The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-κB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-κB and their subsequent regulation of IFN-β and the IL-12 family of cytokines.
TLR3, TLR4 and retinoic acid-inducible gene 1 (RIG-1)/melanoma differentiation associated gene 5 (MDA-5) activation by double stranded (ds) RNA mimic polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) and synthetic ds-β-DNA respectively, mediated IFN-β as well as TNF-α and IL-8 synthesis in monocyte-derived DCs. Using the pharmacological inhibitor of conventional PKCs (cPKCs), Gö6976, we demonstrated that this family of kinases was involved in TLR3, TLR4 and RIG-1/ MDA-5 signaling pathways leading to the production of IFN-β but not of TNF-α and IL-8. Further analysis with the use of specific kinase inactive cPKC isoforms and siRNA targeted to PKCα, we established that PKCα was the isoform involved in the TLR3 signaling pathway. In the case of TLR3, we show that PKCα exerts its effect downstream of TRIF and TBK1. Moreover, we show that inactivation of PKCα specifically inhibits the activation of IRF-3 and not that of NF-κB.
Through biochemical analysis, we assessed the contribution of PKCα in the critical events of IRF-3 activation: a) phosphorylation b) homodimerization c) nuclear translocation d) DNA-binding and e) recruitment of creb-binding protein (CBP). We conclude that inhibition of cPKCs severely hinders the association of IRF-3 with CBP.
Overall, these data revealed the critical role of cPKCs in TLR-dependent and - independent pathways leading to IFN-β synthesis.
The selective targeting of IRF-3 by cPKCs prompted us to study the possible implications of cPKCs in the transcriptional control of IL-12 family members, some of which are regulated by IRF3. Indeed, recent studies have emerged demonstrating the
essential role of IRF-3 in IL-12p35 and IL-27p28 gene expression (1;2). Likewise, we investigated the role of cPKCs in the regulation of LPS- and poly(I:C)-induced expression of IL-12(p40/p35), IL-23(p40/p19) and IL-27(p28/EBI3) in monocyte-derived DCs. Treatment of monocyte-derived DCs with Gö6976 down-regulated LPS- and poly(I:C)-induced IL-12 and IL-27 synthesis while it did not alter IL-23 production.
Next, we showed that impaired IL-12 and IL-27 synthesis was due to repressed IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4 whereas IL-23p19 and IL-27EBI3 gene expression were not modified. Reporter gene assays demonstrated that cPKCs are involved in LPS- and poly(I:C)-induced IL-12p35 and IL-27p28 promoter activity. Finally, experiments in bone marrow-derived DCs from IRF-3-/- and wild type mice showed that IL-23 synthesis does not require IRF-3 activation. We conclude that cPKCs through the control of IRF-3 activity are critically involved in the regulation of IL-12 and IL-27 synthesis downstream of TLR3 and TLR4 while they do not participate in IRF-3-independent IL-23 synthesis.
On whole, we demonstrated a novel function for cPKCs in the regulation of IRF-3 and IRF-3 dependent gene expression, specifically IFN-β, IL-12 and IL-27. In light of the important and divergent roles of IFN-β and IL-12 family of cytokines on the development of T helper (Th) Th1, Th2, Th17-mediated immune responses, cPKCs represent a potential target for therapeutic immunomodulation. This modulation needs to be carefully administered due to the complex interplay of the IL-12 family members in immunity.
I. The role of dendritic cells in immune responses
I.1 Innate and adaptive immunity
The immune system is a mechanism of protection used by vertebrates and invertebrates to detect and control and/or eradicate invading pathogenic organisms.
Engagement of the immune system triggers a cascade of events culminating in a rapid and appropriate immune response.
In vertebrates the immune system consists of two arms, innate and adaptive. The innate arm of immunity uses a set of germ line encoded receptors, called pathogen recognition receptors (PRRs), to recognize pathogen associated molecular patterns (PAMPs) of microorganisms (3). Due to the presence of PRRs on a variety of cell types such as epithelial, endothelial and antigen-presenting cells (APCs), innate immune responses occur rapidly. The adaptive arm of immunity uses somatically generated antigen receptors on T and B cells. Re-arrangement of these receptors gives rise to a great diversity and specificity of the adaptive immune response (3;4).
Innate and adaptive responses are dichotomous entities while also being interdependent. Innate responses are critical in early detection of pathogens (danger signals), as well as the initiation and skewing of the adaptive response by major histocompatability complex (MHC) and up-regulation of co-stimulatory molecules and production of effector cytokines (5;6).
I.2 Human and murine dendritic cell subsets
In the following chapter, the intersection of innate and adaptive immunity will be discussed in the context of two major players: dendritic cells (DCs) and lymphocytes.
DCs are professional antigen-presenting cells which represent an essential link between innate and adaptive immunity. DCs circulate in the blood in an immature state characterized by high endocytic and phagocytic capacity. They are also capable of localizing into tissues where they capture antigen and phenotypically change to up- regulate co-stimulatory and MHC molecules. The maturing DCs migrate to the lymphoid organs in response to chemotractive cytokines, where they initiate the activation of T
cells (7) (figure 1). Human DCs can be expanded from CD 34+ bone marrow progenitor cells (8-11). These stem cells can give rise to common myeloid precursors (CMPs), mono/CMP and common lymphoid precursors (CLPs). CMPs yield interstitial DCs and Langerhans cells whereas CLPs differentiate into plasmacytoid DCs (pDCs) described as CD11c- CD123hi and in the presence of granulocyte-macrophage colony stimulatory factor and interleukin-4, mono/CMPs further differentiate into myeloid DCs (mDCs) described as CD11c+ CD123lo (12-14). In response to PAMPs such as lipopolysaccharide (LPS), unmethylated bacterial CpG DNA and ds viral DNA, mDCs produce the Th1- inducing cytokine IL-12 (12). Human pDCs can be generated from CD11c- CD123hi progenitors in the presence of the IL-3. Upon viral or unmethylated bacterial CpG DNA recognition these DCs produce copious amounts of type I IFN-α and -β (15;16).
In mice, DCs are separated into two subpopulations: CD8a- CD11b+ and CD8a+ CD11b- which can further be categorized based on CD4 expression (17;18). The murine myeloid DC (CD8a- CD11b+) population found in the spleen and lymphoid organs do not produce biologically active IL-12p70 thereby leading to the induction of T helper (Th) 0
or Th2 responses (19-21). Similarly, interferon producing cells (IPCs) were found in mice. Mouse IPCs (mIPCs) are phenotipically characterized as CD8a+ CD11b- B220+ MHC II lo CD4+. These IPCs are most abundant in spleen, bone marrow and lymph nodes but also present in the blood, lung and liver (22). Upon activation by CpG DNA or virus, mIPCs produce large amounts of IL-12 and IFN-α to induce Th1 responses via enhanced co-stimulatory molecule expression and increased surface MHC II expression (figure 2)(20;22).
I.2.1 Antigen presentation
Antigen presentation by DCs represents an essential process required for the interaction of innate and adaptive immunity. DCs are capable of presenting peptides of foreign and self proteins at the cell surface to activate CD4+ and CD8+ T cells (23).
Peptide presentation is orchestrated by MHC molecules which are comprised of two branches: MHC class I and MHC class II.
Processing of MHC class I antigens is a multi-step process consisting of:
cleavage, transport, MHC binding and transport of the MHC I-peptide complex to the cell surface for presentation to T cells (24). Cleavage of endogenous proteins (ranging from 3-22 amino acids) occurs via proteosomes in the cytoplasm which are actively transported from the cytosol to the endoplasmic reticulum (ER) lumen by the transporter associated with antigen presentation (TAP) (25;26). Once transported into the lumen of the ER, the peptides bind to the MHC class I molecules which then translocates via the Golgi apparatus to the cell surface where they present the antigenic peptides to CD8+ cytotoxic T-lymphocytes (26;27).
Presentation of exogenous peptides is accomplished by the MHC class II pathway which also occurs multistepwise: i) uptake of exogenous peptides (phagocytosis, endocytosis, pinocytosis of macropinocytosis) ii) de novo synthesis and assembly of MHC class II molecules in the ER iii) transfer to the Golgi apparatus then sorted to the endocytic pathway. Peptides are degraded inside an acidic endosome where the peptides are able to bind the MHC class II molecules. The MHC class II-peptide complex then traffics to the surface of DCs to activate CD4+ T lymphocytes (24;28).
