 allow pDC to recognize viruses and initiate signals leading to IRF7 phosphorylation, which is responsible for IFN-α production, as well as NF-κB phosphorylation, which is responsible for TNF-α and IL-6 induction. During HIV-infection, lower pDC numbers are found, along with impaired IFN-α production. Chronic immune activation is also found, with patients paradoxically displaying an IFN-signature, indicating the presence of IFN-α, and higher serum TNF-α and IL-6. To see if pDC are affected in vivo by IFN-α or TNF-α, we compared baseline p-STAT1 and p-NF-κB in pDC in HIV-patients vs healthy controls. At baseline, HIV-patient vs. healthy pDC showed a trend towards elevated p-STAT1, while p-NF-κB levels were similar. HIV-patient pDC phosphorylated STAT1 and NF-κB less than healthy controls in response to IFN-α and TNF-α, respectively. In response to viruses, HIV-patient pDC phosphorylated STAT1 more than healthy controls. Despite normal production of TNF-α and IL-6, HIV-patient pDC were unable to phosphorylate NF-κB in response to viruses, suggesting that other mechanisms exist for TNF-α and IL-6 induction. In conclusion, impaired NF-κB phosphorylation by HIV-patient pDC suggest that they are immunologically exhausted and this can lead to a number of downstream effects on protein expression. In contrast, enhanced p-STAT1 in response to viruses can drive the IFN-signature seen in HIV-patients.

Interferon-α inhibits CD4 T cell responses to interleukin-7 and interleukin-2 and selectively interferes with Akt signaling.

Persistent type I IFN production occurs during chronic viral infections, such as HIV disease. As type I IFNs have antiproliferative activity, it is possible that chronic exposure to these cytokines could adversely affect T cell homeostasis. We investigated the capacity of IFN-α to impair T cell proliferation induced by the homeostatic cytokine, IL-7, or another common γ-chain cytokine, IL-2, in cells from healthy human donors. We found that IL-7- or IL-2-induced proliferation of CD4(+) T cells was partially inhibited in the presence of IFN-α. The CD4(+) T cells that were exposed to IFN-α also displayed attenuated induction of IL-2 and CD40L following TCR stimulation. Analyses of signaling pathways indicated that IL-7 and IL-2 induced a delayed and sustained P-Akt signal that lasted for several days and was partially inhibited by IFN-α. In contrast, IL-7-induced P-STAT5 was not affected by IFN-α. Furthermore, IFN-α had no detectable effect on P-Akt that was induced by the chemokine SDF-1. Both inhibitors of P-Akt and P-STAT5 blocked IL-7-induced T cell proliferation, confirming that both signaling pathways are important for IL-7-induced T cell proliferation. These results demonstrate that IFN-α can selectively inhibit cytokine-induced P-Akt as a potential mechanism to disrupt homeostasis of T lymphocytes.

Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material.

Pathophysiology of the pancreas after oral infection of genetically diverse mice with coxsackievirus B4-E2.

Coxsackievirus B4 strain E2 (CVB4-E2) and its association with type 1 diabetes (T1D) have been studied in experimental in vitro and in vivo murine models. CVB4-E2, known to be pancreotropic and diabetogenic in nature, is associated with acute pancreatitis in mice but shows differences in the induction of glycemia after intraperitoneal (i.p.) infection. Therefore, the aim of this work was to study the outcome of oral infection with CVB4-E2 in five mouse strains with different genetic backgrounds: two outbred (Swiss albino, CD1), two inbred (SJL, NOD) and one transgenic (NOD.SCID). Survival rates, fasting blood glucose, histopathology, viral titres and persistence were studied in selected organs and stool samples. Viral protein (VP1), proinflammatory cytokines, and interferon alpha (IFN-α) were analyzed by immunohistochemistry. We observed mortality only in infected NOD and NOD.SCID mice, with differing survival rates implying initial innate protection in the NOD.SCID mice and low virus clearance with replicating virus titres in the studied organs and stool up to day 40 post infection (p.i.). Independent of the mouse strain hyperglycemia, proinflammatory cytokines and histopathological changes were absent in the endocrine pancreas of infected mice. Only the pancreata of the dead NOD.SCID mice showed inflammation even in presence of IFN-α. Host-dependent viral RNA persistence was observed in all outbred mice. In conclusion, oral infection with CVB4-E2, despite the known affinity of this strain towards the pancreatic tissue and the presence of replicating virus, conferred total protection to the endocrine pancreas in all mice and failed to induce the proinflammatory cytokines studied by us.

