Poxviruses: Capturing Cytokines and Chemokines

Cytokines play a critical role in the regulation of immune responses and constitute important targets for virus immune evasion mechanisms. One strategy used by large DNA viruses is to encode proteins that mimic cytokines or cytokine receptors, which modulate the activity of cytokines during infection. Poxviruses encode a unique set of proteins that are secreted from the infected cell and function as soluble cytokine receptors or binding proteins and sequester tumor necrosis factor, interleukin-1β, or chemokines. Characterization of these poxvirus proteins is providing information on virus pathogenesis, the function of cytokines, and new strategies for immune modulation and therapeutic intervention.     

Biological functions and therapeutic opportunities of soluble cytokine receptors

Cytokines control the immune system by regulating the proliferation, differentiation and function of immune cells. They activate their target cells through binding to specific receptors, which either are transmembrane proteins or attached to the cell-surface via a GPI-anchor. Different tissues and individual cell types have unique expression profiles of cytokine receptors, and consequently this expression pattern dictates to which cytokines a given cell can respond. Furthermore, soluble variants of several cytokine receptors exist, which are generated by different molecular mechanisms, namely differential mRNA splicing, proteolytic cleavage of the membrane-tethered precursors, and release on extracellular vesicles. These soluble receptors shape the function of cytokines in different ways: they can serve as antagonistic decoy receptors which compete with their membrane-bound counterparts for the ligand, or they can form functional receptor/cytokine complexes which act as agonists and can even activate cells that would usually not respond to the ligand alone. In this review, we focus on the IL-2 and IL-6 families of cytokines and the so-called Th2 cytokines. We summarize for each cytokine which soluble receptors exist, were they originate from, how they are generated, and what their biological functions are. Furthermore, we give an outlook on how these soluble receptors can be exploited for therapeutic purposes.     

The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.     

Cytokine soluble receptors in perinatal and early neonatal life

BACKGROUND: In contrast to cellular receptors, soluble receptors do not enhance the cellular activation because they do not have transmembranic and cytoplasmic parts, acting thereby as endogenous regulatory mechanisms against systemic functions of cytokines. AIM: To measure serum concentrations of the soluble interleukin-2 receptor (sIL2R), soluble interleukin-4 receptor (sIL4R), soluble interleukin-6 receptor (sIL6R), and soluble tumor necrosis factor-alpha receptor I and soluble tumor necrosis factor-alpha receptor II, during the perinatal and early neonatal period, in order to evaluate their role in activation of immune response in labor and the first days postpartum. METHODS: Soluble receptor serum concentrations were determined by enzyme-linked immunosorbent assay, in 45 healthy, full-termed neonates during the first and fifth days after birth, in 25 of their mothers (MS), in 25 samples of umbilical cords (UC) and in 25 healthy adult donors age-matched with the mothers (controls). RESULTS: Soluble receptor serum concentrations showed considerable changes during labor and early neonatal life, being significantly higher both in MS (except sIL6R) and in neonatal sample UC, first and fifth days after birth, compared with controls (p<0.0001). Neonatal serum sIL2R and sIL6R increased significantly from birth to the fifth day, while the remaining receptors showed a rapid increase in the first day (p<0.0001), declining significantly thereafter (p<0.0001). CONCLUSION: Our findings suggest that the elevated concentrations of all studied soluble cytokine receptors reflect the activation of immune response, and represent also regulatory protective mechanisms for mother and fetus-neonate against the systemic function of cytokines during labor and early neonatal life.     

