An update on enterovirus 71 infection and interferon type I response

Enteroviruses are members of Pichornaviridae family consisting of human enterovirus group A, B, C, and D as well as nonhuman enteroviruses. Hand, foot, and mouth disease (HFMD) is a serious disease which is usually seen in the Asia‐Pacific region in children. Enterovirus 71 and coxsackievirus A16 are two important viruses responsible for HFMD which are members of group A enterovirus. IFN α and β are two cytokines, which have a major activity in the innate immune system against viral infections. Most of the viruses have some weapons against these cytokines. EV71 has two main proteases called 2A and 3C, which are important for polyprotein processing and virus maturation. Several studies have indicated that they have a significant effect on different cellular pathways such as interferon production and signaling pathway. The aim of this study was to investigate the latest findings about the interaction of 2A and 3C protease of EV71 and IFN production/signaling pathway and their inhibitory effects on this pathway.     

Cigarette smoke attenuation of poly I:C-induced innate antiviral responses in human PBMC is mainly due to inhibition of IFN-beta production

The cellular response to dsRNA or its synthetic analog polyinosinic-polycytidylic acid (poly I:C) results in IRF-3-, IRF-7- and NF-kB-mediated activation of type 1 IFNs and pro-inflammatory cytokines critical for innate antiviral immune responses. To investigate whether cigarette smoke compromises type 1 IFN signaling in humans, peripheral blood mononuclear cells (PBMCs) from non-smoking individuals were treated with smoke-conditioned media (SCM) and stimulated with poly I:C. We observed a marked attenuation of IRF-3 and NF-kB activation in PBMCs exposed to SCM compared to control PBMCs. Similarly, PBMCs from smokers or splenocytes from smoke-exposed mice also displayed marked reduction of poly I:C-induced antiviral responses compared with either non-smokers or sham-exposed mice. Cigarette smoke was found to block the production of type I IFNs following poly I:C treatment and inhibit subsequent STAT1 activation. Finally, we confirmed that inhibition of IFN-beta, but not IFN-alpha, predominantly contributes to the cigarette smoke-mediated suppression of innate antiviral responses. These findings provide novel mechanistic insights to the susceptibility of cigarette smokers to viral infections.     

Modulation of innate immunity in human pancreatic islets infected with enterovirus in vitro

Present knowledge of innate immunity in infected cells relies on studies of cell lines and animal models. In this study, primary human pancreatic islets of Langerhans were used to study virus-host interactions in a model of the possible induction of type 1 diabetes by enterovirus (EV). Human islets were infected with a strain of EV isolated at onset of type 1 diabetes, or exposed to synthetic dsRNA (poly(I:C)), used commonly to mimic viral infection. Induction of innate immunity and the effect of the female sex hormone 17β-estradiol, known to have cell-protective effects, on islet chemokine secretion were investigated. 17β-Estradiol reduced EV-but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets, suggesting that separate signaling pathways of the innate immune response are triggered by EV and poly(I:C), respectively. Infection with EV increased the gene-expression of toll-like receptor 3, interferon-β, and the intracellular helicase MDA5, involved in antiviral innate immunity, multi-fold over time, whereas poly(I:C) increased the expression of these genes transiently. The induced expression pattern was similar in all donors, but the expression levels varied greatly. Pre-exposure to poly(I:C) blocked viral replication in islets from 56% of the donors. These data provide insight on the innate immune responses induced by EV in human islets, and show that this can be modulated by 17β-estradiol, and suggest an important difference between virus- and poly(I:C)-induced signaling.     

Induction of iNOS expression and antimicrobial activity by interferon (IFN)-beta is distinct from IFN-gamma in Burkholderia pseudomallei-infected mouse macrophages