Ribavirin improves the IFN-γ response of natural killer cells to IFN-based therapy of hepatitis C virus infection

Ribavirin (RBV) is an important component of interferon (IFN)-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBV's mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by signal transducer and activator of transcription (STAT)1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56(dim) NK cells with cytotoxic effector functions and the frequency of CD56(bright) NK cells with the capacity to produce IFN-γ decreased (P = 0.049 and P = 0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P < 0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-α. This was associated with an increase in IFN-γ production but not cytotoxicity of NK cells during subsequent IFN-α-based therapy. The frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders. RBV enhances the pSTAT4 and IFN-γ response of NK cells to IFN-α-stimulation.

Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα+ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα+ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα− production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67+-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67+-pDC precursors, none of these being IFNα+ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors.

Ribavirin Pretreatment Improves the IFN-γ Response of Natural Killer Cells to IFN-based Therapy of Hepatitis C Virus Infection

Ribavirin is an important component of interferon-based and direct antiviral treatment regimens of hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host interferon response, has been proposed as a mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by STAT1- and STAT4-phosphorylation, respectively. Upon continued exposure to IFN-α, an increase in STAT1 levels limits STAT4 access at the IFN-α/β receptor and results in decreased IFN-γ production. Accordingly, NK cell IFN-γ production decreases during IFN-α-based therapy. To assess whether ribavirin improves the IFN-γ response of NK cells in the presence of IFN-α, NK cells were studied prospectively in 22 HCV patients with and 32 patients without 4 weeks of ribavirin monotherapy, and during subsequent PegIFN/ribavirin therapy. During ribavirin monotherapy, the frequency of IFN-γ producing NK cells decreased (p = 0.001) in correlation with a decrease in their ex vivo pSTAT4 levels ((Rho = 0.58, p = 0.019). However, in vitro or in vivo exposure of NK cells to ribavirin improved their pSTAT4 response to subsequent stimulation with IFN-α (P < 0.01). Furthermore, HCV patients who had received ribavirin-pretreatment showed a greater frequency of IFN-γ-producing NK cells during subsequent PegIFN/ribavirin therapy than those who had not been pretreated (P < 0.05). Finally, the frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders. Conclusions: Ribavirin improved the pSTAT4 and IFN-γ response of NK cells to IFN-α-treatment, which may contribute to an improved second-phase virological response.

Monocyte Activation by Interferon α Is Associated With Failure to Achieve a Sustained Virologic Response After Treatment for Hepatitis C Virus Infection

Interferon α (IFN-α) and ribavirin can induce a sustained virologic response (SVR) in some but not all hepatitis C virus (HCV)-infected patients. The mechanism of effective treatment is unclear. One possibility is that IFN-α differentially improves the functional capacity of classic myeloid dendritic cells (mDCs) by altering expression of surface molecules or cytokines. Others have proposed that antigen-presenting cell activation could be paradoxically detrimental during HCV infection because of the production by monocytes of substances inhibitory or toxic to plasmacytoid dendritic cells. We examined responses to in vitro IFN-α treatment of peripheral blood leukocyte samples from a retrospective treatment cohort of nearly 200 HCV-seropositive patients who had undergone antiviral therapy with ribavirin and pegylated IFN. We analyzed the variable responses of antigen-presenting cell subsets to drug. We found that patients achieving SVR were no more likely to have robust mDC activation in response to IFN-α than those who did not achieve SVR. Rather, patients achieving SVR were distinguished by restrained monocyte activation in the presence of IFN-α, a factor that was second in importance only to IL28B genotype in its association with SVR. These results suggest that interindividual variability in the response of monocytes to IFN-α is an important determinant of treatment success with IFN-α-based regimens.

