Blocking Interferon β Stimulates Vascular Smooth Muscle Cell Proliferation and Arteriogenesis

Increased interferon (IFN)-β signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFNβ on collateral artery growth in mice have been reported. The mechanisms of IFNβ-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFNβ-regulated genes. Immunohistochemically, the IFNβ receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFNβ resulted in an attenuated proliferation, cell-cycle arrest, and increased expression of cyclin-dependent kinase inhibitor-1A (p21). The growth inhibitory effect of IFNβ was attenuated by inhibition of p21 by RNA interference. IFNβ-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFNβ-signaling. RNA interference of the IFNβ receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFNβ signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1^−/− mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration 1 week after femoral artery ligation was improved in IFNAR1^−/− mice compared with wild-type mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFNβ inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFNβ stimulates VSMC proliferation and collateral artery growth.     

Thymosin Beta 4 Protects Mice from Monocrotaline-Induced Pulmonary Hypertension and Right Ventricular Hypertrophy

Pulmonary hypertension (PH) is a progressive vascular disease of pulmonary arteries that impedes ejection of blood by the right ventricle. As a result there is an increase in pulmonary vascular resistance and pulmonary arterial pressure causing right ventricular hypertrophy (RVH) and RV failure. The pathology of PAH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. Current therapies are limited to reverse the vascular remodeling. Investigating a key molecule is required for development of new therapeutic intervention. Thymosin beta-4 (Tβ4) is a ubiquitous G-actin sequestering protein with diverse biological function and promotes wound healing and modulates inflammatory responses. However, it remains unknown whether Tβ4 has any protective role in PH. The purpose of this study is to evaluate the whether Tβ4 can be used as a vascular-protective agent. In monocrotaline (MCT)-induced PH mouse model, we showed that mice treated with Tβ4 significantly attenuated the systolic pressure and RVH, compared to the MCT treated mice. Our data revealed for the first time that Tβ4 selectively targets Notch3-Col 3A-CTGF gene axis in preventing MCT-induced PH and RVH. Our study may provide pre-clinical evidence for Tβ4 and may consider as vasculo-protective agent for the treatment of PH induced RVH.     

A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension

Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.     

Intranasal Administration of Interferon Beta Attenuates Neuronal Apoptosis via the JAK1/STAT3/BCL-2 Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

Neonatal hypoxic-ischemic encephalopathy (HIE) is an injury that often leads to detrimental neurological deficits. Currently, there are no established therapies for HIE and it is critical to develop treatments that provide protection after HIE. The objective of this study was to investigate the ability of interferon beta (IFNβ) to provide neuroprotection and reduce apoptosis after HIE. Postnatal Day 10 rat pups were subjected to unilateral carotid artery ligation followed by 2.5 hr of exposure to hypoxia (8% O₂). Intranasal administration of human recombinant IFNβ occurred 2 hr after HIE and infarct volume, body weight, neurobehavioral tests, histology, immunohistochemistry, brain water content, blood–brain barrier permeability, enzyme-linked immunosorbent assay, and Western blot were all used to evaluate various parameters. The results showed that both IFNβ and the Type 1 interferon receptor expression decreases after HIE. Intranasal administration of human recombinant IFNβ was able to be detected in the central nervous system and was able to reduce brain infarction volumes and improve neurological behavior tests 24 hr after HIE. Western blot analysis also revealed that human recombinant IFNβ treatment stimulated Stat3 and Bcl-2 expression leading to a decrease in cleaved caspase-3 expression after HIE. Positive Fluoro-Jade C staining also demonstrated that IFNβ treatment was able to decrease neuronal apoptosis. Furthermore, the beneficial effects of IFNβ treatment were reversed when a Stat3 inhibitor was applied. Also an intraperitoneal administration of human recombinant IFNβ into the systemic compartment was unable to confer the same protective effects as intranasal IFNβ treatment.     