Cross-presentation is a process by which exogenous proteins are expressed by MHC class I. This process occurs when internalized exogenous antigen escapes lysosomal degradation and enters the cytosol and is then transported into the ER by TAP and binds to MHC class I molecules or when the peripheral phagosomes is composed of the ER membrane that contains MHC class I molecules (29;30). Likewise, the presentation of endogenous peptides on MHC class II molecules occurs by autophagy and the direct import of cystolic proteins into lysosomes (31;32).
I.2.2 Co-stimulatory functions
DC- T cell interactions occur in a three step process where all of the steps are required for full activation of naïve CD4+ T cells. Signal 1 is the presentation of antigen by the DC via MHC to the T cell receptor (TCR), signal 2 is co-stimulation and signal 3 is the production of immuno-modulatory cytokines, represented in figure 3 (33). CD80 (B7.1) and CD86 (B7.2) are two important co-stimulatory molecules of the B7 family
that engage the ligand CD28 of naïve T cells. This signal enhances and sustains T cell activation and leads to IL-2 secretion which induces T cell proliferation. B7.1 and B7.2 are also known to bind to a surface molecule cytotoxic T lymphocyte antigen 4 (CTLA-4) found on T cells during late differentiation. However B7-CTLA-4 interactions result in a negative signal decreasing IL-2 production and leads to memory T cell induction (34).
Another important co-stimulatory molecule that is up-regulated on activated T cells is CD40Ligand (CD40L). CD40L can interact with CD40 of DCs and B cells. CD40- CD40L interactions between DCs and T cells increase cytokine production by DCs such as IL-12. The ability of DCs to completely activate T cells depends on the activation status of the DCs. DCs that present antigen without the subsequent co-stimulatory signals give rise to anergic or tolerized T cells (35) (figure 3).
Tolerance is the specific ability of host immunity to not respond to antigens that is induced by DCs exposed to PAMPs which sub-optimally activate the immunomodulatory capacity of DC (36). Antigen presentation by DCs without co- stimulation (signal 2) induces anergic T cells. T cell anergy can also be induced by CTLA-4 or due to other suboptimal conditions in TCR engagement (36;37).
I.2.3 Regulation of Th cell responses
DCs instruct effector function, memory, anergic T cell development or programmed cell death. Upon maturation by direct PRR triggering or environmental signals, DCs up-regulate MHC and co-stimulatory molecules and secrete soluble cytokines to initiate and define T cell effector function. Maturing DCs migrate from the periphery to the lymph nodes where they present antigen to naïve T cells. The potent stimulatory capacity of DCs is determined by various factors such as: antigen dose and type, strength of TCR signals, co-stimulatory molecules and cytokine production (38).
In humans and mice, Th effector functions are divided into Th1, Th2, Th17
subtypes which are characterized by their cytokine profiles (39;40). Th1 and Th2
differentiation is driven by two potent Th cell polarizing cytokines secreted by DCs: IL- 12 and IL-4 which drive Th1 and Th2 differentiation respectively. In addition to the differentiation capacity of Th skewing cytokines, the subtypes can further be
characterized by transcription factor requirements and signaling cascades. Generally, Th1 cells promote cell-mediated responses by the production of interferon gamma (IFNγ).
IL-12 triggers a signal transducer and activator of transcription (STAT)-4-mediated induction of T-box transcription factor for Th1 cell commitment. T-box expressed in T cells (T-bet) is responsible for chromatin remodeling of IFN-γ, IL-12Rβ2 expression and its own expression in an autocrine fashion (41). Th2 cells induce humoral responses with the production of IL-4, IL-5, IL-10 and IL-13 (33). STAT-6-mediated IL-4 signaling leads to the induction of the transcription factor GATA-3 which regulates the IL-4/IL- 5/IL-13 panel of Th2 cytokines (41;42).
Several differences have been recently identified between mouse and man in the induction of Th17 cells. In mice, Th17 represents a distinct subset of cells that are generated from naïve T cells in the presence of the synergistic effect of TGF-β/IL-6 that are mutually exclusive of Tregs which are induced by TGF-β alone. In response to this cytokine milieu, these cells up-regulate the IL-23R which makes them responsive to IL- 23, potentiating Th17 proliferation and survival (40). This subset of cells is capable of secreting the pro-inflammatory family of cytokines IL-17A and IL-17F (IL-17A will further be referred to as IL-17). The Th17 population of T cells requires the transcription factor retinoic acid-related orphan receptor gamma t (RORγt) for differentiation (43). In humans, Th17 cells have recently been characterized. IL-23 induced in vivo the differentiation of naïve T cells as well as IL-23R+ memory T cells into Th17 cells that produced IL-17A, IL-17F and IL-22 and interestingly IFN-γ. This subset along with other T helper lineages depended on IL-2 for survival. In vitro, these cells were promoted by the synergy of IL-23 and IL-1β (44). Th17 cells have been associated with several autoimmune diseases in humans such as rheumatoid arthritis and multiple sclerosis (45). It has also been implicated in murine models of Collagen-Induced Arthritis (CIA) and Experimental Autoimmune Encephalomyelitis (EAE) (46;47). A proper balance of Th1, Th2 and Th17 must be obtained and regulated for an effective immune response. These processes are controlled by negative- and cross-regulation.
IFNγ secreted by Th1 cells cross-regulates Th2 differentiation by down-regulating Th2
skewing cytokines and vice versa. Likewise, IL-4, IFN-γ, IL-27 and IL-12 suppress Th17
differentiation (44). The IL-23/IL-17 axis is also associated with protection against
bacterial infections. Using a model of Klebsiella pneumoniae lung infection, it was demonstrated that IL-23 deficiency (IL-23p19-/- and IL-12p40-/-) and IL-17R deficiency increased the susceptibility of the mice to bacterial dissemination. The phenotype was characterized by decreased neutrophil recruitment due to abolished chemoattractant cytokine production and abrogated IL-17 production (48;49).
Homeostasis of the immune response is controlled by the function of regulatory T cells (Tregs). Tregs can be divided into three distinct populations: natural Treg, Tr1 and Th3. Treg cells (CD4+CD25+Foxp3+) develop in the thymus and migrate into the periphery to suppress the activation of self-reactive T cells. Tr1 and Th3 are generated from naïve T cells in the periphery after antigen encounter and DC direction. Tr1
preferentially produce IL-10 while TGF-β production characterizes Th3 cells (50). The differentiation of the various Th and Treg subtypes is represented in figure 4.
II. Pathogen recognition receptors
II.1 Interleukin 1 receptor family
The IL-1 receptor (IL-1R) family consists of 10 members of which some are important for host defense. The 10 family members have many structural similarities in their extracelllular and cytoplasmic domains. These receptors are described as membrane-spanning proteins that contain immunoglobulin (Ig)-like ligand-binding domains and/or cytoplasmic domains with or without carboxyl-terminal tails.
Of these members, the type I IL-1R has been well characterized. The IL-1R is a heterodimeric molecule comprised of the IL-1R and the IL-1R accessory protein (IL- 1RAcP). Signaling through the IL-1R can occur through the binding of either IL-1α or IL-1β which exert identical biological effects. These two IL-1 molecules bind to the same extracellular ligand-binding domain of the IL-1R which then leads to the recruitment of IL-1RAcP. IL-1R- IL-1RAcP heterodimerization is required for IL-1 signaling (51;52). Next, the receptor complex recruits the adaptor molecule myeloid differentiation factor 88 (MyD88) to its cytoplasmic Toll/IL-1R resistance (TIR) domain.
The ensuing signaling cascade involves the recruitment of interleukin receptor associated
kinase 1 (IRAK1), TNF-α associated factor 6 (Traf6) and NF-κB which regulates the expression of many pro-inflammatory genes (52;53). Huang et al went even further to show the role of MEKK3 in the signaling cascade of IL-1R in mouse embryonic fibroblasts (54). The same MyD88/IRAK1/Traf6/NF-κB signaling cascade was delineated for the heterodimeric IL-18 receptor complex consisting of IL-18R and the IL- 18R accessory protein, which is required for IL-18 signaling. Signaling via these two receptors leads to the induction of the inflammatory cytokines IL-1 and IL-18 respectively. IL-1 is important in activating innate immune responses while IL-18 is implicated in Th1 differentiation and augmenting NK proliferation and activity (55).
