ARTHRITIS & RHEUMATISM Vol. 54, No. 2, February 2006, pp 408–420 DOI 10.1002/art.21571 © 2006, American College of Rheumatology REVIEW The Type I Interferon System in Systemic Lupus Erythematosus Lars Rönnblom,1 Maija-Leena Eloranta,2 and Gunnar V. Alm3 Introduction the type I IFN system and summarize the present knowledge of its involvement in SLE and some other autoimmune diseases. Therapeutic consequences of this information and some unanswered questions will be discussed. The type I IFN system. The type I IFN system can be defined as the type I IFN genes and proteins, the inducers of type I IFN production, the cells producing type I IFNs, as well as the target cells affected by the type I IFNs. The IFN proteins and genes. The human type I IFNs constitute a family of related proteins acting on the same receptor, the type I IFN receptor (IFNAR). The IFN␣ subtypes are quantitatively most important in this family and have a high degree of homology but unique activity profiles. They are products of 13 different genes clustered on chromosome 9, while single genes exist for IFN␤, IFN, IFN, and IFN/ (7). Recently, a new class of type I IFN–like molecules with 3 members, IFN 1–3, was identified (8,9). These novel IFNs do not use the IFNAR and are therefore sometimes classified as type III IFNs. Type II IFN has only a single member, IFN␥, which is unrelated to either type I or type III IFNs and signals through a separate receptor. IFN inducers and their recognition. Enveloped viruses are the classic type I IFN inducers, but some bacteria and protozoa can also trigger IFN production. The critical activators of IFN gene transcription are DNA or RNA that is recognized by either cell membrane– associated or intracellular receptors (Figure 1). The Toll-like receptors (TLRs) are a family of pattern recognition molecules that are activated by a wide spectrum of microbial components (10,11). TLR-3 mediates IFN␣ production induced by double-stranded RNA (dsRNA), whereas TLR-7 and TLR-8 are involved in recognition of single-stranded RNA (ssRNA) IFN inducers. In contrast, unmethylated CpG-rich DNA triggers IFN production via TLR-9. TLR-4, a receptor for lipopolysaccharide, can also mediate induction of IFN␤ expression by certain viral proteins. Most TLRs signal via the myeloid differentiation factor 88 (MyD88) Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease characterized by a large number of aberrations in the immune system, typically resulting in the formation of autoantibodies against nucleic acid and associated proteins. In order to better understand the disease mechanisms and develop efficient therapies for SLE, new technologies such as microarray analysis have been applied to cells and tissues from patients with SLE. Studies of the gene expression profile in SLE have revealed that the majority of patients have the dominant pattern of type I interferon (IFN)–inducible gene expression, commonly referred to as an IFN signature (1,2). These discoveries are consistent with earlier observations that patients with SLE have increased serum levels of IFN␣ and cellular expression of the IFN␣-inducible protein MxA (3). The observation that an SLE syndrome can develop during longterm IFN␣ treatment of patients with chronic infections and malignant diseases (4) further supports a critical role for the type I IFN system in the etiopathogenesis of SLE (5,6). Several investigations also suggest that type I IFNs may be important in autoimmune diseases other than SLE and point to the possibility that the type I IFN system is a key actor when tolerance is lost and autoreactivity appears. Here, we present a short overview of Supported in part by the Swedish Research Council, the Swedish Rheumatism Foundation, the King Gustaf V 80-Year Foundation, and the Uppsala University Hospital Research and Development Fund. 1 Lars Rönnblom, PhD, MD: Uppsala University, Uppsala, Sweden; 2Maija-Leena Eloranta, PhD: Uppsala University and Swedish University of Agricultural Sciences, Uppsala, Sweden; 3Gunnar V. Alm, PhD, MD: Swedish University of Agricultural Sciences, Uppsala, Sweden. Drs. Rönnblom and Alm have received consulting fees (less than $10,000 per year) from Miltenyi Biotec. Address correspondence and reprint requests to Lars Rönnblom, PhD, MD, Department of Medical Sciences, Section of Rheumatology, Uppsala University Hospital, SE-75185 Uppsala, Sweden. E-mail: firstname.lastname@example.org. Submitted for publication June 30, 2005; accepted in revised form October 25, 2005. 