Arsenic programmes cellular genomic-immunity through miR-2909 RNomics

It is widely recognized that human cells are equipped with innate antiviral-RNA armour involving the production of type I interferons and APOBEC3G (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G) gene-product. Although arsenic has been shown to have paradoxical effect on one arm of this armour involving APOBEC3G, the exact molecular mechanism of its action in this regard is far from clear. The present study, addressed to explore as to how arsenic programmes this innate antiviral-RNA cellular-sensing pathway, clearly revealed that arsenic programmes this innate cellular antiviral genomic response through its inherent capacity to initiate cellular miR-2909 RNomics pathway, involving not only the modulation of APOBEC3G gene but also KLF4 (Kruppel-like factor 4) dependent regulation of gene coding for IKBKε (Inhibitor of nuclear factor kappa-B kinase subunit epsilon) which in turn modulates RIG-I (retinoic acid-inducible gene 1) pathway responsible for the production of IFNβ (interferon beta) through restriction of CYLD (Cylindromatosis) deubiqutinating activity. This restricted inhibitory enzyme activity of CYLD upon NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) also ensures sustained expression of miR-2909. Our results for the first time show that cellular miR-2909 RNomics may constitute an innate genomic armour to promote as well as restrict retroviral infection.     

High Basal Expression of Interferon-Stimulated Genes in Human Bronchial Epithelial (BEAS-2B) Cells Contributes to Influenza A Virus Resistance

Respiratory epithelial cells play a key role in influenza A virus (IAV) pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV−host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77) IAVs in normal primary human bronchial epithelial (NHBE) and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK) cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7), stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK) inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo.     

Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene

Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory tract infection for which no vaccine or antiviral treatment is available. The RSV NS1 protein seems to antagonize the host interferon (IFN) response; however, its mechanism is unknown. Here, we used a plasmid-borne small interfering RNA targeting the NS1 gene (siNS1) to examine the role of NS1 in modulating RSV infection. RSV replication was reduced in A549 cells, but not IFN-deficient Vero cells, transfected with siNS1. siNS1 induced upregulated expression of IFN-beta and IFN-inducible genes in A549 cells. siNS1-transfected human dendritic cells, upon RSV infection, produced elevated type-1 IFN and induced differentiation of naive CD4+ T cells to T helper type 1 (TH1) cells. Mice treated intranasally with siNS1 nanoparticles before or after infection with RSV showed substantially decreased virus titers in the lung and decreased inflammation and airway reactivity compared to controls. Thus, siNS1 nanoparticles may provide an effective inhibition of RSV infection in humans.     

Effect of chemokine receptor CX3CR1 deficiency in a murine model of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory illness in infants and young children worldwide, making it a high priority for development of strategies for prevention and treatment. RSV can cause repeat infections throughout life, with serious complications in elderly and immunocompromised patients. Previous studies indicate that the RSV G protein binds through a CX3C chemokine motif to the host chemokine receptor, CX3CR1, and modulates the inflammatory immune response. In the current study, we examined the contribution of CX3CR1 to the immune response to RSV infection in mice. CX3CR1-deficient mice showed an impaired innate immune response to RSV infection, characterized by substantially decreased NK1.1(+) natural killer, CD11b(+), and RB6-8C5(+) polymorphonuclear cell trafficking to the lung and reduced IFNγ production compared with those in wildtype control mice. Leukocytes from CX3CR1-deficient mice were poorly chemotactic toward RSV G protein and CX3CL1. These results substantiate the importance of the RSV G CX3C-CX3CR1 interaction in the innate immune response to RSV infection.     

Effects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D--glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.     

A role for IL-18 in neutrophil activation

IL-18 expression and functional activity has been identified in several autoimmune and infectious diseases. To clarify the potential role of IL-18 during early innate immune responses, we have explored the capacity of IL-18 to activate neutrophils. Human peripheral blood-derived neutrophils constitutively expressed IL-18R (alpha and beta) commensurate with the capacity to rapidly respond to IL-18. IL-18 induced cytokine and chemokine release from neutrophils that was protein synthesis dependent, up-regulated CD11b expression, induced granule release, and enhanced the respiratory burst following exposure to fMLP, but had no effect upon the rate of neutrophil apoptosis. The capacity to release cytokine and chemokine was significantly enhanced in neutrophils derived from rheumatoid arthritis synovial fluid, indicating differential responsiveness to IL-18 dependent upon prior neutrophil activation in vivo. Finally, IL-18 administration promoted neutrophil accumulation in vivo, whereas IL-18 neutralization suppressed the severity of footpad inflammation following carrageenan injection. The latter was accompanied by reduction in tissue myeloperoxidase expression and suppressed local TNF-alpha production. Together, these data define a novel role for IL-18 in activating neutrophils and thereby promoting early innate immune responses.     