The protective effects of MSC‐EXO against pulmonary hypertension through regulating Wnt5a/BMP signalling pathway

The aim of the study was to explore the mechanism of mesenchymal stem cell‐derived exosomes (MSC‐EXO) to protect against experimentally induced pulmonary hypertension (PH). Monocrotaline (MCT)‐induced rat model of PH was successfully established by a single intraperitoneal injection of 50 mg/kg MCT, 3 weeks later the animals were treated with MSC‐EXO via tail vein injection. Post‐operation, our results showed that MSC‐EXO could significantly reduce right ventricular systolic pressure (RVSP) and the right ventricular hypertrophy index, attenuate pulmonary vascular remodelling and lung fibrosis in vivo. In vitro experiment, the hypoxia models of pulmonary artery endothelial cell (PAEC) and pulmonary vascular smooth muscle cell (PASMC) were used. We found that the expression levels of Wnt5a, Wnt11, BMPR2, BMP4 and BMP9 were increased, but β‐catenin, cyclin D1 and TGF‐β1 were decreased in MSC‐EXO group as compared with MCT or hypoxia group in vivo or vitro. However, these increased could be blocked when cells were transfected with Wnt5a siRNA in vitro. Taken together, these results suggested that the mechanism of MSC‐EXO to prevent PH vascular remodelling may be via regulation of Wnt5a/BMP signalling pathway.     

Notch Signaling Change in Pulmonary Vascular Remodeling in Rats with Pulmonary Hypertension and Its Implication for Therapeutic Intervention

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1–4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1–2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.     

Novel Mechanisms of Sildenafil in Pulmonary Hypertension Involving Cytokines/Chemokines,MAP Kinases and Akt

Pulmonary arterial hypertension (PH) is associated with high mortality due to right ventricular failure and hypoxia, therefore to understand the mechanism by which pulmonary vascular remodeling initiates these processes is very important. We used a well-characterized monocrotaline (MCT)-induced rat PH model, and analyzed lung morphology, expression of cytokines, mitogen-activated protein kinase (MAPK) phosphorylation, and phosphatidylinositol 3-kinase-Akt (PI-3k-Akt) pathway and nuclear factor (NF)-κB activation in order to elucidate the mechanisms by which sildenafil''s protective effect in PH is exerted. Besides its protective effect on lung morphology, sildenafil suppressed multiple cytokines involved in neutrophil and mononuclear cells recruitment including cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2α/β, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-1α, lipopolysaccharide induced CXC chemokine (LIX), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-1α, and MIP-3α. NF-κB activation and phosphorylation were also attenuated by sildenafil. Furthermore, sildenafil reduced extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK activation while enhanced activation of the cytoprotective Akt pathway in PH. These data suggest a beneficial effect of sildenafil on inflammatory and kinase signaling mechanisms that substantially contribute to its protective effects, and may have potential implications in designing future therapeutic strategies in the treatment of pulmonary hypertension.     

RNA‐binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral activity

The type I interferon (IFN‐I, IFN‐α/β)‐mediated immune response is the first line of host defense against invading viruses. IFN‐α/β binds to IFN‐α/β receptors (IFNARs) and triggers the expression of IFN‐stimulated genes (ISGs). Thus, stabilization of IFNARs is important for prolonging antiviral activity. Here, we report the induction of an RNA‐binding motif‐containing protein, RBM47, upon viral infection or interferon stimulation. Using multiple virus infection models, we demonstrate that RBM47 has broad‐spectrum antiviral activity in vitro and in vivo. RBM47 has no noticeable impact on IFN production, but significantly activates the IFN‐stimulated response element (ISRE) and enhances the expression of interferon‐stimulated genes (ISGs). Mechanistically, RBM47 binds to the 3''UTR of IFNAR1 mRNA, increases mRNA stability, and retards the degradation of IFNAR1. In summary, this study suggests that RBM47 is an interferon‐inducible RNA‐binding protein that plays an essential role in enhancing host IFN downstream signaling.     