408 TYPE I INTERFERON IN SLE Figure 1. Overview of the activation of natural interferon (IFN)– producing cells (also termed plasmacytoid dendritic cells) by interferogenic immune complexes and virus. Viral DNA or RNA are recognized by either intracellular molecules (e.g., RNA is recognized by the helicase retinoic acid inducible gene I [RIG-I]) or endosomal Toll-like receptor 7 (TLR-7), TLR-8, and TLR-9. Immune complexes containing CpG-rich DNA or single-stranded RNA (and associated proteins) derived from dying cells can, after Fc␥ receptor IIa (Fc␥RIIa)– mediated endocytosis, activate endosomal TLRs 7–9. Via these receptors, nucleic acids activate the kinases tumor necrosis factor (TNF) receptor–associated factor (TRAF) family member–associated NF-B activator (TANK)–binding kinase 1 (TBK1) and IKK, which in turn phosphorylate (P) transcription factors, especially IFN regulatory factor 3 (IRF-3), IRF-5, and IRF-7. The IRFs, together with additional transcription factors (not shown), initiate expression from both type I IFN and IRF genes. Signaling via the type I IFN receptor (IFNAR) involves activation of the receptor-associated kinases Tyk-2 and Jak-1, which phosphorylate the transcription factors Stat-1 and Stat-2. The latter associate with IRF-9 and form a complex (ISGF3) that interacts with IFN-stimulated response elements (ISRE) in the promoters of hundreds of IFN-stimulated genes (ISG). Several other pathways used by IFNAR are not shown. CARD ⫽ caspase activation and recruitment domain; MyD88 ⫽ myeloid differentiation factor 88; IRAK-1 ⫽ interleukin-1 receptor–associated kinase 1. adaptor protein, but MyD88-independent pathways also exist. Thus, TLR-3 exclusively uses Toll/interleukin-1 (IL-1) receptor domain–containing adaptor-inducing IFN␤ (TRIF) and not MyD88 as the adaptor. Furthermore, TLR-4 acts on IFN␤ genes via a TRIF and TRIF-related adaptor molecule pathway (12). The adaptor molecules recruit protein kinases, notably IKK and tumor necrosis factor (TNF) receptor–associated factor 409 family member–associated NF-B activator (TANK)– binding kinase 1 (TBK1). The latter protein kinases phosphorylate IFN regulatory factor 3 (IRF-3), IRF-5, and IRF-7, which form enhanceosomes together with other transcription factors, such as CREB protein/p300, activating transcription factor 2/c-Jun, and NF-B (13). Here, mostly IFN␤ and IFN␣ gene expression has been studied, and the activation of type III IFNs and the other members of the type I IFN family remains to be elucidated. There are also TLR-independent intracellular pathways mediating type I IFN production induced by RNA that enters the cytosol. One pathway involves protein kinase R, which is activated by dsRNA (14). In addition, caspase activation and recruitment domain– containing helicase retinoic acid inducible gene I (RIG-I) has been implicated in the intracellular recognition of viral RNA, involving TANK, TBK1, and IKK in the activation of IRFs (15). Producers. Most cells can produce small amounts of IFN␣/␤ in response to certain RNA viruses. In contrast, natural IFN␣-producing cells (NIPCs), also termed immature plasmacytoid dendritic cells (PDCs), produce extremely large amounts of IFN␣ (⬃1 ⫻ 109 molecules in 12 hours) in response to many different microorganisms (16). NIPCs/PDCs are infrequent in the circulation (⬍1% of peripheral blood mononuclear cells [PBMCs]) and can be recruited to sites of inflammation. The cells express, for instance, class II major histocompatibility complex (MHC), CD4, CD40, CD83, high levels of the IL-3 receptor (CD123), and 2 specific markers termed blood dendritic cell antigen 2 (BDCA-2) and BDCA-4, but lack the costimulatory molecules CD80 and CD86 (17,18). The BDCA-2 molecule represents an endocytic type II C-type lectin, which might function as an antigen-capturing molecule (19). NIPCs/PDCs also express Fc␥ receptor IIa (Fc␥RIIa) (20) and TLR-1, TLR-6, TLR-7, TLR-9, and TLR-10 (21), which explains their capacity to sense a wide variety of different molecules in the external milieu and respond to potential danger signals. Monocyte/macrophages and myeloid/monocytederived dendritic cells produce type I IFN in response to certain RNA viruses, such as influenza and Sendai viruses, and the dsRNA poly(I-C) (22,23). These cells express TLRs 3, 4, 7, and 8 but produce IL-12 instead of type I IFN upon exposure to TLR-7/8 agonists (22,23). IFNAR-mediated effects. The IFNAR consists of 2 polypeptide chains, IFNAR-1 and IFNAR-2. Interaction of type I IFN with the IFNAR results in activation of the receptor-associated kinases Tyk-2 and Jak-1, 410 RÖNNBLOM ET AL Table 1. Immunomodulatory effects of type I interferon* Target cell Dendritic cell NIPC/PDC Monocyte/macrophage Th cell Tc cell B cell NK cell Effect Survival, maturation, and activation, with increased cross-presentation to Tc cells and stimulation of Th and B cells via, e.g., enhanced expression of costimulatory molecules and class I/II major histocompatibility complex. Prolonged survival, increased type I IFN production. Up-regulation of TLR-1, TLR-2, TLR-3, and TLR-7. Modulates antimicrobial activity, stimulates iNOS expression. Differentiation to myeloid dendritic cells and inhibition of their IL-12 production. Promotes the Th1 pathway by, e.g., increased expression of IL-12 receptor, IFN␥, and T-Bet. Enhances activation and survival of naive and memory T cells. Increased cytotoxic activity, prolonged survival. Enhanced activation and differentiation of plasmablasts to plasma cells, immunoglobulin class switch, and antibody production. Enhanced cytotoxicity and IFN␥ production. * Tc ⫽ cytotoxic T; NIPC/PDC ⫽ natural interferon-␣ (IFN␣)–producing cell/plasmacytoid dendritic cell; TLR-1 ⫽ Toll-like