IL-1 signaling is subject to negative regulation by decoy receptors (type II IL-1R) and IL-1R antagonists (Single immunoglobulin IL-1 receptor related; T1/ST2; IL-1R accessory protein-like related and three immunoglobulin domain-containing IL-1R- related) that once bound fail to initialize the IL-1R signaling cascade (52;56-58).
Likewise, IL-18R signaling is negatively controlled in the same manner. IL-18R binding-protein acts as a sink for IL-18 binding (52). The host regulation of these pathways is paramount to limiting the deleterious effects of IL-1 in chronic inflammation and limiting IL-18-induced Th1 differentiation (figure 5).
II.2 Toll-like receptors
Another structurally similar group of receptor molecules essential for host defense against infectious pathogens are toll-like receptors (TLRs). Together these evolutionarily conserved molecules make up the superfamily of receptors that comprise the host’s arsenal of defense.
The first Toll proteins were discovered and characterized in Drosophila melanogaster. Drosophila has two immune signaling pathways, Toll and Immune deficiency (IMD). Drosophila Toll (dToll) plays a dual role. dToll is essential for dorsal-ventral axis orientation in fly embryos and later involved in fungal defense in adult flies (59). In embryonic development, dToll is activated by spätzle which recruits two adaptor molecules, Tube and Pelle which in turn lead to the degradation of Cactus (a Drosophila homologue of IκB) that allows the release and nuclear translocation of the
transcription factors Dorsal or Dorsal-related immunity factor (Dif) (60). In turn, these transcription factors induce the expression of ventral proteins twist and snail (61). In its second role, dToll initiates antifungal and anti-Gram-positive bacterial responses in adult flies (62). In adult flies, the spätzle-induced dToll signaling culminates in the activation and nuclear translocation of Doral and Dif with the latter being the predominate transactivator of the antifungal gene Drosomycin. The IMD pathway imparts Gram- negative bacterial immune responses in adult flies. By way of peptidoglycan recognition proteins (a family of microbial recognitionproteins/enzymes), adult flies are able to distinguish between Gram-negative and Gram-positive bacteria via their peptidoglycan modifications. The IMD pathway-induction of antibacterial peptides, diptericin, cecropin, drosocin and attacin are all transactivated by the nuclear factor Relish (61;63;64).
II.2.1 Toll-like receptor expression and function
In humans, 10 members of the TLR family have been identified, characterized and categorized. Each TLR has a distinct specificity and a diverse and complex signaling pathway that yields pathogen specific responses (59). TLRs are responsible for pathogen recognition, signal transduction and tailoring of the innate immune response ultimately dictating the acquired response as well. Due to the grand scale of the assigned task, TLRs are expressed on a multitude of cells from immune to epithelial cells. The TLR repertoire on cells aids in its effector function. TLRs can be characterized along many lines such as: PAMP specificity, TLR expression, interface with the environment and TLR activation- cytokine profiles (65). Similar to IL-1R, TLRs have extracellular, transmembrane and cytoplasmic domains (66). The extracellular domain is comprised of leucine-rich repeats (LRR) which distinguishes them from IL-1R members. It is with this domain that TLRs recognize pathogens in a PAMP-specific manner. Upon receptor ligation, the signal is transduced by the intracellular TIR domain. The TIR domain of TLRs is a protein-protein interaction domain that recruits signaling mediators to initiate immune responses (66). These adaptor molecules also contain TIR domains as well as
death domains. The TIR-TIR interactions of TLRs and adaptor molecules convey TLR signaling specificity.
II.2.2 Description of Toll-like receptors
TLR2: TLR2 is capable of recognizing a large array of microbial and bacterial components. It recognizes peptidoglycan (PGN) from Gram-positive bacteria like Staphylococcus aureus, lipoproteins (triacylated) and lipopeptides (diacylated) from various bacterial sources, glycophosphatidylinositol anchors of Trypanosoma cruzi, lipoarabinomannan from Mycobacterium tuberculosis, porins form Neisseria meningitides and the yeast cell-wall component zymosan (67). The broad range ligand- specificity of TLR2 is due to its ability to dimerize with other TLRs. Dimerization with either TLR1 or TLR6 is how TLR2 is able to discriminate between diacylated and triacylated lipoproteins and induce cytokine production in response to these ligands.
Specifically, TLR2/TLR6 dimers are responsible for recognizing diacylated lipoproteins such as synthetic mycoplasmal macrophage-activating liopopeptide 2 (MALP-2). TLR2-
/- and TLR6-/- embryonic fibroblasts are unresponsive to MALP-2 but reconstitution of these two TLRs restores MALP-2 responsiveness as assessed by IL-6 production (68;69).
Triacylated lipoproteins like synthetic Pam3CysteinSerineLysine4 (PAM3CSK4) are preferentially recognized by the TLR2/TLR1 dimer as TLR1 deficient macrophage responses were partially impaired (67;69).
TLR3: Viral infection and replication in cells results in the generation of dsRNA during the lifecycle of the virus which stimulates immune cells. dsRNA, synthetic dsRNA mimic poly(I:C) and small interfering RNA (siRNA) are all capable of initiating TLR3 signaling (69;70). TLR3 is localized in endosomal compartments and its maturation/acidification is a prerequisite of TLR3 signaling (71). Biochemical and functional analysis of the TLR3 ectodomain reveal that it is a horseshoe shaped solenoid with multiple leucine-rich repeats. This domain is largely glycosylated on one face but the glycosylation-free face allows for ligand-binding, accessory protein binding and oligomerization. The proposed model suggests that a TLR3 monomer binds dsRNA then homodimerizes via disulfide bonds to activate signal transduction (72;73). Compelling
evidence has demonstrated the TLR3 role in the recognition of poly(I:C). Human embryonic kidney (HEK) 293 cells co-transfected with TLR3 and a NF-κB reporter gene show poly(I:C)-induced NF-κB activation which is abrogated when the cells are transfected with a kinase inactive form of TLR3. Likewise, RAW 264.7 macrophages demonstate poly(I:C)-induced NF-κB activation (74). TLR3 deficient mice show decreased poly(I:C) responsiveness (69;70). dsRNA was shown to phosphorylate Thr759 and Tyr858 of the cytoplasmic domain of TLR3, two essential sites necessary for complete TLR3-mediated activation of IRF-3 and NF-κB. Thr759 initiated the phosphorylation of IκB and subsequent release and nuclear translocation of NF-κB while Tyr858 mediated its complete phosphorylation and transactivation capabilities (75). Sarker et al demonstrated a similar two-step model for IRF-3 activation that requires the same phosphotyrosine sites. Tyr858 activated Tank-binding kinase 1(TBK1) and induced IRF-3 activation (partial phosphorylation, dimerization and nuclear translocation) while mutated forms of TBK1 do not permit the complete phosphorylation of IRF-3 necessary for gene transcription (76;77).
TLR4: Lipopolysaccharide (LPS), an integral component of Gram-negative bacteria outer membranes, have immunostimulatory capacity on phagocytic cells via its hydrophobic domain, lipid A (69). Several strains of hyporesponsive mice have been generated and shown to have mutations in their TLR4 gene. C3H/HeJ mice have a missense mutation in which a proline is substituted for a histidine. C57BL/10ScCr mice lack the TLR4 gene as a consequence of chromosomal deletion. Similarly, C57BL/6.KB2 mice contain inactive TLR4 due to a deletion of exon II, resulting in a premature stop codon (78;79). As a result of positional cloning of the LPS locus and through the generation of TLR4 knockout mice, investigators were able to confirm TLR4 as the key receptor in LPS recognition. Subsequently, these mice strains are more susceptible to Gram-negative infection. In a different approach, Bihl et al. showed that TLR4 overexpression in transgenic mice, correlates with increased splenocyte proliferation after in vitro LPS challenge and increased resistance to in vivo challenge of salmonella typhimurium. These mice were hypersensitive to in vivo LPS challenge further demonstrating the role of TLR4 in the recognition of LPS (79). The presence of TLR4 alone is not enough to confer LPS responsiveness. LPS physically interacts with a
signaling complex composed of TLR4, CD14 and an extracellular accessory protein, myeloid differentiation 2 (MD-2). Results from MD-2-/- mice have abrogated LPS- induced signaling. In the current model of LPS signaling the LPS-binding protein captures plasma LPS and associates with the TLR4 receptor complex in which all components are required (67;80).
