Exposure to cigarette smoke suppresses IL-15 generation and its regulatory NK cell functions in poly I:C-augmented human PBMCs

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Rα in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Rα production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.     

Impairment of human NK cell cytotoxic activity and cytokine release by cigarette smoke

NK cells play essential roles in innate host defense against microbial infections and tumor surveillance. Although evidence suggests that smoking has adverse effects on the immune system, little is known about whether smoking compromises NK cell effector functions. In this study, we show that cigarette smoke-conditioned medium (SCM) dose-dependently inhibits in vitro IFN-gamma production by polyinosinic:polycytidylic acid (poly I:C)-activated PBMC and NK cells isolated from nonsmoking individuals. Similarly, SCM attenuated poly I:C-induced TNF-alpha production by PBMC and NK cells. The inhibitory effect of cigarette smoke on TNF-alpha production was reversible. PBMC and NK cells isolated from smokers displayed significant reduction of IFN-gamma and TNF-alpha secretions compared with nonsmokers in response to poly I:C activation. We further observed that SCM attenuated NK cell cytotoxic activity, which was associated with decreased up-regulation of perforin expression. Attenuated cytotoxic activity was also observed in PBMCs isolated from smokers. Finally, anti-IL-12 mAb-blocking data revealed that an attenuation of IFN-gamma production by PBMC was indirect, likely via attenuation of IL-12 production, and the effect on NK cells was IL-12-independent. Our data indicate that cigarette smoke compromises function of human NK cells. This may contribute to a higher incidence of viral infections and cancer among smokers.     

Polyinosinic-polycytidylic acid-mediated stimulation of human gammadelta T cells via CD11c dendritic cell-derived type I interferons

The recognition of pathogen-associated molecular patterns (PAMPs) by the innate immune system is a crucial step in inducing effective immune responses. Double-stranded RNA [mimicked by polyinosinic-polycytidylic acid (poly(I:C)], synthesized by various types of viruses, represents one important member of these immunostimulatory microbial components. Here we report that poly(I:C) has potent gammadelta T-cell costimulatory capacity. Within peripheral blood mononuclear cells, poly(I:C)-stimulated gammadelta T cells expressed increased levels of CD69 and exhibited significantly enhanced antigen-mediated proliferation in response to isopentenylpyrophosphate (IPP). Among several recombinant cytokines tested, type I interferons (IFN-alpha, IFN-beta) and interleukin-15 (IL-15) showed a similar activation pattern of gammadelta T cells. gammadelta T-cell clones and purified gammadelta T cells did not respond to poly(I:C), indicating indirect effects of this compound. Depletion of CD11c(+) dendritic cells (DC), which express Toll-like receptor 3 (TLR3), known to recognize poly(I:C), abrogated poly(I:C)-mediated stimulation of gammadelta T cells. In addition, the supernatant of poly(I:C)-treated CD11c(+) DC was able to mimic the stimulatory effects of poly(I:C) on gammadelta T cells. Experiments with neutralizing antibodies indicated that type I IFNs, but not IL-15, contributed to the poly(I:C)-mediated activation of gammadelta T cells. In conclusion, gammadelta T-cell activation by immunostimulatory double-stranded RNA, such as poly(I:C), is indirectly mediated via type I IFNs derived from TLR3-expressing CD11c(+) DCs. These results suggest that upon confrontation with certain viruses, gammadelta T cells can be rapidly activated by type I interferons and may contribute to effective antiviral responses.     

An antiserum against Atlantic salmon IFNa1 detects IFN and neutralizes antiviral activity produced by poly I:C stimulated cells

Type I interferons (IFNs) play a crucial role in innate immune responses against virus infections in vertebrates. Two IFNs (IFNa1 and IFNa2) have previously been cloned from Atlantic salmon. In the present work a polyclonal antiserum, which was generated against salmon IFNa1 was used to study its production in cells by immunoblot detection and neutralization of antiviral activity. The antiserum was first confirmed to detect and neutralize the antiviral activity of recombinant salmon IFNa1 produced in HEK293 cells. The antiserum also detected IFNa1 and neutralized 95–98% of the antiviral activity in supernatants of poly I:C stimulated salmon TO cells. This suggests that IFNa1/IFNa2 are the major IFNs produced by poly I:C stimulated TO cells. The antiserum neutralized most of the IFN activity in poly I:C stimulated head kidney leucocytes from three of five individuals, but in stimulated leucocytes from the other two individuals only 75% of the antiviral activity was neutralized. This shows that although IFNa1/IFNa2 are major IFNs secreted by poly I:C stimulated leucocytes, these cells can also produce additional molecules with IFN-like activity.     

Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice

Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)‐6, monocyte chemoattractant protein‐1, tumor necrosis factor, and IFN‐γ. EV 71 infection abolished both poly (I:C)‐ and CB3‐induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71‐infected RAW264.7 cells produced significantly lower amount of type I IFNs than non‐infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over‐expression systems in both mice and RAW264.7 cells. The 3C over‐expression, however, did not interfere with poly (I:C)‐induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein. J. Med. Virol. 84:1779–1789, 2012. © 2012 Wiley Periodicals, Inc.     

IFN-λs

For decades, type I IFNs have been considered indispensable and unique antiviral mediators for the activation of rapid innate antiviral protection. However, the recent discovery of type III IFNs is challenging this paradigm. Since their identification in 2002/2003 by two independent groups, type III IFNs or IFN-λs, also known as IL-28/29, have been the subject of increased study with consequent recognition of their importance in virology and immunology. Initial reports suggested that IFN-λs functionally resemble type I IFNs. Although IFN-λs and classical type I IFNs (IFN-α/β) utilize distinct receptor complexes for signaling, both types of IFNs activate similar intracellular signaling pathways and biological activities, including the ability to induce antiviral state in cells, and both type I and type III IFNs are induced by viral infection. However, different antiviral potency, pattern of their induction and differential tissue expression of their corresponding receptor subunits suggest that the type I and type III IFN antiviral systems do not merely duplicate each other. Recent studies have started to reveal unique biological activities of IFN-λs in and beyond innate antiviral 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.     