Burkholderia pseudomallei is a causative agent of melioidosis. This Gram-negative bacterium is able to survive and multiple inside both phagocytic and nonphagocytic cells. We previously reported that exogenous interferons (both type I and type II) enhanced antimicrobial activity of the macrophages infected with B. pseudomallei by up-regulating inducible nitric oxide synthase (iNOS). This enzyme thus plays an essential role in controlling intracellular growth of bacteria. In the present study we extended our investigation, analysing the mechanism(s) by which the two types of interferons (IFNs) regulate antimicrobial activity in the B. pseudomallei-infected macrophages. Mouse macrophage cell line (RAW 264.7) that was exposed simultaneously to B. pseudomallei and type I IFN (IFN-beta) expressed high levels of iNOS, leading to enhanced intracellular killing of the bacteria. However, neither enhanced iNOS expression nor intracellular bacterial killing was observed when the macrophages were preactivated with IFN-beta prior to being infected with B. pseudomallei. On the contrary, the timing of exposure was not critical for the type II IFN (IFN-gamma) because when the cells were either prestimulated or co-stimulated with IFN-gamma, both iNOS expression and intracellular killing capacity were enhanced. The differences by which these two IFNs regulate antimicrobial activity may be related to the fact that IFN-gamma was able to induce more sustained interferon regulatory factor-1 (IRF-1) expression compared with the cells activated with IFN-beta.     

Listeria monocytogenes induces an interferon‐enhanced activation of the integrated stress response that is detrimental for resolution of infection in mice

Type I interferons (IFNs) induce a detrimental response during Listeria monocytogenes (L. monocytogenes) infection. We were interested in identifying mechanisms linking IFN signaling to negative host responses against L. monocytogenes infection. Herein, we found that infection of myeloid cells with L. monocytogenes led to a coordinated induction of type I IFNs and activation of the integrated stress response (ISR). Infected cells did not induce Xbp1 splicing or BiP upregulation, indicating that the unfolded protein response was not triggered. CHOP (Ddit3) gene expression was upregulated during the ISR activation induced by L. monocytogenes. Myeloid cells deficient in either type I IFN signaling or PKR activation had less upregulation of CHOP following infection. CHOP‐deficient mice showed lower expression of innate immune cytokines and were more resistant than wild‐type counterparts following L. monocytogenes infection. These findings indicate that L. monocytogenes infection induces type I IFNs, which activate the ISR through PKR, which contributes to a detrimental outcome in the infected host.     

Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN‐β production capable of improving dendritic cell function

Viral double‐stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine–polycytidylic acid (poly I:C) and polyadenylic–polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN‐β autocrine loop, dsRNA‐elicited IFN‐β production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN‐β at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte‐derived DCs. IFN‐β produced by poly I:C‐activated human cancer cells increased the capacity of monocyte‐derived DCs to stimulate IFN‐γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3^?/? mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1^?/? mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN‐β production and contribute to the antitumoral response.     

The effect of types I and III interferons on adrenocortical cells and its possible implications for autoimmune Addison's disease

Autoimmune Addison's disease (AAD) is caused by selective destruction of the hormone‐producing cells of the adrenal cortex. As yet, little is known about the potential role played by environmental factors in this process. Type I and/or type III interferons (IFNs) are signature responses to virus infections, and have also been implicated in the pathogenesis of autoimmune endocrine disorders such as type 1 diabetes and autoimmune thyroiditis. Transient development of AAD and exacerbation of established or subclinical disease, as well as the induction of autoantibodies associated with AAD, have been reported following therapeutic administration of type I IFNs. We therefore hypothesize that exposure to such IFNs could render the adrenal cortex susceptible to autoimmune attack in genetically predisposed individuals. In this study, we investigated possible immunopathological effects of type I and type III IFNs on adrenocortical cells in relation to AAD. Both types I and III IFNs exerted significant cytotoxicity on NCI‐H295R adrenocortical carcinoma cells and potentiated IFN‐γ‐ and polyinosine‐polycytidylic acid [poly (I : C)]‐induced chemokine secretion. Furthermore, we observed increased expression of human leucocyte antigen (HLA) class I molecules and up‐regulation of 21‐hydroxylase, the primary antigenic target in AAD. We propose that these combined effects could serve to initiate or aggravate an ongoing autoimmune response against the adrenal cortex in AAD.     

Autoimmunity is a type I interferon-deficiency syndrome corrected by ingested type I IFN via the GALT system.