ERK phosphorylation and tumor necrosis factor-alpha production by monocytes are persistent in response to immobilized IgG

Engagement of Fcγ receptors on leukocytes by immune complexes induces both cytokine production and immune complex internalization. The relationship between these processes is unclear. In many disease states, Fcγ receptors encounter their ligands in deposited forms that cannot be readily internalized. In this study, we examined the kinetics of ERK1/2 phosphorylation and TNF-α secretion in primary human monocytes in response to soluble heat-aggregated IgG or surface-bound IgG, to mimic soluble immune complexes and tissue-deposited IgG, respectively. Soluble aggregated IgG induced transient signaling, leading to peak phosphorylation of ERK1/2 by 15 min and peak TNF-α levels by 1 h, whereas surface-bound IgG caused sustained responses over the course of several hours. Treatment with the vacuolar ATPase inhibitor bafilomycin led to increased persistence of ERK1/2 phosphorylation in response to aggregated IgG. When monocytes were incubated with both soluble aggregated IgG and surface-bound IgG simultaneously, ERK1/2 phosphorylation was transient. These results suggest that Fcγ receptor internalization is an important step in termination of inflammatory signaling, and that small immune complexes can exert an overall down-modulatory effect when encountered in the presence of immobilized IgG.     

Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses

Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease.     

SOCS2-induced proteasome-dependent TRAF6 degradation: a common anti-inflammatory pathway for control of innate immune responses

Pattern recognition receptors and receptors for pro-inflammatory cytokines provide critical signals to drive the development of protective immunity to infection. Therefore, counter-regulatory pathways are required to ensure that overwhelming inflammation harm host tissues. Previously, we showed that lipoxins modulate immune response during infection, restraining inflammation during infectious diseases in an Aryl hydrocarbon receptor (AhR)/suppressors of cytokine signaling (SOCS)2-dependent-manner. Recently, Indoleamine-pyrrole 2,3- dioxygenase (IDO)-derived tryptophan metabolites, including L-kynurenine, were also shown to be involved in several counter-regulatory mechanisms. Herein, we addressed whether the intracellular molecular events induced by lipoxins mediating control of innate immune signaling are part of a common regulatory pathway also shared by L-kynurenine exposure. We demonstrate that Tumor necrosis factor receptor-associated factor (TRAF)6--member of a family of adapter molecules that couple the TNF receptor and interleukin-1 receptor/Toll-like receptor families to intracellular signaling events essential for the development of immune responses--is targeted by both lipoxins and L-kynurenine via an AhR/SOCS2-dependent pathway. Furthermore, we show that LXA?- and L-kynurenine-induced AhR activation, its subsequent nuclear translocation, leading SOCS2 expression and TRAF6 Lys47-linked poly-ubiquitination and proteosome-mediated degradation of the adapter proteins. The in vitro consequences of such molecular interactions included inhibition of TLR- and cytokine receptor-driven signal transduction and cytokine production. Subsequently, in vivo proteosome inhibition led to unresponsiveness to lipoxins, as well as to uncontrolled pro-inflammatory reactions and elevated mortality during toxoplasmosis. In summary, our results establish proteasome degradation of TRAF6 as a key molecular target for the anti-inflammatory pathway triggered by lipoxins and L-kynurenine, critical counter-regulatory mediators in the innate and adaptive immune systems.     

The soluble IL-6 receptors: serum levels and biological function.

IL-6 exerts its biological activities through interaction with specific receptors expressed on the surface of target cells. IL-6 binds first to a low-affinity (10(-9) M) subunit, a 80 kDa glycoprotein also called gp80 or IL-6R alpha. The IL-6/IL-6R alpha complex recruits the signal-transducing b subunit, a 130 kDa glycoprotein called gp130. The association of gp130 with IL-6 and IL-6R alpha leads to the formation of the high-affinity IL-6 receptor complex, to the linkage of two gp130 subunits and to signal transduction. Soluble forms of both receptors have been described and found in biological fluids. Soluble cytokine receptors are generated by either proteolytic cleavage of their membrane moiety or by alternative splicing. Both mechanisms have been described for sIL-6R and sgp130 formation. Interestingly, the association of IL-6 with the soluble form of IL-6R alpha is capable of eliciting a biological response in cells that express only the membrane gp130. This type of activation, called "trans-signalling", renders virtually all cells capable of responding to IL-6/sIL-6R alpha complexes, making for a large new spectrum of IL-6 activities, ranging from the control of the immune response to involvement in pathological states. In this review the biological activities of IL-6 will be considered in the light of new knowledge concerning the association of IL-6 and the soluble IL-6 receptors.     