TLR5: The principal component of flagella is a 55 kDa protein, flagellin that is on the surface of broad array of both Gram-positive and Gram-negative bacteria. The rod- like structures are used by the bacteria as a means of motility; it is also a virulence factor that is recognized by TLR5 (68). To demonstrate the role of TLR5 as the ligand for flagella, experiments using Chinese hamster ovary cells stably expressing TLR5 transfected with a NF-κB reporter gene were shown to induce NF-κB in response to purified flagella and various flagellated bacteria. Furthermore, TLR5-stimulating activity was elicited by introducing the flagella gene into a non-flagellated bacterium and abrogated by deleting the flagella genes from flagellated bacterium (81). TLR5 is expressed on a variety of cells from monocytes and DCs but is also very abundant on the basolateral side of intestinal epithelial cells and represent a mode of detection of bacterial infection of the intestinal tract (70). Interestingly, several flagellated bacteria such as Helicobacter pylori and Campylobacter jejuni have been shown to evade immuno- recognition by TLR5. H. pylori is the primary cause of gastritis, peptic ulcer disease and gastric cancer while C. jejuni infection of the small and large intestines is the cause of diarrhea. These bacteria are capable of immuno-evasion due to mutations in their flagellin gene that abolishes their immunostimulatory capacity while not effecting their motility (82).
TLR7 and TLR8: These two structurally similar receptors are localized in endosomal/lysosomal compartments and recognize guanosine (G) - and uridine (U) - rich single stranded RNA (GU-rich ssRNA) as well as synthetic antiviral compounds (70).
Imiquimod and R-848 are part of the imidazoquinoline family of compounds that have potent antiviral and antitumor properties (69). Recognition of GU-rich ssRNA, Imiquimod and R-848 by pDCs results in copious amounts of IFN-α while they elicit IL- 12 production by monocyte-derived DC. Macrophages from TLR7 knockout mice did not respond to imiquimod or R-848 and the splenocytes did not proliferate. Furthermore,
TLR7 knockout bone marrow-derived DC did not show R-848-induced up-regulation of co-stimulatory molecules (69). TLR7 and TLR8 transfected HEK-293 cells responded to human immunodeficiency virus-1-derived ssRNA (83). Taken together, these experiments demonstrate the essential role of TLR7 and TLR8 in the recognition of ssRNA and imidazoquinolines.
TLR9: A distinct feature of bacterial DNA is the abundance of unmethlylated CpG dinucleotides which in vertebrates is methylated and has a lower frequency of CpG motifs (84). It is by this means that the host can distinguish between self and non-self CpG DNA. CpG DNA or synthetic CpG DNA/TLR9 interactions occur in endosomal compartments that similar to TLR3, require maturation/acidification of the endosome for TLR9-induced cellular responses (68;69). The immunostimulatory potential 5’- CpG – 3’ sequences is species specific such that GACGTT motifs are more stimulatory for mouse cells while GTCGTT is a potent human immunomodulator. HEK 293 cells transfected with either human or murine TLR9 were only responsive to their cognate CpG motifs (69). CpG DNA/TLR9 interactions activates B cell proliferation, induces macrophage activation and cytokine production as well as DC maturation. In response to CpG, TLR9 deficient mice failed to produce inflammatory cytokines, there was no proliferative activity of splenocytes and their pDCs remained immature (70). This reveals the essential role of TLR9 in CpG DNA signaling. The immunogenic nature of CpG DNA is reflected by the up-regulation of co-stimulatory molecules, the production of IFN-α by pDCs and increased antigen presentation (68).
TLR10: TLR10 is the only remaining orphan human TLR which shares a common locus on the same chromosome as TLR1 and TLR2 (85). Insight into the expression, function and ligand of TLR10 has been hampered by the lack of a homologue in rodents. In spite of this fact some groups have made headway in tackling the answers to these questions. Recently, new data has been brought into view in the way of TLR10 cellular expression, mRNA, protein expression and signaling. TLR10 mRNA and protein levels have been shown in B cells, B cell lines and pDCs. Likewise the expression of TLR10 was also confirmed on B cells and pDCs from tonsil. Co-immunoprecipitation experiments in HEK 293 cells transfected with HA-tagged TLR10 revealed that TLR10 is capable of homodimerization as well as forming heterodimers with TLR1 and TLR2 and
does not associate with the accessory protein MD-2. Mutations in the TIR domain of TLR10 or co-transfection with dominant negative MyD88 abrogated the NF-κB reporter gene activity. Interestingly, mutation or knockdown with siRNA targeting TRIF, TRAM of TIRAP has no effect on the signaling capabilites of TLR10. These experiments as well as a direct interaction of the fusion protein with MyD88 by co-immunoprecipitation show that TLR10 in humans is functional and is MyD88-dependent (86). Since the rat genome contains a possible functional TLR10 gene as opposed to mice which do not, further research may enable a more thorough examination of signaling parameters and identification of the TLR10 ligand.
TLR11: Although in humans TLR11 contains many stop codons that does not yield a full-length protein, it is present in mice. Through extensive studies in TLR11-/- mice, it has been shown that this receptor is responsible for the recognition of uropathogenic Escherichia coli and a profiling-like protein from Toxoplasma gondii (87).
Curiously, TLR11 in mice responds to pathogens important in human disease while the receptor is not expressed in humans. Recently it was demonstrated that TLR5 functions in the urinary tract of humans and could be the receptor for the recognition of uropathogenic Escherichia coli (70) (figure 6).
It should also be mentioned that TLRs are capable of recognizing endogenous proteins also such as heat shock proteins in the case of TLR4. A detailed list of TLR- agonists pairs can be seen on Table 1.
II.3 Intracellular pathogen receptors
II.3.1 Protein kinase RNA-dependent
PKR: Protein kinase RNA-dependent (PKR) is a major mediator of antiviral responses. PKR contains two dsRNA binding motifs in the amino terminus and a serine/threonine kinase catalytic domain in the carboxyl terminus. In the inactive state PKR is in a closed conformation, upon binding of dsRNA, undergoes a conformational change and auto-phoshorylation revealing the ATP-binding and dimerization domains (88). Activated PKR interferes with viral replication by phoshorylating the α subunit of
eukaryotic translation initiation factor 2 (eIF2α) leading to general inhibition of protein synthesis consequently, the synthesis of viral proteins. The various strategies used by viruses to alter PKR activity affirm the important contribution of this kinase in the antiviral state. Vero E6 cells, which carry a PKR with a mutated kinase domain, are more susceptible to viral infection and are delayed in clearing the infections. This attenuated PKR is unable to auto-phosphorylate and subsequently phosphorylate eIF2α (89). PKR-/- animals were very sensitive to infection with vesicular stomatitis virus and influenza virus and defective in IFN signaling in response to these viruses and dsRNA (5). In all, this demonstrates the importance of PKR in virus- and dsRNA- induced signaling pathway.
II.3.2 Nucleotide-binding oligomerization domain
NOD: Nucleotide-binding oligomerization domain (NOD) 1 and NOD2 represent a class of receptors responsible for cytosolic surveillance and sensing of intracellular bacterial PAMPs. NODs contain an N-terminal caspase-activating and recruiting domain (CARD) domain, central nucleotide-binding site (NBS) and a C-terminal LRR domain which classifies them as NBS-LRR family of receptors. The NBS-LRR family can be subdivided by differences in the N-terminal domains: NODs possess CARD domains, NALPs contain pyrin domains and NAIPs have baculovirus inhibitor of apoptosis protein repeat domains (90). NOD1 (also designated (CARD4) contains one N-terminal CARD domain, found in epithelial cells of the intestinal tract and is responsible for the recognition of the PGN structure meso-diaminopimelate (meso-DAP) of Gram-negative bacteria (90;91). Synthetic meso-DAP represents the minimal immunostimulatory structure for NOD1 triggering. NOD2 (also designated CARD15) is mainly expressed on monocytes, macrophages and epithelial cells of the intestinal tract. NOD2 although having significant homology to NOD1, contains two N-terminal CARD domains (90).