Type I interferons (IFN-alpha/beta), products of the innate immune system, can modulate immune function whereas proinflammatory IFN-gamma (type II IFN), a product of the acquired immune system upregulates inflammation and enhances cell mediated immunity. We have proposed a unifying hypothesis of the origin of autoimmunity as a type I IFN immunodeficiency syndrome involving inadequate regulation of the acquired immune system product IFN-gamma by the IFN-alpha/beta innate immune system. The common theme of ingested type I IFNs in autoimmunity is inhibition of proinflammatory type II IFN systemically or at the target organ. In multiple sclerosis (MS) and insulin-dependent diabetes mellitus (IDDM) at the target organ, and in rheumatoid arthritis (RA) as a regulator of other proinflammatory cytokines, IFN-gamma is the nexus of inflammation in autoimmunity. Ingested type I IFNs counteract type II IFN, overcome the relative lack of type I IFN activity, and ameliorate autoimmunity. The administration of type I IFNs (IFN-alpha/beta) via the gut offers an exciting alternative to systemic application for overcoming the type I IFN immunodeficiency in autoimmunity. Successful use of ingested type I IFN in three separate prototypical autoimmune diseases suggests a broad antiinflammatory therapeutic profile for this technology.     

Type III interferons are expressed by Coxsackievirus‐infected human primary hepatocytes and regulate hepatocyte permissiveness to infection

Hepatitis is a common and potentially fatal manifestation of severe Coxsackievirus infections, particularly in newborn children. Little is known of the immune‐mediated mechanisms regulating permissiveness to liver infection. It is well established that type I interferons (IFNs) play an important role in the host innate immune response to Coxsackievirus infections. Recent studies have highlighted a role for another IFN family, the type III IFNs (also called IFN‐λ), in anti‐viral defence. Whether type III IFNs are produced by hepatocytes during a Coxsackievirus infection remains unknown. Moreover, whether or not type III IFNs protects hepatocytes from a Coxsackievirus infection has not been addressed. In this study, we show that primary human hepatocytes respond to a Coxsackievirus B3 (CVB3) infection by up‐regulating the expression of type III IFNs. We also demonstrate that type III IFNs induce an anti‐viral state in hepatocytes characterized by the up‐regulated expression of IFN‐stimulated genes, including IFN‐stimulated gene (ISG15), 2′‐5′‐oligoadenylate synthetase 2 (OAS2), protein kinase regulated by dsRNA (PKR) and myxovirus resistance protein 1 (Mx1). Furthermore, our study reveals that type III IFNs attenuate CVB3 replication both in hepatocyte cell lines and primary human hepatocytes. Our studies suggest that human hepatocytes express type III IFNs in response to a Coxsackievirus infection and highlight a novel role for type III IFNs in regulating hepatocyte permissiveness to this clinically relevant type of virus.     

RNA recognition and signal transduction by RIG-I-like receptors

Summary:  Viral infection is detected by cellular sensor molecules as foreign nucleic acids and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Recent identification of cytoplasmic viral sensors, such as retinoic acid-inducible gene-I-like receptors (RLRs), highlights their significance in the induction of antiviral innate immunity. Moreover, it is intriguing to understand how they can discriminate endogenous RNA from foreign viral RNA and initiate signaling cascades leading to the induction of type I IFNs. This review focuses on the current understanding of the molecular machinery underlying RNA recognition and subsequent signal transduction by RLRs.     

Inhibition of DC-SIGN-mediated transmission of human immunodeficiency virus type 1 by Toll-like receptor 3 signalling in breast milk macrophages

The majority of cells in early/colostrum milk are breast milk macrophages (BrMMø) expressing dendritic cell (DC)‐specific intercellular adhesion molecule 3 (ICAM3) grabbing nonintegrin (DC‐SIGN), and the expression level of DC‐SIGN on BrMMø will determine cell‐to‐cell human immunodeficiency virus type 1 (HIV‐1) transmissibility. Thus, one of the strategies to prevent vertical transmission of HIV‐1 through breast‐feeding is to find a way to suppress DC‐SIGN expression on BrMMø. As for the expression of Toll‐like receptors (TLRs) in BrMMø, TLR3 was always seen in BrMMø but not in peripheral blood monocytes (PBMo). Also, the expression of TLR3 was slightly enhanced in BrMMø when the cells were treated with interleukin (IL)‐4. Moreover, when TLR3 was stimulated with its specific ligand, the double‐stranded RNA (dsRNA) poly(I:C), DC‐SIGN expression on BrMMø was reduced even in the IL‐4‐mediated enhanced state. Some reduction may be caused by type I interferons (IFNs), such as IFN‐α/β, secreted from BrMMø. Indeed, both IFNs, particularly IFN‐β, showed a strong capacity to suppress the enhancement of DC‐SIGN expression on IL‐4‐treated BrMMø and such TLR3‐mediated DC‐SIGN suppression was partially abrogated by the addition of anti‐IFN‐α/β‐receptor‐specific antibodies. As expected, DC‐SIGN‐mediated HIV‐1 transmission to CD4‐positive cells by BrMMø was inhibited by either poly(I:C) stimulation or by treatment with type I IFNs. These findings suggest a possible strategy for preventing mother‐to‐child transmission (MTCT) of HIV‐1 via breast‐feeding through TLR3 signalling.     