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.

Cytotoxic activity of ex-vivo generated IFNα-induced monocyte-derived dendritic cells in brain glioma patients

Recent studies have revealed that besides the important role in triggering the adoptive antitumor immunity, dendritic cells (DCs) possess direct cytotoxic antitumor activity. Here, we investigated brain glioma patient monocyte-derived DCs generated in the presence of IFNα and GM-CSF (IFN-DCs). These DCs were characterized by reduced cytotoxic activity against TRAIL-resistant HEp-2 cells. The impairment of DC cytotoxic function was observed mainly in high-grade glioma patients and associated with poor survival. The dysfunction of patient DC cytotoxicity was partially restored under in vitro pretreatment of DCs with double-stranded human DNA as well as rIL-2. In contrast to healthy donors, IFN-DCs in a part of high-grade glioma patients also failed to lyse primary autologous or allogeneic glioma cells.Our findings point to possible contribution of DC impairment in tumor pathogenesis in brain glioma and justify the necessity to evaluate and correct DC cytotoxic function when exploring DCs as cancer vaccines in glioma.

Stroke prevention in the Stroke Belt

<h3>Background</h3> Juvenile-onset systemic lupus erythematosus (JSLE) is a multisystem autoimmune disease characterised by autoantibodies directed against nuclear autoantigens. In the presence of JSLE sera, an imbalance of pro- and anti-apoptotic factors results in an increase in neutrophil apoptosis, a potential source of autoantigens. Type 1 and 2 interferons are increased in JSLE serum, however the specific effect of IFN alpha on neutrophil apoptosis has not been elucidated. <h3>Objectives</h3> Investigate the effect of IFN alpha on neutrophil apoptosis. <h3>Methods</h3> Neutrophils isolated from healthy control adult donors were incubated unstimulated, or IFN alpha (10ng) for ∼20hrs. GMCSF is known to delay neutrophil apoptosis, and was used as a positive control. The percentage of apoptotic neutrophils were measured using flow cytometry via annexin V FITC staining. Western blot analysis was used to measure caspase 3 activation. Cleaved caspase is the active protein and therefore caspase dependant apoptosis would result in increased caspase 3 expression from its inactive form (procaspase 3). <h3>Results</h3> IFN alpha was observed to reduce neutrophil apoptosis. Flow cytometry (% annexin V positive cells, mean ± SEM) demonstrated 50%±4.9% apoptosis following IFNα exposure (n=5) compared to 74%±8% unstimulated (n=5, <i>p</i>=0.016). GMCSF was observed to delay neutrophil apoptosis (55%±4.4). Western blot analysis showed reduced caspase cleavage upon IFNα incubation. This was demonstrated by the observation the procaspase 3/caspase 3 ratio to be increased in the presence of IFNα (5.4±3.5, n=4) as compared to unstimulated neutrophils (2.4±1.8, n=4, <i>p</i>=0.149) and GMCSF stimulated (5.81±2.9, n=4). <h3>Conclusions</h3> Modulation of neutrophil apoptosis by inflammatory cytokines may be associated with the outcome of inflammation. Understating the relationship between type 1 interferons and neutrophil apoptosis is important in systemic autoimmune diseases such as JSLE. We have shown that IFN alpha reduces neutrophil apoptosis which is either directly or indirectly due to reduced caspase 3 activation. The implications for this interaction on development and progression of inflammatory autoimmune diseases is currently being investigated paying particular focus on the both type 1 and type 2 interferons. <h3>References</h3> Bengtsson, A.A., Sturfelt, G., Truedsson, L., Blomberg, J., Alm, G., Vallin, H., Rönnblom, L. (2000) Activation of type I interferon system in systemic lupus erythematosus correlates with disease activity but not with antiretroviral antibodies. Lupus. 9, 664-71. Bouts, Y.M., Wolthuis, D.F, Dirkx, M.F., Pieterse, E., Simons, E.M., van Boekel, A.M., Dieker, J.W., van der Vlag, J. (2012) Apoptosis and NET formation in the pathogenesis of SLE. Autoimmunity. 45, 597-601. Kim, T., Kanayama, Y., Negoro, N., Okamura, M., Takeda, T., Inoue, T. (1987) Serum levels of interferons in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 70, 562-69. Midgley, A., McLaren, Z., Moots, R.J., Edwards, S.W., Beresford, M.W. (2009). The role of neutrophil apoptosis in juvenile-onset systemic lupus erythematosus. Arthritis Rheum. 60, 2390-2401 Sakamoto, E., Hato, F., Kato, T., Sakamoto, C., Akahori, M., Hino, M., Kitagawa, S. (2005). Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2. J. Leukoc. Biol. 78, 301-9. <h3>Disclosure of Interest</h3> None declared