Various experimental models have identified GlcNAc-MurNAc-dipeptide (MDP) of Gram-negative and Gram-positive bacteria as the specific bacterial ligand for NOD2.
Furthermore, synthetic MDP was used to identify muramyl dipeptide of bacterial PGN as the minimal essential NOD2-inducing moiety. Mutations in NOD2 have been associated with Crohn’s disease. The most frequent NOD2 mutation involves a frameshift that
results in MDP-insentive cells. These cells are unable to initiate MDP-induced NF-κB activation (92;93). In addition, peripheral blood mononuclear cells from normal- or heterozygous- NOD2 allele patients displayed LPS- and MDP-induced NF-κB DNA- binding as well as activation of NF-κB-dependent genes. Conversely, these outcomes of NOD2 signaling were absent in homozygous-NOD2 allele patients in response to MDP while LPS responses remained intact (92). Taken together, these results demonstrate the role of NODs in the detection of intracellular bacterial pathogens and that they recognize specific moieties non-related to the structures recognized by TLRs.
II.3.3 Retinoic acid inducible gene I
RIG-1/MDA-5: Retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5) are members of the DExD/H box RNA helicase family that function in antiviral immune responses to intracellular viral dsRNA and synthetic dsRNA, poly(I:C). These cytosolic helicases both contain N-terminal caspase recruitment domains (CARD) involved in protein-protein interactions and a helicase domain that unwinds dsRNA in an ATP-dependent manner (94). RIG-1 and MDA-5 represent the cytosolic pathway of type I interferon (IFN) gene induction. RIG-1 is involved in the recognition of 5’-triphosphates of Flaviviridae, Paramyxoviridae, Orhomyxoviridae and Rhabdoviridae RNA viruses while MDA-5 senses picornoviruses (95). Triggering of RIG-1 and MDA-5 leads to conformational changes to promote CARD-mediated downstream signaling events leading to NF-κB and IRF-3 activation (96). As a consequence of being inducible by type I IFN, RIG-1 and MDA-5 are subject to positive-feedback regulation (97). Functional disruption of these two intracellular receptors by siRNA, generation of mutant receptors, generation of deficient mice or by viral evasion mechanisms exhibit diminished type I IFN induction (94). Indeed, RIG-1 deficient mice were defective in responding to negative-sense ssRNA viruses as well as positive-sense ssRNA viruses. On the other hand, MDA-1 deficient mice were defective in responding to positive-sense ssRNA viruses. It is noteworthy to mention the existence of a third helicase homologous to RIG-1 and MDA-5. Lgp2 also detects RNA but lacks a caspase signaling domain which renders it a negative regulator of RIG-1 and MDA-5
(95). RIG-1 and MDA-5 use the downstream adaptor IFN-β promoter stimulator protein (IPS)-1 (98-101). IPS-1 is characterized by an N-terminal CARD domain that mediated its RIG-1 and MDA-5 CARD-CARD interaction. In the C-terminal there is a hydrophobic transmembrane region that localizes the protein on the surface of the outer mitochondrial membrane. The two domains of IPS-1 are essential for its function as mutations abolish IPS-1-mediated signals (99;101). This pathway can activate NF-κB via the signaling complex IPS-1/TRAF2/TRAF6/TAK1/IKKβ (101). IRF-3 and IRF-7 activation results from the IPS-1/TRAF3/TBK1 complex (102). RIG-1 and MDA-5 are essential cytosolic receptors in the recognition of viral RNA and synthetic dsRNA that signal in a TLR-independent fashion to activate NF-κB, IRF-3 and IRF-7 and induce I IFN production (figure 7).
II.4.1 Toll-like receptor/interleukin-1 signaling pathways
TLR signaling occurs through a conserved IL-1R pathway that promotes the expression of pro-inflammatory and anti-viral genes. To date, five adaptor molecules have been associated with TLR signaling: molecule myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like (MAL) also called Tir domain-containing adaptor protein (TIRAP), Tir domain-containing adaptor inducing IFN-β (TRIF) also known as Tir domain-containing adaptor molecule (TICAM) 1, TRIF-related adaptor molecule (TRAM) also known as Tir domain-containing adaptor molecule (TICAM) 2 and SARM recently shown to be a negative regulator of TRIF-dependent cytokine and chemokine induction (103;104). The adaptor molecules link the TLR members to downstream NF- kB, MAPK and IRF pathways.
TLRs can be broadly grouped by categorizing them based on their recruitment of the adaptor molecule MyD88 which is an intracellular adaptor molecule that contains a TIR domain that mediates TIR-TIR interactions with the various TLRs (70). In its N- terminus, Myd88 contains a death domain (DD) by which is able to recruit and bind other proteins in a DD-DD fashion (105). TLR2 via MAL-MyD88 and TLR5, 7, 8 and 9 via MyD88 follow a similar pathway. Briefly, upon engagement of their respective ligands, these TLRs recruit MyD88 to the receptor via TIR-TIR interactions which then associates
with IL-1R-associated kinase (IRAK) members through death domain interactions. The phosphorylation of IRAK 1 and IRAK 4 leads to their dissociation from the receptor complex followed by TNF receptor associated factor (TRAF) 6 association. From TRAF6, two divergent signaling pathways ensue. One pathway initiating NF-κB activation and the other mitogen-activated protein (MAP) kinase activation that cooperate in the production of inflammatory cytokines (70;105). This signaling pathway can be negatively regulated by IRAK-M which lacks kinase activity. IRAK-M has been shown to prevent IRAK1/4 receptor dissociation by preventing the phosphorylation-dependent dissociation of stabilization of the TLR/MyD88/IRAK complex thereby disrupting TLR signaling (106).
TLR3 is the only TLR that signals purely in a MyD88-independent manner.
TLR3 is located in an endosomal compartment and requires the acidification of the endosome for its signaling (71). The sole adaptor molecule utilized by TLR3 is TRIF.
This finding was substantiated by the use of dominant negative TRIF, siRNA-mediated TRIF knockdown and the generation of TRIF deficient mice, resulting in the severe impairment in the activation of transcription factors and the induction of cytokines (107;108). TLR3 engagement leads to TRIF recruitment followed by its interaction with the noncanonical IKKs: TBK1 and IKKi (109). TBK1 and IKKi have been shown to directly phosphorylate IRF-3 on critical serine sites necessary for IRF-3 activation.
Likewise, TBK1-/- was associated with inhibited IRF-3 activation while IKKi-/- also displayed impaired IRF-3 activation albeit to a lesser extent (110;111). On the contrary, TBK1/IKKi double deficient cells had a complete abolition of IRF-3 activity, suggesting a supplemental role for IKKi in the activation of IRF-3 (112). The formation of a TBK1/IKKi/NAP1(NAK-associated protein 1)/TRAF3 complex leads to the activation and nuclear translocation of IRF-3. Similarly, TRIF can interact with receptor-interacting protein (RIP) 1 and TRAF6 to induce NF-κB activation (113). RIP1-/- mice have no NF- κB activation in response to poly(I:C) revealing the essential role of the kinase RIP1 in TLR3-mediated NF-κB activation. Furthermore, RIP3 negatively regulates this pathway by competing with RIP1 for the binding to TRIF through their RIP homotypic interaction motif (114). Recently, Pin1, a peptidyl prolyl isomerase targeting phosphor-Serine/Thr-
Pro containing proteins has been identified. Pin1 is a negative regulator of TLR- dependent and -independent IRF-3 activation and consequent IFN-β production (115).
TLR4 is unique from the other TLRs in that it possesses both a MyD88- dependent and independent pathway. TLR4 signaling utilizes four adaptor molecules:
MyD88, MAL, TRIF and TRAM. In MyD88-dependent signaling, LPS triggering of TLR4 recruits MAL which acts as a bridging molecule for MyD88 recruitment. MAL- MyD88 then interacts and activates IRAK1 and IRAK 4 subsequently activating TRAF6.