SLAM and CD31: signaling molecules involved in cytokine secretion during the development of innate and adaptive immune responses

Immune cells are modulated through the crosslinking of receptors named "immunoreceptors". Ligation of immunoreceptors by their ligands induces a tyrosine-phosphorylation signal that is essential for cell activation or inhibition. Physiologically, immunoreceptor triggering is not enough for cell activation, and stimulation of co-receptors is necessary for antigen-evoked cytokine production. Thus, signal transduction pathways mediated by proteins that regulate cytokine secretion are critical to achieve an effective immune response of the host, where the balance between positive and negative signaling allows effective immune responses, preventing tolerance and autoimmunity. This review deals with recent studies based on the role of the receptor signaling lymphocytic activation molecule (SLAM), a signaling protein that modulates cytokine secretion by immune cells, and the transmembrane glycoprotein CD31, which plays multiple roles in cellular signaling events by modulating the balance between inhibitory and stimulatory signals to immune cells. Recent studies have shed light on the ability of these molecules to transmit different signals that regulate the ability of innate and adaptive immune cells to synthesize stimulatory and inhibitory cytokines.     

Double-stranded RNA induces shedding of the 34-kDa soluble TNFR1 from human airway epithelial cells via TLR3-TRIF-RIP1-dependent signaling: roles for dual oxidase 2- and caspase-dependent pathways

TNF, an important mediator of inflammatory and innate immune responses, can be regulated by binding to soluble TNF receptors. The 55-kDa type 1 TNFR (TNFR1), the key receptor for TNF signaling, is released to the extracellular space by two mechanisms, the inducible cleavage and shedding of 34-kDa soluble TNFR1 (sTNFR1) ectodomains and the constitutive release of full-length 55-kDa TNFR1 within exosome-like vesicles. The aim of this study was to identify and characterize TLR signaling pathways that mediate TNFR1 release to the extracellular space. To our knowledge, we demonstrate for the first time that polyinosinic-polycytidylic acid [poly (I:C)], a synthetic dsRNA analogue that signals via TLR3, induces sTNFR1 shedding from human airway epithelial (NCI-H292) cells, whereas ligands for other microbial pattern recognition receptors, including TLR4, TLR7, and nucleotide-binding oligomerization domain containing 2, do not. Furthermore, poly (I:C) selectively induces the cleavage of 34-kDa sTNFR1 ectodomains but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. RNA interference experiments demonstrated that poly (I:C)-induced sTNFR1 shedding is mediated via activation of TLR3-TRIF-RIP1 signaling, with subsequent activation of two downstream pathways. One pathway involves the dual oxidase 2-mediated generation of reactive oxygen species, and the other pathway is via the caspase-mediated activation of apoptosis. Thus, the ability of dsRNA to induce the cleavage and shedding of the 34-kDa sTNFR1 from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated.     

Trans-signaling by cytokine and growth factor receptors

Although ligand-induced dimerization or oligomerization of receptors is a well established mechanism of growth factor signaling, increasing evidence indicates that biological responses are often mediated by receptor trans-signaling mechanisms involving two or more receptor systems. These include G protein-coupled receptors, cytokine, growth factor and trophic factor receptors. Greater responsiveness and inhibitory signaling responses are provided when different signaling pathways merge through receptor trans-signaling.     

Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.     

The role of soluble receptors in cytokine biology: the agonistic properties of the sIL-6R/IL-6 complex

Cytokines perform ever-increasing roles in both, the regulation of general homeostasis and in orchestrating the immune response during disease. To ensure that control of the cytokine network is tightly regulated, nature has developed a series of systems designed for this purpose. In this respect, researchers have placed considerable emphasis on identifying and characterising the regulatory properties of soluble cytokine receptors. These proteins bind their ligands with similar affinities to those of their cognate transmembrane receptors and are effective at prolonging the circulating half-life of cytokines they bind. However, it is the individual capacity of these soluble receptors to act as either antagonists or agonists which has been the principal focus of most research studies. This review provides an overview of the activities of soluble cytokine receptors, but primarily concentrates on those that possess agonistic properties.