IFN-α Induces APOBEC3G, F, and A in Immature Dendritic Cells and Limits HIV-1 Spread to CD4+ T Cells

Cytokines and IFNs, such as TNF-α and IFN-α, upregulate costimulatory molecules in monocyte-derived dendritic cells (MDDCs), enabling effective Ag presentation to T cells. This activation of MDDCs is often accompanied by upregulation of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) (A3) family proteins that are able to restrict HIV-1 replication in MDDCs by inducing hypermutations in the viral genome. In this study, we show that TNF-α upregulates costimulatory molecules and are able to restrict HIV-1BaL replication in MDDCs without significant induction of A3G, A3A, or A3F. Conversely, low quantities of IFN-α failed to upregulate costimulatory molecules, did not induce IL-12p40 or migration, but significantly induced A3G, A3A, and A3F mRNA expression and restricted viral replication in MDDCs. We also showed that transmission of HIV-1 from MDDCs to autologous T cells was significantly reduced in the presence of IFN-α. Sequence analyses detected the induction of high frequency of G-to-A hypermutations in the env genes from HIV-1BaL-infected MDDCs treated with low quantities of IFN-α2b. These findings show that low quantities of IFN-α can induce functional A3 family proteins and restrict HIV-1 replication in MDDCs while keeping an immature nonmigratory phenotype, supporting further investigations of modalities that enhance retroviral restriction factors. In addition, the findings highlight the role of IFN-α as a double-edged sword in HIV-1 infection, and we show that IFN-α can be powerful in reducing HIV-1 infection both in MDDCs and T cells.

Phenotypic properties of transmitted founder HIV-1

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

Interferon alpha inhibits antigen-specific production of proinflammatory cytokines and enhances antigen-specific transforming growth factor beta production in antigen-induced arthritis