TAK1 then phosphorylates IκB leading to its ubiquitination and proteosome-mediated degradation thereby liberating NF-κB for nuclear translocation and gene induction (70;113). MAL-MyD88 mediates early NF-κB activation. However, TLR4 also signals using a MyD88- independent pathway. In this instance, the adaptor TRAM co-localizes with TLR4 to induce LPS-mediated signals. TRAM is specifically involved in TLR4- mediated MyD88- independent signaling pathway. This finding was demonstrated by the abrogation of TLR4-mediated MyD88- independent cytokine production in TRAM deficient mice (116). TRAM was shown to be a myristoylated protein localized in the plasma membrane and Golgi apparatus. The myristoylation of TRAM allows for its insertion into the lipid bilayer but when phosphorylated dissociates from the membrane.
The activation of TRAM depends on the PKCε-induced phosphorylation. The PKCε- mediated phosphorylation of Ser16 represents a key process required for its function (117;118). The role of TRAM is to recruit TRIF to the TLR4 signaling complex (119).
Similar to TLR3, TLR4 signaling via TRIF leads to the recruitment and activation of the kinases TBK1/IKKi/NAP1/TRAF3 and subsequent activation and nuclear translocation of IRF-3. TRIF also forms another complex containing TRAF6 and RIP1 to mediate late phase NF-κB activation.
As outlined above, TLR3 and TLR4 signaling leads to the activation of the transcription factors IRF-3, NF-κB and AP-1 members in a MyD88-independent manner.
The activation of these transcription factors enables TLR3 and TLR4 to mediate the production of type I IFNs, IFN-inducible proteins as well as inflammatory cytokines (120). TLR3 and TLR4 are not alone in their ability to induce type I IFNs as TLR7, 8 and 9 also mediate type I IFN synthesis upon ligand recognition. MyD88-dependent induction of type I IFNs is unique to TLRs 7, 8 and 9. In humans, TLR7, 8 and 9-
induced type I IFN occurs in a special subset of DCs called pDCs which are characterized by their ability to produce large amounts of IFNα. In pDCs, TLR7 and TLR9 signaling utilizes MyD88 which can interact with IRAK4 to induce the activation of NF-κB while for the induction of IFNα, the signaling complex consists of MYD88/IRAK1/IRF-7. The necessity for IRAK1 in the induction of IRF-7 in this pathway was demonstrated by the use of kinase inactive mutants of IRAK1 and IRAK4. These experiments showed that IRAK1 directly interacted with IRF-7 and was required for its activation and nuclear translocation and transactivation of the IFNα4 promoter (121). Decreased IFN-α production was also observed in IRF-7-/-, IRAK1-/- and IRAK4-/- (although less drastic) cells revealing a possible contributory role of IRAK4 in the activation of IRAK1 (97)(figure 8).
III. Type I interferon
III.1 The role of type I interferon in immune responses
Type I IFNs comprising IFN beta (-β) and IFN alpha (-α) family are central to the innate immunity. The pleiotropic nature of type I IFNs has been attributed with a potent antiviral state, inhibition of cellular proliferation, anti-tumor and immunomodulatory properties (122;123). Type I IFNs encompass a large group of related cytokines that function through a common IFNα/β receptor. In humans, there is a single gene for IFN-β while there are multiple genes for IFN-α (IFN-α1 to -α13 (124). Type I IFNs are produced by epithelial cells, fibroblasts, DCs, macrophages and monocytes in response to viral as well as non-viral pathogens. Type I IFNs are produced early upon infection to trigger early non-specific defense mechanisms such as induction of genes that inhibit viral replication and increase the cytotoxic effect of NK cells. Late phase type I IFN promotes CTL responses, enhance antibody production, apoptosis and facilitate in Th1- skewing of naïve T cells (5;124). The production of IFN-α genes can be divided into early and late phases. The early phase occurs in a de novo protein synthesis-independent
manner while late production is protein synthesis-dependent. IFN-α subtype profiles are differentially induced depending on the virus and they are regulated by different IRFs.
Several TLRs have been identified that lead to the induction of type I IFNs. In human monocyte-derived DCs, the pathogen-induced production of type I IFNs is described as a two-step induction model. In these cells, TLR3 and TLR4 lead to the rapid and robust induction of IFN-β. This early IFN-β synthesis then signals in an autocrine/paracrine loop resulting in a positive feedback loop potentiating the production of IFN-β while simultaneously inducing the production of IFNα subtypes, the synthesis of other IRF members and the induction of interferon stimulated genes (ISG) (125) (figure 9). This two-step model was clarified by the use of IFNα/β receptor-, IRF-9 and IFN-β-deficient mouse embryonic fibroblasts which exhibited dramatically impaired viral-induced IFN-α gene expression (126). On the other hand, in human pDCs, IFN- α synthesis induced by TLR7, TLR8 and TLR9 occurred in an IFNα/βR-dependent and - independent manner, as they are able to directly activate constitutive IRF-7 (127;128).
The early induction of IFN-β and IFN-α subtypes aid in controlling the pathogen at the onset of infection until the adaptive arm of the response is directed and initiated.
In vitro generated DC, derived from human monocytes or mouse bone-marrow precursors expressed high levels of co-stimulatory molecules, MHC class I and II and increase T cell proliferative capacity in response to IFN-α/-β treatment. DC isolated from mouse spleen underwent a similar maturation after IFN-α/-β treatment in vivo or in vitro (129).
IFNs are also essential mediators in the host’s defense against oncogenesis.
IFN-β- or IFNα/β receptor- deficient mouse embryonic fibroblasts resulted in transformed colonies that could induce tumors in nude mice (130).
III.2 Transcriptional control of the interferon-β gene
The IRF family members are essential transcription factors for type I IFNs expression. The family members have a conserved amino terminal DNA-binding domain (DBD) containing five tryptophan repeats. The DBD allows IRFs to associate with 5’-
AANNGAAA-3’ consensus or IRF specific sequences of gene. The IRF association domain in the C-terminus mediates homo- or heteromeric protein-protein interactions with other transcription factors (131)(figure 10). This C-terminal IAD is not present in IRF-1 nor IRF-2. IRF-1, 3, 5 and 7 have been described as positive regulators of type I IFN gene expression while IRF-2 can act as a negative regulator (97). IRF-3 and IRF-7 are famously known as key factors in the induction of type I IFN in response to viral infection and poly(I:C) treatment, as a result they have been extensively studied. IRF-3 is constitutively expressed in the cytosol where it is in a latent, inactive state. Inactive IRF- 3 is in a closed conformation in which the N-terminal and C-terminal domains bind to each other (132). Upon viral activation IRF-3 undergoes multiple post-translational modifications leading to the apparition of a slower migrating form of IRF-3 in SDS- PAGE (133). IRF-3 is activated by phosphorylation of several Ser (385, 386, 396, 398, 402 and 405) and Thr (Thr404) sites (134). Following phosphorylation, IRF-3 undergoes a conformational change. Point mutations of Ser385 and Ser386 results in the abolition of phosphorylation-dependent dimerization of IRF-3 (131;135). Likewise, it was shown that Ser396 represents the minimal phospho acceptor site required for IRF-3 activation (136). The functional outcomes of the remaining phosphorylation sites have yet to be described. IRF-3 phosphorylation is followed by homodimerization or heterodimerization and nuclear translocation. Early phase IFN-β induction is mediated by IRF-3 homodimers whereas late phase and the induction of IFN-α is mediated by IRF- 3/IRF-7 or IRF-7/IRF-7 dimers (137). In contrast, IRF-7 is expressed in very low amounts in most cells but strongly induced upon type I IFN mediated signaling (137;138). IFN-β and IFN-α signaling through the IFNα/β receptor induces the formation of the IFN-stimulated gene factor 3 (ISGF3). ISGF3 is a hetero-trimeric complex of STAT1/STAT2/IRF-9 that binds to the ISRE of the IRF-7 promoter to induce its transcription as demonstrated in pull-down assays (139). IRF-7 has a similar history as IRF-3. It resides inactive in the cytosol and is subject to multiple phosphorylation, dimerization, nuclear translocation and gene induction (125;140).