Deletion of STAT5a/b in Vascular Smooth Muscle Abrogates the Male Bias in Hypoxic Pulmonary Hypertension in Mice: Implications in the Human Disease

Chronic hypoxia typically elicits pulmonary hypertension (PH) in mice with a male-dominant phenotype. There is an opposite-sex bias in human PH, with a higher prevalence in women, but greater survival (the “estrogen paradox”). We investigated the involvement of the STAT5a/b species, previously established to mediate sexual dimorphism in other contexts, in the sex bias in PH. Mice with heterozygous or homozygous deletions of the STAT5a/b locus in vascular smooth muscle cells (SMCs) were generated in crosses between STAT5a/b^fl/fl and transgelin (SM22α)-Cre^+/+ parents. Wild-type (wt ) males subjected to chronic hypoxia showed significant PH and pulmonary arterial remodeling, with wt females showing minimal changes (a male-dominant phenotype). However, in conditional STAT5^+/− or STAT5^−/− mice, hypoxic females showed the severest manifestations of PH (a female-dominant phenotype). Immunofluorescence studies on human lung sections showed that obliterative pulmonary arterial lesions in patients with idiopathic pulmonary arterial hypertension (IPAH) or hereditary pulmonary arterial hypertension (HPAH), both male and female, overall had reduced STAT5a/b, reduced PY-STAT5 and reduced endoplasmic reticulum (ER) GTPase atlastin-3 (ATL3). Studies of SMCs and endothelial cell (EC) lines derived from vessels isolated from lungs of male and female IPAH patients and controls revealed instances of coordinate reductions in STAT5a, STAT5b and ATL3 in IPAH-derived cells, including SMCs and ECs from the same patient. Taken together, these data provide the first definitive evidence for a contribution of STAT5a/b to the sex bias in PH in the hypoxic mouse and implicate reduced STAT5 in the pathogenesis of the human disease.     

Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling

Type I interferons (IFNs) activate differential cellular responses through a shared cell surface receptor composed of the two subunits, IFNAR1 and IFNAR2. We propose here a mechanistic model for how IFN receptor plasticity is regulated on the level of receptor dimerization. Quantitative single-molecule imaging of receptor assembly in the plasma membrane of living cells clearly identified IFN-induced dimerization of IFNAR1 and IFNAR2. The negative feedback regulator ubiquitin-specific protease 18 (USP18) potently interferes with the recruitment of IFNAR1 into the ternary complex, probably by impeding complex stabilization related to the associated Janus kinases. Thus, the responsiveness to IFNα2 is potently down-regulated after the first wave of gene induction, while IFNβ, due to its ∼100-fold higher binding affinity, is still able to efficiently recruit IFNAR1. Consistent with functional data, this novel regulatory mechanism at the level of receptor assembly explains how signaling by IFNβ is maintained over longer times compared with IFNα2 as a temporally encoded cause of functional receptor plasticity.     

Involvement of DNA-PKcs in the Type I IFN Response to CpG-ODNs in Conventional Dendritic Cells in TLR9-Dependent or -Independent Manners

CpG-ODNs activate dendritic cells (DCs) to produce interferon alpha (IFNα) and beta (IFNβ). Previous studies demonstrated that Toll-like receptor 9 (TLR9) deficient DCs exhibited a residual IFNα response to CpG-A, indicating that yet-unidentified molecules are also involved in induction of IFNα by CpG-A. Here, we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) but not Ku70 deficient BMDCs showed defective IFNα and IFNβ responses to CpG-A or CpG-B. Loss of both DNA-PKcs and TLR9 further reduced the IFNα response to CpG-A. These DNA-PKcs and TLR9 effects were mediated by their downstream Akt/mTORC1 pathway and downstream events IRAK1 and IKKα. Loss of DNA-PKcs, TLR9, MyD88 or IRAK4 impaired phosphorylation of Akt(S473), S6K, S6, IRAK1, or IKKα in BMDCs in response to CpG-ODNs. The residual IFNα and IFNβ in DNA-PKcs-deficient BMDCs were partially responsible for the induction of IL-6 and IL-12 by CpG-ODNs and their stimulatory effect was blocked by IFNAR1 neutralizing antibodies. Further analysis indicated that CpG-ODN associated with DNA-PKcs and Ku70, and induced DNA-PKcs’s interaction with TRAF3. Intriguingly, DNA-PKcs but not Ku70 expression level was reduced in TLR9-deficient BMDCs. Taken together, our data suggest that DNA-PKcs is an important mediator in the type I IFN response to CpG-ODNs in TLR9-dependent or -independent fashions.     