IntroductionInterferon alpha (IFN-α) has a complex role in autoimmunity, in that it may both enhance and prevent inflammation. We have previously shown that the presence of IFN-α at sensitization protects against subsequent antigen-triggered arthritis. To understand this tolerogenic mechanism, we performed a descriptive, hypothesis-generating study of cellular and humoral responses associated with IFN-α-mediated protection against arthritis.MethodsArthritis was evaluated at day 28 in mice given a subcutaneous injection of methylated bovine serum albumin (mBSA), together with Freund adjuvant and 0 to 5,000 U IFN-α at days 1 and 7, followed by intraarticular injection of mBSA alone at day 21. The effect of IFN-α on mBSA-specific IgG1, IgG2a, IgG2b, IgA, and IgE was evaluated by enzyme-linked immunosorbent assay (ELISA). Cytokines in circulation and in ex vivo cultures on mBSA restimulation was evaluated with ELISA and Luminex, and the identity of cytokine-producing cells by fluorescence-activated cell sorting (FACS) analysis.ResultsAdministration of IFN-α protected mice from arthritis in a dose-dependent manner but had no effect on antigen-specific antibody levels. However, IFN-α did inhibit the initial increase of IL-6, IL-10, IL-12, and TNF, and the recall response induced by intraarticular mBSA challenge of IL-1β, IL-10, IL-12, TNF, IFN-γ, and IL-17 in serum. IFN-α decreased both macrophage and CD4+ T cell-derived IFN-γ production, whereas IL-17 was decreased only in CD4+ T cells. Ex vivo, in mBSA-restimulated spleen and lymph node cell cultures, the inhibitory effect of in vivo administration of IFN-α on proinflammatory cytokine production was clearly apparent, but had a time limit. An earlier macrophage-derived, and stronger activation of the antiinflammatory cytokine transforming growth factor beta (TGF-β) was observed in IFN-α-treated animals, combined with an increase in CD4+ T cells producing TGF-β when arthritis was triggered by mBSA (day 21). Presence of IFN-α at immunizations also prevented the reduction in TGF-β production, which was induced by the intraarticular mBSA injection triggering arthritis in control animals.ConclusionsAdministration of IFN-α has a profound effect on the cellular response to mBSA plus adjuvant, but does not affect antigen-specific Ig production. By including IFN-α at immunizations, spleen and lymph node cells inhibit their repertoire of antigen-induced proinflammatory cytokines while enhancing antiinflammatory TGF-β production, first in macrophages, and later also in CD4+ T cells. On intraarticular antigen challenge, this antiinflammatory state is reenforced, manifested as inhibition of proinflammatory recall responses and preservation of TGF-β levels. This may explain why IFN-α protects against antigen-induced arthritis.

Ribavirin Does Not Impair the Suppressive Activity of Foxp3+CD4+CD25+Regulatory T Cells

Ribavirin is an antiviral drug used in combination with pegylated interferon-α (IFN-α) for the treatment of hepatitis C virus (HCV) infection. Recently, ribavirin was reported to inhibit the suppressive activity of regulatory T (Treg) cells. In the present study, we re-evaluated the effect of ribavirin on Foxp3+CD4+CD25+ Treg cells from normal donors. First, we examined the expression of CTLA-4 and CD39, which are known to play a role in the suppressive function of Treg cells. We found that ribavirin treatment did not modulate the expression of CTLA-4 and CD39 in Treg cells. We also studied the effect of ribavirin on Treg cells in the presence of IFN-α; however, the expression of CTLA-4 and CD39 in Treg cells was not changed by ribavirin in the presence of IFN-α. Next, we directly evaluated the effect of ribavirin on the suppressive activity of Treg cells in the standard Treg suppression assay, by co-culturing CFSE-labeled non-Treg CD4+ T cells with purified Treg cells. We found that ribavirin did not attenuate the suppressive activity of Treg cells. Taken together, while ribavirin reversed Treg cell-mediated suppression of effector T cells in the previous study, we herein demonstrate that ribavirin does not impair the suppressive activity of Treg cells.

The Presence of Alpha Interferon at the Time of Infection Alters the Innate and Adaptive Immune Responses to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating and costly diseases to the swine industry worldwide. Overall, the adaptive immune response to PRRS virus (PRRSV) is weak, which results in delayed elimination of virus from the host and inferior vaccine protection. PRRSV has been shown to induce a meager alpha interferon (IFN-α) response, and we hypothesized that elevated IFN-α levels early in infection would shorten the induction time and increase elements of the adaptive immune response. To test this, we measured both antibody and cell-mediated immunity in pigs after the administration of a nonreplicating human adenovirus type 5 vector expressing porcine IFN-α (Ad5-pIFN-α) at the time of PRRSV infection and compared the results to those for pigs infected with PRRSV alone. Viremia was delayed, and there was a decrease in viral load in the sera of pigs administered the Ad5-pIFN-α. Although seroconversion was slightly delayed in pigs receiving Ad5-pIFN-α, probably due to the early reduction in viral replication, little difference in the overall or neutralizing antibody response was seen. However, there was an increase in the number of virus-specific IFN-γ-secreting cells detected in the pigs receiving Ad5-pIFN-α, as well as an altered cytokine profile in the lung at 14 days postinfection, indicating that the presence of IFN-α at the time of infection can alter innate and adaptive immune responses to PRRSV.