The enhancer region of the IFN-β promoter has been extensively characterized as containing four overlapping regulatory cis elements that are designated as positive regulatory domain (PRD)-I, -II, -III and –IV (141-143). Promoter region analysis of the
Ifnβ gene revealed that PRDII and PRDIV are bound by NF-κB and the heterodimer of activating transcription factor 2 (ATF-2) and c-Jun, respectively, and that these elements cooperate with PRDI and PRDIII in the induction of the IFN-β promoter (144). The PRDI and PRDIII elements were shown to be bound by distinct members of the IRF family with much attention focused on IRF-3, which binds to the PRDIII-I composite site on the IFN-β promoter and is required for IFN-β gene transcription (145-147).
Optimal transcription of the IFN-β gene depends on the assembly of higher-order multicomponent transcription factor complex. This multicomponent transcription factor complex termed an “enhanceosome” is essential for the recruitment of the basal transcriptional machinery to the promoter. The importance of the enhanceosome structure for transcriptional synergy was demonstrated by mechanistic studies. Merika et al demonstrated that synergistic activation of IFN-β requires the precise arrangement of the transcription factors to the PRD sites of the enhancer region. The transcription factor/PRD interactions occurs stereo specifically as inversion or re-arrangement of the sequences results in decreased promoter activation (142;146).
HMGI(Y) belongs to a family of proteins that are essential architectural components that bind to the minor groove of AT-rich DNA, controls the assembly, stability, function and subsequent disassembly of the enhanceosome (148). Viral infection leads to high mobility group I(Y) (HMGI(Y))-induced chromatin remodeling of the ifnβ gene. Next, the enhanceosome is formed: IRF-3 homodimer, NF-κB, and the heterodimer ATF-2/c-Jun bind their DNA-specific sequences and then recruit the co- activators Creb-binding protein (CBP)/p300, p300/CBP-associated factor (PCAF) and general control-of-amino acid synthesis 5 (GCN5), all of which have acetyltransferase activities. Histones H3 and H4 of the nucleosome are hyper-acetylated by CBP/p300 and GCN5 after which the BRM-associated factor (BAF) is responsible for displacing the nucleosome. Transcription is initiated when transcription factor (TF) II D /TFIIB/TFIIA/upstream stimulatory activity/RNA polymerase II complex gain access to the promoter (142;148-150). IFN-β enhanceosome formation and gene transcription is represented in figure 11.
III.3 Biological effects of type I interferon
III 3.1 Control of viral infection
In an effort to prevent viral replication, dissemination or combat persistent viral infection, the host has developed a complex defense network. The initial response involves cells of the innate system such as cytotoxic lymphocytes and natural killer cells which destroy infected cells via perforin and granzymes. Following cellular infection a second line of defense exists. Intracellular receptors recognizing RNA (TLR3, PKR, RIG-1/MDA-5) are triggered to induce interferons to thwart viral infections from spreading. Likewise, infected cells can be prompted to undergo apoptosis (5). The importance of the IFN system is emphasized by the fact that many pathogenic viruses have developed a means of evading cellular recognition or hijacking the signaling cascade leading to IFNα/β. Viruses such as influenza A, herpes simplex and human immunodeficiency virus have well documented subversion mechanisms ranging from interfering with antigen presentation, down-regulation of co-stimulatory molecules, latency and targeting of immune cells, to promote viral replication (151-154).
III 3.2 Role in autoimmunity
Type I IFNs are cytokines that exhibit important biological effects such as antiviral, anti-proliferative and immunomodulatory activities. Unfortunately, the effects of type I IFNs can be aberrant as in the case of autoimmune disease. Type I IFNs have been identified as factors in the initiation and/or exacerbation of autoimmune disorders.
Autoimmune disease has been defined as a clinical syndrome caused by the activation of T and B cells or both, in the absence of an on-going infection or other cause.
Systemic lupus erythematosus (SLE) is a remitting disease characterized by virus- associated flares/relapses that result in the breakdown of peripheral tolerance after excessive type I IFN exposure (155). The sera of SLE patients have elevated levels of type I IFNs and PBMCs and leukocytes from these patients display an “interferogenic signature” meaning increased ISG expression (156). Treatment of PBMCs of healthy
donors with SLE sera activated DCs and drove the differentiation of monocytes to mature DCs that also displayed the “interferogenic signature”. Likewise these DCs activated and expanded auto-reactive T cells and induced auto-reactive plasma B cells. This excessive production of type I IFNs is attributed to the stimulation of the abundant pDCs present in the skin of SLE patients. Type I IFNs are able to increase CD8+ T cell cytotoxicity and enhanced auto-reactive B cell responses (155;156).
Psoriasis is the most prevalent immune-mediated skin disease in adults. There are a multitude of triggering factors that include physical injury, IFN treatment and infection.
Histologically, psoriatic lesions are characterized by thickening of the epidermis, elongated epidermal undulations, epidermal infiltrates of CD8+ T cells and myeloid DCs and pDCs, accumulation of neutrophil aggregates and blood vessel dilation. pDCs are a main source of IFN-α, are abundant in lesions and therefore proposed as a critical early initiator of cellular inflammation in the formation of psoriatic lesions (157). It was further demonstrated that injections of rIFN-α leads to psoriasis and that it also activated IFNα/β signaling in keratinocytes. To investigate the role of innate immunity in triggering pathogenic T-cell cascades leading to psoriasis, Gilliet et al investigated the effects of TLR7 signaling in psoriatic lesions. Exacerbation and spreading of the lesion was observed with the TLR7 agonist, imiquimod, treatment. Following treatment, MxA levels were increased most likely due to the stimulation of the high pDC infiltrates (158).
It is the sum of these cellular interactions that create the profile and clinical phenotype of autoimmunity (159).
IV. Interleukin 12
IV.I Role of interleukin 12 in immune responses
IL-12 is a pro-inflammatory cytokine world renowned for its ability to stimulate cellular immunity. Targeted or natural mutations occurring in IL-12 or in the components of its receptor have underscored the importance of this cytokine in the control of IFN-γ production by T and NK cells, in the development of the Th1 cellular differentiation and in the resistance to infections by intracellular organisms (160). The
IL-12 family of cytokines has now grown to encompass five subunits: p35, p40, p19, p28 and Epstein-Barr virus-induced molecule (EBI3) (161). The five subunits are able to homo- or heterodimerize among themselves which render them biologically active or antagonistic as in the case of p40 homodimers. The biologically active forms of IL- 12(p35/p40), IL-23(p40/p19) and IL-27(p28/EBI3) are summarized in figure 12. The use of genetically deficient animals or neutralizing antibodies elucidated the requirement of IL-12p70(p35/p40), IL-23(p19/p40) and IL-27(p28/EBI3) for resistance against many infectious agents and in the induction of organ-specific autoimmunity (161;162).
Structurally and functionally similar, these cytokines display distinct roles in T-helper cell responses. This family of cytokines, produced by monocytes, macrophages and DC, are tightly controlled and susceptible to multiple factors such as bacterial, viral and parasitic infection, CD40-CD40L interactions and cytokines (160;163). Herein, we will discuss the structure, receptor requirements, transcriptional control and biological function of the IL-12 family of cytokines.
IV.2 Interleukin 12 family members
IV.2.1 Interleukin 12
The IL-12 molecule is a secreted covalently linked 70 kDa heterodimer (IL- 12p70) consisting of the subunits p35 and p40 which are regulated by separate mechanisms at the transcriptional and post-transcriptional level by various TLR- triggering microbial, viral and parasitic components. The p35 sequence is homologous to that of IL-6 while p40 is homologous to the extracellular portion of the IL-6 receptor alpha chain. The p40 subunit is synthesized in excess to the p35 subunit while the latter is unable to be secreted without dimerization with other subunits such as p40 and EBI3 in the same cell. Although p35 is capable of dimerizing with EBI3, the biological function of this molecule remains to be described (161). IL-12p70 signals through the membrane bound IL-12 receptor (IL-12R) which is formed by two chains: IL-12Rβ1 and IL-12Rβ2.
IL-12R ligation by IL-12p70 activates the Janus kinase-STAT (Jak-STAT) pathway, predominately via STAT4 homodimer activation. Ultimately, IL-12 induces the
differentiation of naïve T cells into IFN-γ producing Th1 cells and the activation of antigen-presentation of DCs (164). Originally autoimmune disorders were attributed to aberrant IL-12 production but since the discovery of IL-23 and IL-27 these initial observations have been re-evaluated.