Wnt5a attenuates hypoxia-induced pulmonary arteriolar remodeling and right ventricular hypertrophy in mice

Hypoxic pulmonary hypertension (HPH), which is characterized by pulmonary arteriolar remodeling and right ventricular hypertrophy, is still a life-threatening disease with the current treatment strategies. The underlying molecular mechanisms of HPH remain unclear. Our previously published study showed that Wnt5a, one of the ligands in the Wnt family, was critically involved in the inhibition of hypoxia-induced pulmonary arterial smooth muscle cell proliferation by downregulation of β-catenin/cyclin D1 in vitro. In this study, we investigated the possible functions and mechanisms of Wnt5a in HPH in vivo. Recombinant mouse Wnt5a (rmWnt5a) or phosphate buffered saline (PBS) was administered to male C57/BL6 mice weekly from the first day to the end of the two or four weeks after exposed to hypoxia (10% O₂). Hypoxia-induced pulmonary hypertension was associated with a marked increase in β-catenin/cyclin D1 expression in lungs. Right ventricular systolic pressure and right ventricular hypertrophy index were reduced in animals treated with rmWnt5a compared with PBS. Histology showed less pulmonary vascular remodeling and right ventricular hypertrophy in the group treated with rmWnt5a than with PBS. Treatment with rmWnt5a resulted in a concomitant reduction in β-catenin/cyclin D1 levels in lungs. These data demonstrate that Wnt5a exerts its beneficial effects on HPH by regulating pulmonary vascular remodeling and right ventricular hypertrophy in a manner that is associated with reduction in β-catenin/cyclin D1 signaling. A therapy targeting the β-catenin/cyclin D1 signaling pathway might be a potential strategy for HPH treatment.     

Effect of Different Interferonα2 Preparations on IP10 and ET-1 Release from Human Lung Cells

Background

Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity.

Methods

Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL).

Results

IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells.

Conclusions

Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.     

Bridging the Species Divide: Transgenic Mice Humanized for Type-I Interferon Response

We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These “HyBNAR” (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.     

Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

Background

Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo.

Methodology/Principal Findings

We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)⁺ transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (∼20 µm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP⁺ BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP⁺ cells that localized to the pulmonary vasculature were α-smooth muscle actin⁺ and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner.

Conclusions/Significance

These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.     

Impaired Expression of Type I and Type II Interferon Receptors in HCV-Associated Chronic Liver Disease and Liver Cirrhosis

Purpose

Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients.

Methods

Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting.

Result

HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients.

Conclusion

HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.     

Type I Interferon Controls Propagation of Long Interspersed Element-1

Type I interferons (IFN) including IFNα and IFNβ are critical for the cellular defense against viruses. Here we report that increased levels of IFNβ were found in testes from mice deficient in MOV10L1, a germ cell-specific RNA helicase that plays a key role in limiting the propagation of retrotransposons including Long Interspersed Element-1 (LINE-1). Additional experiments revealed that activation of LINE-1 retrotransposons increases the expression of IFNβ and of IFN-stimulated genes. Conversely, pretreatment of cells with IFN suppressed the replication of LINE-1. Furthermore, the efficacy of LINE-1 replication was increased in isogenic cell lines harboring inactivating mutations in diverse elements of the IFN signaling pathway. Knockdown of the IFN receptor chain IFNAR1 also stimulated LINE-1 propagation in vitro. Finally, a greater accumulation of LINE-1 was found in mice that lack IFNAR1 compared with wild type mice. We propose that LINE-1-induced IFN plays an important role in restricting LINE-1 propagation and discuss the putative role of IFN in preserving the genome stability.     

Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFNα Response

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNα and β) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNα production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNα production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNα by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNα induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNα production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNα induction.