Role of antiviral antibodies in resistance against coxsackievirus B3 infection: interaction between preexisting antibodies and an interferon inducer.

An experimental model of coxsackievirus B3 infection in newborn mice was utilized to examine the protective role of antiviral antibodies and an interferon inducer, polyinosinic acid-polycytidylic acid [poly(I:C)]. Subcutaneous administration to the infected mice of specific antiviral antibodies resulted in significant protection against coxsackievirus B3 infection. Antibody-treated animals had shortened viremia, early clearance of virus from tissues, and a reduced mortality rate. Dose response to antibodies was clearly demonstrated. However, the time of antibody administration in relation to the infection cycle was important. The protection was observed if antibodies were given before infection (24 h) or shortly after (2 h) infection. Administration of antibodies 24 h after infection resulted in no protection. The interferon inducer poly(I:C) prolonged the survival time of the infected mice, but this protective effect was incomplete and could only be demonstrated in animals treated before infection (24 h) or shortly after (2 h) infection. Enhanced protection against lethal coxsackievirus B3 infection was achieved in animals treated with a combination of antiviral antibodies and poly(I:C). These data confirm that antibody-mediated immunity plays a significant role in resistance against coxsackievirus B3 infection and suggest that antiviral antibodies may interact with poly(I:C) or work independently to produce an enhanced protective effect.     

B‐cell‐activating factor expressions in salivary epithelial cells after dsRNA virus infection depends on RNA‐activated protein kinase activation

B‐cell‐activating factor (BAFF) plays a key role in promoting activation of autoimmune B cells. This cytokine may be expressed in and secreted by salivary gland epithelial cells (SGEC) after stimulation with type I IFN or viral or synthetic dsRNA. Because this BAFF expression depends only in part on endosomal TLR and type I IFN, we investigated whether other dsRNA sensors could be implicated in BAFF expression. Using human SGEC, we confirmed the partial dependence of BAFF expression on TLR‐3 by replicating the partial inhibition of BAFF expression observed upon endosomal inhibition using TLR‐3 or Toll/IL‐1R domain‐containing protein inducing IFN‐β silencing mRNA, but not with TLR‐7 silencing mRNA. Melanoma differentiation‐associated gene 5 silencing mRNA had no effect on BAFF expression, but retinoic acid‐inducible gene I silencing mRNA had a slight effect observed following infection with dsRNA reovirus‐1. Inhibition of RNA‐activated protein kinase (PKR) by 2‐aminopurine completely abolished both BAFF mRNA and protein production after reovirus‐1 infection and poly(I:C) stimulation through NF‐κB and p38 MAPK pathways, with the latter implicated only after poly(I:C) stimulation. Thus, PKR is the dsRNA sensor implicated in BAFF induction in SGEC after dsRNA stimulation. In autoimmune diseases, PKR may be an interesting target for preventing BAFF following the induction of innate immunity.     

Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells

The objective of this study was to examine the expression of Toll-like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid [poly(I:C)] induces an antiviral response in these cells. Fluorescence-activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)-kappaB and production of the proinflammatory cytokine interleukin (IL)-6 and the chemokine IL-8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)-beta mRNA expression (7-fold). Poly(I:C) stimulation also up-regulated mRNA expression of the antiviral chemokine IFN-gamma inducible protein 10 (IP10), myxovirus resistance gene A and 2',5'-oligoadenylate synthetase (5-, 10- and 9-fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti-TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)-mediated IFN-beta expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection.     