IV.2.2 Interleukin 23
Another IL-6 homologue, p19, is able to associate with p40 to form the novel covalently bonded heterodimeric cytokine IL-23 (165). IL-23 signals through a receptor complex consisting of IL-12Rβ1 chain and the IL-23 receptor (IL-23R) subunit (165;166). Similar to the IL-12R, signaling through the IL-12Rβ1-IL-23R complex leads to Jak-STAT activation but like many differences amongst the two cytokines, IL-23 preferentially induces STAT3/STAT4 heterodimers (161). This receptor is expressed on DC as well as activated T and NK cells (164). IL-23 drives the development of Th17 T cells and specifically targets the proliferation and cytokine profile of memory T cells in humans and mouse (47). IL-12 and IL-23 can be induced by similar PAMPs like LPS, CpG and CD40L while some are specific to IL-23 induction such as PGN, Bordetella pertussis and PGE2 (a product of arachidonic acid metabolism which has potent immunomodulatory effects). The production of IL-12 and IFN-γ has the ability to suppress the function of Th17 cells. IL-12p35, IL-12Rβ2, IFN-γ and STAT1 deficient mice show increased autoimmunity (47).
The IL-23-IL-17 axis has a crucial role on EAE and CIA concluded from the observation that IL-23 deficient mice are resistant to EAE and CIA (47). The use of anti- IL-23p19 and anti-IL-17 neutralizing antibodies led to complete protection and partial amelioration, respectively, of EAE symptoms (167). IL-23 is implicated in various carcinomas of multiple organs as compared normal tissue. These samples had elevated p19 and p40 mRNA expression as well as IL-17 levels while p19-/- and p40-/- mice were resistant to tumor induction as compared to control mice (168).
IV.2.3 Interleukin 27
IL-27 is another recently identified cytokine of the IL-12 family. It is composed of the p28 and EBI3 subunits that are homologous to IL-6 and IL-12p40 respectively. In humans, EBI3 binds non-covalently with p28 to induce the secretion of IL-27 as p28 is unable to be secreted as monomer or homodimer (162). The signal transducing receptor is the IL-27 receptor (IL-27R). WSX-1 (or T cell cytokine receptor TCCR) together with glycoprotein 130 (gp130) constitutes a functional IL-27 receptor. Indeed, transfection of WSX-1 or gp130 alone in mouse myeloid precursor Ba/F3 cells or mouse fibroblasts NIH3T3 cells was insufficient to render them responsive to IL-27. Only cells expressing both subunits were able to mediate cellular responses to IL-27 (169). IL-27 produced mainly by macrophages, monocytes and dendrititc cells mediated JAK activation and phosphorylation of STAT1 and STAT3. gp130 is ubiquitously expressed in immune an non-immune cells while WSX-1 is expressed primarily on lymphoid cells with highest expression in naïve T and NK cells (170). The IL-27R sensitizes naïve T cells to the early phase Th1 commitment and the proliferative effects of IL-27. In response to various bacteria and intracellular pathogens, IL-27, in synergy with IL-12 also triggers the production of IFN-γ of naïve T cells (162;169;170). Similarly, IL-27 synergized with IL-2 and IL-12 to enhance IFN-γ production from these cells (162). WSX-1/ TCCR deficient mice showed impaired Th1 responses (i.e. IFN-γ production), increased susceptibility to infection, decreased bacterial clearance and IgG2a class switching when challenged in vivo with protein antigen or intracellular pathogens (170). Conversely, there is also evidence that IL-27 can inhibit inflammatory processes. Infection with intracellular pathogens such as Toxoplasma gondii and Trypanosoma cruzi leads to the production of inflammatory cytokines, T cell activation and T cell proliferation. WSX-1-
/- and EBI3-/- mice infected with T. gondii or T. cruzi were competent to mount this type of response in order to control parasite replication but surprisingly these mice were unable to downregulate this normally protective Th1 response and developed severe inflammatory disease. WSX-1-/- and EBI3-/- mice exhibited increased IFN-γ production, highly activated T cells, increased T cell proliferation and hyperactive NK and NKT cells as compared to wild-type counterparts (171;172). These results identify IL-27 as a potent
antagonist of T cell-mediated hyperactivity by negatively regulating T cell effector functions.
Recent studies detailed a more far reaching immunoregulatory role of IL-27.
Myelin oligodendrocyte glycoprotein immunization of mice leads to the development of EAE, the murine equivalent of the human autoimmune disease multiple sclerosis. In this model, IL-27Rα-/- mice developed severe EAE characterized by hyperproliferation of Th17 cells. In this setting, it was revealed that IL-27 was able to neutralize the IL-6- induced T cell hyperproliferation through direct effect on effector T cells. The absence of IL-27Rα on these cells made them unresponsive to the supressive effects of IL-27.
This suppression was independent of Treg function (173;174).
Although IL-12, IL-23 and IL-27 show some functional overlap, these cytokines are non-redundant and functionally unique. Due to the temporal and sequential expression of these cytokines it is plausible that they act at different stages of T cell co- stimulation to influence effector cells of innate and adaptive immunity. Tentatively, the induction of IL-27 is thought to be an early inducer of Th1 commitment and differentiation that would then be followed by expansion and stabilization of Th1
responses by IL-12 (161). Interestigly, IL-27 appears to play a dual role in immunity by impacting both Th1 and Th2 immune responses (175;176). The interplay and cross- regulation of this family of cytokines exemplifies the complexity of signals that create and maintain immune responses or lead to pathology (figure 13).
IV.3 Transcriptional control of interleukin 12 family members
IV.3.1 IL-12p35
In humans and mice, the p35 subunit of IL-12 is tightly regulated and is controlled at the transcriptional and post-transcriptional levels. IL-12p35 gene regulation is complicated by the fact that it contains alternative transcription start sites and atypical post-translational processing mechanisms leading to the synthesis of various isoforms.
This tight regulation makes the p35 subunit the rate limiting step in the synthesis of bioactive IL-12p70. The 5’-flanking region of the human p35 gene has been cloned from
mononcytes and EBV-transformed B cell lines and shown to contain two distinct transcription start sites, a TATA-containing element and a CpG island upstream of the promoter, respectively (177). Gene knockout mice and over-expression approaches have identified IRF-1 and NF-κB as important transcription factors for LPS-induced and IFN-γ primed p35 induction (178). The structural rearrangement of chromatin (nucleosome disruption) is a prerequisite for high levels of gene expression. Chromatin remodeling allows access to the DNA by transcription factors necessary in controlling the expression of genes. Sensitivity to nuclease digestion reflects DNA accessibility of this region. The IL-12p35 gene contains several nucleosomes which mask nuclease hypersensitive regions before LPS/IFN-γ treatment. Indirect end-labeling experiments in human monocytic cells, THP-1, and human DCs enabled the visualization of an inducible nucleosome-free region of the p35 gene spanning nt -450 to nt -280 in response to LPS/IFN-γ stimulation Digestion with restriction enzymes further characterized the remodeled region of p35 to 120 to 167 bp which corresponds to nucleosome 2. Using 5’ deletion mutants of the p35 promoter and reporter gene assays it was further delineated that this inducible region (between nt-396 and -241) contains binding sites for the transcription factor SP1. This study elegantly shows that LPS/IFN-γ induces the transient and rapid remodeling of nucleosome 2, revealing the binding sites for Sp1 leading to p35 mRNA synthesis (179).
Goriely et al went on further to demonstrate the key role of NF-kB (p50/p65 and p65/p65) and IRF-3 in p35 promoter activity and gene expression (1;180). The aforementioned experiments give us a schematic of the IL-12p35 promoter that contains binding sites for the transcription factors Sp1, NF-κB, IRF-1 and IRF-3 which can be activated in response to various TLRs and in response to IFN-γ.
IV.3.2 IL-12p40
The molecular mechanisms of IL-12p40 gene expression have been extensively studied and some important mediators have been identified (178). The IL-12p40 promoter is assembled in four tightly positioned nucleosomes which are modified upon cellular activation with various TLR stimuli. Differences in nucleosome remodeling and access to key transcription factor binding sites are due in part to differences in TLR
