The effect of IFN-ß 1a on expression of MDA5 and RIG-1 in multiple sclerosis patients.

The aims of this study were to compare mRNA levels of melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene 1 (RIG-1) in multiple sclerosis (MS) patients in comparison to the healthy controls as well as investigating the effects of IFN-ß 1a on the expression of these molecules. In this study, mRNA levels of MDA5 and RIG-1 in peripheral leukocytes of 30 new cases of MS patients and 35 healthy controls were evaluated using the real-time-PCR method. mRNA levels of MDA5 and RIG-1 were determined in the MS patients 6 months after treatment with standard doses of IFN-ß 1a. mRNA levels of MDA5 and RIG-1 were significantly decreased in the MS patients in comparison to the healthy controls. The analysis also revealed that IFN-ß 1a therapy leads to the upregulation of RIG-1, but not MDA5, in the total MS patients and the female group. MS patients suffer from insufficient expression of MDA5 and RIG-1, and IFN-ß 1a therapy results in the upregulation of RIG-1 in the patients, especially in the female patients. Thus, it seems that IFN-ß 1a not only decreased pathogenic inflammatory responses but also modulated the expression of RIG-1 to protect the patients from infectious diseases and upregulation of IFN-I in a positive feedback.

The RIG-I pathway is involved in peripheral T cell lymphopenia in patients with dermatomyositis.

BACKGROUND: Peripheral T cell lymphopenia is a clinical phenomenon in some patients with dermatomyositis (DM). Patients with T cell lymphopenia are more susceptible to life-threatening infections. However, the pathogenesis of T cell lymphopenia remains unclear. In this study, we aimed to determine retinoic acid-inducible gene I (RIG-I) expression in peripheral T lymphocytes and explore the correlation between RIG-I and T cell lymphopenia in DM. METHODS: The mRNA and protein expression levels of RIG-I were determined in peripheral T lymphocytes of 26 treatment-naive DM patients by q-PCR and Western blot. The apoptosis of peripheral T lymphocytes was detected by flow cytometry. The associations between RIG-I expression levels and clinical characteristics were investigated. In Jurkat cell, we examined the relationship between RIG-I and cell apoptosis following RIG-I overexpression or activation by specific ligand (pppRNA). The CRISPR/Cas9 gene editing system was used for RIG-I knockout. Fas and caspase 3 were identified by Western blot. CCK8 colorimeter was performed to monitor cell proliferation. RESULTS: In DM patients, we observed the peripheral T lymphocyte count decreased notably while the apoptosis of T lymphocytes increased significantly compared with healthy control. RIG-I expression levels in peripheral T cell correlated negatively with T cell count in DM patients. RIG-I protein expression decreased significantly, and the number of T cell increased when disease was improved. In Jurkat cells, increased apoptosis and elevated expression of Fas and cleaved-caspase 3 protein were observed following RIG-I overexpression or RIG-I-specific ligand (pppRNA) activation. Meanwhile, the proliferation of Jurkat cells was markedly reduced. Whereas, neither cell apoptosis nor the cell viability of the RIG-I knockout clones exhibited significant changes following pppRNA activation. CONCLUSION: Our study showed for the first time that negative correlation between the increased RIG-I expression in peripheral T lymphocyte and T cell count in some patients with DM. We demonstrated that highly expressed RIG-I played a critical role in inducing apoptosis and inhibiting proliferation of T lymphocyte in vitro. Therefore, RIG-I-mediated apoptosis may be one of the possible mechanisms of T cell lymphopenia in some patients with DM. These findings expand our existing knowledge on the mechanisms of innate immunity in pathogenesis and provide new therapeutic avenues for DM.

Bronchial mucosal IFN-α/ß and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations.

BACKGROUND: The innate immune system senses viral infection through pattern recognition receptors (PRRs), leading to type I interferon production. The role of type I interferon and PPRs in rhinovirus-induced asthma exacerbations in vivo are uncertain. OBJECTIVES: We sought to compare bronchial mucosal type I interferon and PRR expression at baseline and after rhinovirus infection in atopic asthmatic patients and control subjects. METHODS: Immunohistochemistry was used to detect expression of IFN-α, IFN-ß, and the PRRs: Toll-like receptor 3, melanoma differentiation-associated gene 5, and retinoic acid-inducible protein I in bronchial biopsy specimens from 10 atopic asthmatic patients and 15 nonasthmatic nonatopic control subjects at baseline and on day 4 and 6 weeks after rhinovirus infection. RESULTS: We observed IFN-α/ß deficiency in the bronchial epithelium at 3 time points in asthmatic patients in vivo. Lower epithelial IFN-α/ß expression was related to greater viral load, worse airway symptoms, airway hyperresponsiveness, and reductions in lung function during rhinovirus infection. We found lower frequencies of bronchial subepithelial monocytes/macrophages expressing IFN-α/ß in asthmatic patients during infection. Interferon deficiency at baseline was not accompanied by deficient PRR expression in asthmatic patients. Both epithelial and subepithelial PRR expression were induced during rhinovirus infection. Rhinovirus infection-increased numbers of subepithelial interferon/PRR-expressing inflammatory cells were related to greater viral load, airway hyperresponsiveness, and reductions in lung function. CONCLUSIONS: Bronchial epithelial IFN-α/ß expression and numbers of subepithelial IFN-α/ß-expressing monocytes/macrophages during infection were both deficient in asthmatic patients. Lower epithelial IFN-α/ß expression was associated with adverse clinical outcomes after rhinovirus infection in vivo. Increases in numbers of subepithelial cells expressing interferon/PRRs during infection were also related to greater viral load/illness severity.