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.     

Post‐translational regulation of antiviral innate signaling

The innate immune system initiates immune responses by pattern‐recognition receptors (PRR). Virus‐derived nucleic acids are sensed by the retinoic acid‐inducible gene I (RIG‐I)‐like receptor (RLR) family and the toll‐like receptor (TLR) family as well as the DNA sensor cyclic GMP‐AMP (cGAMP) synthase (cGAS). These receptors activate IRF3/7 and NF‐κB signaling pathways to induce the expression of type I interferons (IFNs) and other cytokines firing antiviral responses within the cell. However, to achieve a favorable outcome for the host, a balanced production of IFNs and activation of antiviral responses is required. Post‐translational modifications (PTMs), such as the covalent linkage of functional groups to amino acid chains, are crucial for this immune homeostasis in antiviral responses. Canonical PTMs including phosphorylation and ubiquitination have been extensively studied and other PTMs such as methylation, acetylation, SUMOylation, ADP‐ribosylation and glutamylation are being increasingly implicated in antiviral innate immunity. Here we summarize our recent understanding of the most important PTMs regulating the antiviral innate immune response, and their role in virus‐related immune pathogenesis.     

The Role of Type I IFNs in Influenza: Antiviral Superheroes or Immunopathogenic Villains?

The important role of interferons (IFNs) in antiviral innate immune defense is well established. Although recombinant IFN-α was approved for cancer and chronic viral infection treatment by regulatory agencies in many countries starting in 1986, no IFNs are approved for treatment of influenza A virus (IAV) infection. This is partially due to the complex effects of IFNs in acute influenza infection. IAV attacks the human respiratory system and causes significant morbidity and mortality globally. During influenza infection, depending on the strain of IAV and the individual host, type I IFNs can have protective antiviral effects or can contribute to immunopathology. In the context of virus infection, the immune system has complicated mechanisms regulating the expression and effects of type I IFN to maximize the antiviral response by both activating and enhancing beneficial innate cell function, while limiting immunopathological responses that lead to exaggerated tissue damage. In this review, we summarize the complicated, but important, role of type I IFNs in influenza infections. This includes both protective and harmful effects of these important cytokines during infection.     

Poly(I.C)-induced interferons enhance susceptibility of SCID mice to systemic candidiasis.

In the absence of any demonstrable T- or B-cell responses, gnotobiotic CB-17 SCID (severe combined immunodeficient) mice not only show innate resistance to acute systemic (intravenous challenge) candidiasis but also manifest innate resistance to systemic candidiasis of endogenous (gastrointestinal tract) origin. Poly(I. C), a potent inducer of interferons (IFNs) in vivo, enhanced the susceptibility of CB-17 SCID mice to acute systemic candidiasis and to systemic candidiasis of endogenous origin, as demonstrated by increased numbers of viable Candida albicans in internal organs after poly(I. C) treatment. The poly(I. C)-enhanced susceptibility of mice to candidiasis was abrogated by in vivo treatment with antibodies to IFN-alpha, -beta, and -gamma. In vivo depletion of natural killer cells from SCID mice did not significantly enhance their susceptibility to systemic candidiasis or abrogate poly(I. C)-enhanced susceptibility. In vivo and in vitro, treatment with poly(I. C) impaired the candidacidal and phagocytic activity of thioglycollate-elicited macrophages from SCID mice. Antibody to IFN-alpha/beta or IFN-beta alone interfered with the ability of poly(I. C) to impair the candidacidal activity of macrophages from SCID mice in vitro. These data suggest that poly(I. C)-induced interferons can impair the candidacidal activity of macrophages in SCID mice and decrease their innate resistance to acute systemic candidiasis and to systemic candidiasis of endogenous origin.     

Double-stranded RNA induces production of RANTES and IL-8 by human nasal fibroblasts

Double-stranded RNA (dsRNA) and the viral RNA mimic, polyinosine-polycytidylic acid (poly(I:C)), are recognized by toll-like receptor 3 (TLR3) that mediates the innate immune response to viral infections. In this study, we investigated the effects of poly(I:C) on the production of chemokines (IL-8, RANTES, and eotaxin), Type I IFNs (IFNalpha and IFNbeta), Th1-cytokines (IL-12 and IFNgamma), and pro-inflammatory cytokines (TNF-alpha and IL-1beta) by human nasal mucosa-derived fibroblasts. Human nasal fibroblasts were treated with poly(I:C), and levels of cytokines and chemokines were measured by ELISA. Incubation with poly(I:C) significantly enhanced the secretion of RANTES and IL-8. However, eotaxin, IL-1beta, TNF-alpha, IFNalpha, IFNgamma, and IL-12 were not secreted from nasal fibroblasts stimulated with poly(I:C). The JNK inhibitor SP600125 and the PI3-kinase inhibitor LY294002 significantly blocked the poly(I:C)-induced release of RANTES and IL-8, whereas the p38 MAP kinase inhibitor SB203580 suppressed poly(I:C)-induced secretion of IL-8, but not RANTES. Nasal fibroblasts play an important role in initiating antiviral responses and inflammation of the nasal cavity by producing chemokines leading to enhanced inflammatory cell recruitment.