Roles of Type I and III Interferons in COVID-19

Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Type I and III interferon (IFN) responses act as the first line of defense against viral infection and are activated by the recognition of viruses by infected cells and innate immune cells. Dysregulation of host IFN responses has been known to be associated with severe disease progression in COVID-19 patients. However, the reported results are controversial and the roles of IFN responses in COVID-19 need to be investigated further. In the absence of a highly efficacious antiviral drug, clinical studies have evaluated recombinant type I and III IFNs, as they have been successfully used for the treatment of infections caused by two other epidemic coronaviruses, SARS-CoV-1 and Middle East respiratory syndrome (MERS)-CoV. In this review, we describe the strategies by which SARS-CoV-2 evades IFN responses and the dysregulation of host IFN responses in COVID-19 patients. In addition, we discuss the therapeutic potential of type I and III IFNs in COVID-19.     

Defective RNA sensing by RIG‐I in severe influenza virus infection

Influenza virus infection causes worldwide seasonal epidemics. Although influenza is usually a mild disease, a minority of patients experience very severe fulminating disease courses. Previous studies have demonstrated a role for type I interferon (IFN) in anti‐viral responses during influenza. So far, however, IFN regulatory factor (IRF)7 deficiency is the only genetic cause of severe influenza described in humans. In this study we present a patient with severe influenza A virus (IAV) H1N1 infection during the 2009 swine flu pandemic. By whole exome sequencing we identified two variants, p.R71H and p.P885S, located in the caspase activation and recruitment domain (CARD) and RNA binding domains, respectively, of DExD/H‐box helicase 58 (DDX58) encoding the RNA sensor retinoic acid inducible gene 1 (RIG‐I). These variants significantly impair the signalling activity of RIG‐I. Similarly, patient cells demonstrate decreased antiviral responses to RIG‐I ligands as well as increased proinflammatory responses to IAV, suggesting dysregulation of the innate immune response with increased immunopathology. We suggest that these RIG‐I variants may have contributed to severe influenza in this patient and advocate that RIG‐I variants should be sought in future studies of genetic factors influencing single‐stranded RNA virus infections.     

Critical role of the endogenous interferon ligand–receptors in type I and type II interferons response

Separate ligand–receptor paradigms are commonly used for each type of interferon (IFN). However, accumulating evidence suggests that type I and type II IFNs may not be restricted to independent pathways. Using different cell types deficient in IFNAR1, IFNAR2, IFNGR1, IFNGR2 and IFN‐γ, we evaluated the contribution of each element of the IFN system to the activity of type I and type II IFNs. We show that deficiency in IFNAR1 or IFNAR2 is associated with impairment of type II IFN activity. This impairment, presumably resulting from the disruption of the ligand–receptor complex, is obtained in all cell types tested. However, deficiency of IFNGR1, IFNGR2 or IFN‐γ was associated with an impairment of type I IFN activity in spleen cells only, correlating with the constitutive expression of type II IFN (IFN‐γ) observed on those cells. Therefore, in vitro the constitutive expression of both the receptors and the ligands of type I or type II IFN is critical for the enhancement of the IFN activity. Any IFN deficiency can totally or partially impair IFN activity, suggesting the importance of type I and type II IFN interactions. Taken together, our results suggest that type I and type II IFNs may regulate biological activities through distinct as well as common IFN receptor complexes.     

Enhancement of anti-Aspergillus T helper type 1 response by interferon-��-conditioned dendritic cells

Although data show the importance of type I interferons (IFNs) in the regulation of the innate and adaptive immunity elicited in response to viral, bacterial and parasitic infections, the functional activities of these cytokines during fungal infections are poorly understood. We examined here the impact of IFN‐β on the response of human monocyte‐derived dendritic cells (DCs) infected in vitro with Aspergillus fumigatus. Having found that A. fumigatus‐infected DCs do not express IFN‐β, we evaluated the effect of the exogenous addition of IFN‐β on the maturation of human DCs induced by the infection with A. fumigatus conidia. Although the phagocytosis of the fungus was not affected by IFN‐β treatment, the expression of CD86 and CD83 induced upon A. fumigatus challenge was enhanced in IFN‐β‐conditioned DCs, which also showed an increased expression of IL‐27 and IL‐12p70, members of IL‐12 family. Through these modifications, IFN‐β improved the capacity of DCs to promote an anti‐Aspergillus T helper type 1 response, as evaluated by mixed leucocyte reaction, which plays a crucial role in the control of invasive aspergillosis. Our results identified a novel effect of IFN‐β on anti‐Aspergillus immune responses which, in turn, might open new perspectives on the use of IFN‐β in immunotherapy for fungal infections aimed at enhancing the immunological functions of DCs.