Transforming growth factor-Beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response

Rhinovirus (RV) infection is a major cause of asthma exacerbations which may be due to a deficient innate immune response in the bronchial epithelium. We hypothesized that the pleiotropic cytokine, TGF-β, influences interferon (IFN) production by primary bronchial epithelial cells (PBECs) following RV infection. Exogenous TGF-β(2) increased RV replication and decreased IFN protein secretion in response to RV or double-stranded RNA (dsRNA). Conversely, neutralizing TGF-β antibodies decreased RV replication and increased IFN expression in response to RV or dsRNA. Endogenous TGF-β(2) levels were higher in conditioned media of PBECs from asthmatic donors and the suppressive effect of anti-TGF-β on RV replication was significantly greater in these cells. Basal SMAD-2 activation was reduced when asthmatic PBECs were treated with anti-TGF-β and this was accompanied by suppression of SOCS-1 and SOCS-3 expression. Our results suggest that endogenous TGF-β contributes to a suppressed IFN response to RV infection possibly via SOCS-1 and SOCS-3.     

Acute osmotic stress affects Tilapia (Oreochromis mossambicus) innate immune responses

Most of the tilapia studies are focused on its osmoregulatory mechanism. Meanwhile, less information is available about its innate immune response on fish faced with hyperosmolality. In the present study, in vivo analyses were carried out to investigate the innate immune response of Oreochromis mossambicus, transferred from freshwater to 25 ppt seawater (SW). In vivo, lysozyme activities of plasma and head kidney (HK) were increased at 1h and at 24h after transfer to SW but decreased at 8h after SW transfer. Surprisingly, the alternative complement pathway in plasma increased 8h after SW transfer. The phagocytic capacity of spleen and HK immune cells increased modestly at 1h and at 4h, after SW transfer, but the respiratory burst activity of immune cells in both HK and spleen shows an increase in superoxide release at 8h after SW transfer. Our results reveal that the transfer of fish from conditions of hypoosmolarity to hyperosmolality significantly enhances plasma lysozyme, ACP activity, and both phagocytic and respiratory burst activity. Taken together, the results indicate that exposure of tilapia to hyperosmotic conditions has immunostimulatory effects on its cellular immune reactions (phagocytosis and respiratory burst activity) and humoral reactions (lysozyme activity and complement activity).     

Poly (I:C), an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells

ABSTRACT: BACKGROUND: Double-stranded RNA (dsRNA) and its mimic, polyinosinic acid: polycytidylic acid [Poly (I:C)], are recognized by toll-like receptor 3 (TLR3) and induce interferon (IFN)-beta in many cell types. Poly (I:C) is the most potent IFN inducer. In in vivo mouse studies, intraperitoneal injection of Poly (I:C) elicited IFN-alpha/beta production and natural killer (NK) cells activation. The TLR3 pathway is suggested to contribute to innate immune responses against many viruses, including influenza virus, respiratory syncytial virus, herpes simplex virus 2, and murine cytomegalovirus. In Chikungunya virus (CHIKV) infection, the viruses are cleared within 7-10 days postinfection before adaptive immune responses emerge. The innate immune response is important for CHIKV clearance. RESULTS: The effects of Poly (I:C) on the replication of CHIKV in human bronchial epithelial cells, BEAS-2B, were studied. Poly (I:C) suppressed cytopathic effects (CPE) induced by CHIKV infection in BEAS-2B cells in the presence of Poly (I:C) and inhibited the replication of CHIKV in the cells. The virus titers of Poly (I:C)-treated cells were much lower compared with those of untreated cells. CHIKV infection and Poly (I:C) treatment of BEAS-2B cells induced the production of IFN-beta and increased the expression of anti-viral genes, including IFN-alpha, IFN-beta, MxA, and OAS. Both Poly (I:C) and CHIKV infection upregulate the expression of TLR3 in BEAS-2B cells. CONCLUSIONS: CHIKV is sensitive to innate immune response induced by Poly (I:C). The inhibition of CHIKV replication by Poly (I:C) may be through the induction of TLR3, which triggers the production of IFNs and other anti-viral genes. The innate immune response is important to clear CHIKV in infected cells.     

Respiratory syncytial virus nonstructural proteins NS1 and NS2 mediate inhibition of Stat2 expression and alpha/beta interferon responsiveness

Respiratory syncytial virus (RSV) subverts the antiviral interferon (IFN) response, but the mechanism for this evasion was unclear. Here we show that RSV preferentially inhibits IFN-alpha/beta signaling by expression of viral NS1 and NS2. Thus, RSV infection or expression of recombinant NS1 and NS2 in epithelial host cells causes a marked decrease in Stat2 levels and the consequent downstream IFN-alpha/beta response. Similarly, NS1/NS2-deficient RSV no longer decreases Stat2 levels or IFN responsiveness. RSV infection decreased human but not mouse Stat2 levels, so this mechanism of IFN antagonism may contribute to viral host range, as well as immune subversion.     

Mycobacterium abscessus glycopeptidolipid prevents respiratory epithelial TLR2 signaling as measured by HβD2 gene expression and IL-8 release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.     

Immunomodulatory effects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune system.

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0 mg (control), 25, 50 or 100 mg kg(-1) chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.