Central Role of the NF-κB Pathway in the Scgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-Induced Airway Inflammation.

Lower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin (Scgb1a1)-expressing bronchiolar epithelial cells in RSV infection. Mice expressing a tamoxifen (TMX)-inducible Cre recombinase-estrogen receptor fusion protein (CreERTM) knocked into the Scgb1a1 locus were crossed with mice that harbor a RelA conditional allele (RelAfl ), with loxP sites flanking exons 5 to 8 of the Rel homology domain. The Scgb1a1CreERTM/+ × RelAfl/fl mouse is a RelA conditional knockout (RelACKO) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelACKO mice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NF-κB-dependent cytokines in response to RSV. In addition, RelACKO mice had reduced expression levels of interferon (IFN) regulatory factor 1/7 (IRF1/7) and retinoic acid-inducible gene I (RIG-I), components of the mucosal IFN positive-feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA polymerase II (Pol II) phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser2 Pol II formation, histone H3K122 acetylation, and cytokine secretion in vitro and in vivo TMX-treated RelACKO mice have less weight loss and reduced airway obstruction/hyperreactivity yet similar levels of IFN-γ production despite higher levels of virus production. These data indicate that the nonciliated Scgb1a1-expressing epithelium is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation and chemokine and mucosal IFN production via the RelA-BRD4 pathway.IMPORTANCE RSV infection is the most common cause of infant hospitalizations in the United States, resulting in 2.1 million children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NF-κB/RelA transcription factor in this subset of epithelial cells. These mice show an impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores and airway hyperreactivity yet increased levels of virus replication. We conclude that RelA-BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection.

RIG-I overexpression decreases mortality of cigarette smoke exposed mice during influenza A virus infection.

BACKGROUND: Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines which participate in clearing viral infections. Cigarette smoke (CS) exposure increases the frequency and severity of respiratory tract infections. METHODS: We generated a RIG-I transgenic (TG) mouse strain that expresses the RIG-I gene product under the control of the human lung specific surfactant protein C promoter. We compared the mortality and host immune responses of RIG-I TG mice and their litter-matched wild type (WT) mice following challenge with influenza A virus (IAV). RESULTS: RIG-I overexpression increased survival of IAV-infected mice. CS exposure increased mortality in WT mice infected with IAV. Remarkably, the effect of RIG-I overexpression on survival during IAV infection was enhanced in CS-exposed animals. CS-exposed IAV-infected WT mice had a suppressed innate response profile in the lung compared to sham-exposed IAV-infected WT mice in terms of the protein concentration, total cell count and inflammatory cell composition in the bronchoalveolar lavage fluid. RIG-I overexpression restored the innate immune response in CS-exposed mice to that seen in sham-exposed WT mice during IAV infection, and is likely responsible for enhanced survival in RIG-I TG mice as restoration preceded death of the animals. CONCLUSIONS: Our results demonstrate that RIG-I overexpression in mice is protective for CS enhanced susceptibility of smokers to influenza infection, and that CS mediated RIG-I suppression may be partially responsible for the increased morbidity and mortality of the mice exposed to IAV. Thus, optimizing the RIG-I response may be an important treatment strategy for CS-enhanced lung infections, particularly those due to IAV.

Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production.

Type I interferons (IFN-α/ß) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11. Since nsp11 is an endoribonuclease (EndoU), the structure function relationship was examined using a series of nsp11 EndoU mutant plasmids. The mutants that impaired the EndoU activity failed to suppress IFN and led to the normal expression of MAVS. Seven single amino acid substitutions (4 in subdomain A and 3 in subdomain B) plus one insertion (frame-shift in nsp11) were then introduced into PRRSV infectious cDNA clones to generate nsp11 mutant viruses. Unfortunately, all EndoU knock-out nsp11 mutant viruses appeared replication-defective and no progenies were produced. Three mutations in EndoU subdomain A expressed the N and nsp2/3 proteins but their infectivity diminished after 2 passages. Taken together, our data show that PRRSV nsp11 endoribonuclease activity is critical for both viral replication and IFN antagonism. More importantly, the endoribonuclease activity of nsp11 demonstrates the substrate specificity towards MAVS and RIG-I (transcripts and proteins) over p65 and IRF3 in the context of gene transfection and overexpression. This is likely a mechanism of nsp11 suppression of type I IFN production.

Expression and functionality of Toll- and RIG-like receptors in HepaRG cells.

BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells. METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection. RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines. CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system.