receptor 1; iNOS ⫽ inducible nitric oxide synthase; IL-12 ⫽ interleukin-12; NK ⫽ natural killer. which phosphorylate the transcription factors Stat-1 and Stat-2 (Figure 1). Stat-1 and Stat-2 associate with IRF-9 and form a complex (ISGF3) that interacts with IFNstimulated response elements in the promoters of hundreds of IFN-stimulated genes (24–26). Several other pathways are, however, also involved in the action of the IFNAR (24). The proteins corresponding to the IFNstimulated genes have a wide spectrum of effects on many different cell types, including inhibition of viral replication and induction of apoptosis in cells infected by at least some viruses. The IFN-stimulated genes also include genes that encode proteins such as RIG-I and IRF-5/7 that are involved in expression of type I IFN genes. By such mechanisms, type I IFNs constitute a major defense system in the innate immune response against viruses. The prominent immunomodulatory actions of IFN␣/␤ have attracted a great deal of interest in recent years (Table 1). Thus, IFN␣/␤ causes dendritic cell maturation and activation, with increased expression of class I and class II MHC molecules, chemokine and chemokine receptors, as well as costimulatory molecules such as CD80, CD86, the B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD), and a proliferation-inducing ligand (APRIL) (27). This facilitates antigen presentation, homing to lymphoid organs, and activation of immune cells. Helper T cells are in this way stimulated and develop along the Th1 pathway, at least in part because type I IFN stimulates expression of the IL-12 receptor and transcription factor T-bet, which counteract the GATA- dependent Th2 default pathway (28,29). Type I IFNs also markedly stimulate development of cytotoxic T cells, in part because of an increased cross-presenting function of dendritic cells (30) and prevention of apoptosis of activated T cells (31,32). Furthermore, the type I IFNs decrease the threshold for activation of B cells via the B cell receptor (BCR) and enhance differentiation, antibody production, and immunoglobulin isotype class switching (33–35). Therefore, the type I IFNs serve as a bridge between innate and adaptive immunity and can be viewed as stress hormones in the immune system, signaling danger and contributing to its activation. However, as discussed below, this also entails an increased risk of immunization by autoantigens. The type I IFN system in SLE Serum IFN␣. It has been known for more than 25 years that patients with SLE have elevated serum IFN␣ levels (36), and that these levels correlate to both disease activity and severity (36–38). There is also a correlation between serum IFN␣ levels and several markers of immune activation typical for SLE, e.g., anti-dsDNA titers, IL-10 levels, and degree of complement activation. From a clinical point of view, there is an association between serum levels of IFN␣ and certain disease manifestations, such as fever, skin rash, and leukopenia. In addition, increased IFN␣ levels are more common early in the disease course compared with later stages of disease, suggesting a role for IFN␣ in the initial events of the disease process. However, some patients with SLE TYPE I INTERFERON IN SLE do not have detectable levels of serum IFN␣ at the time of any investigation—not even during disease flares. Blood leukocytes from such patients nevertheless often display increased levels of the IFN␣-inducible protein MxA, indicating in vivo IFN␣ production (39). It is therefore possible that all patients with SLE have increased type I IFN production at some time point during the disease course. In order to clarify this issue, sensitive IFN␣ assays need to be developed and applied to large patient cohorts that are followed up longitudinally. NIPCs/PDCs. The number of circulating NIPCs/ PDCs in patients with SLE is reduced ⬎70-fold compared with that in normal individuals (40); this observation has been confirmed in several studies (41–43). The remaining cells have an intact IFN␣-producing capacity and express the cell surface antigens BDCA-2 and BDCA-4 (42). Furthermore, a marked increase in the number of functionally active NIPCs/PDCs was noted when PBMCs from SLE patients were cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF) and IFN␣/␥ in vitro, suggesting that there is no intrinsic defect in NIPCs/PDCs from patients with SLE. Consequently, the reduced number of NIPCs/ PDCs in the circulation is probably attributable to recruitment to tissues, and such cells can in fact be demonstrated in both skin biopsy specimens and lymph nodes obtained from patients with SLE (44–47). Chemokines, such as CXCL10/CXCL12 and their corresponding receptors CXCR3/CXCR4, are important in this recruitment of NIPCs/PDCs to tissues (16). Interestingly, IFN␣ messenger RNA (mRNA) and proteinexpressing cells could be detected in biopsy specimens obtained from both lesional and apparently normal skin, indicating a general activation of NIPCs/PDCs in the setting of SLE (44). However, the lymphoid tissues may be the most important compartment for the NIPCs/ PDCs in SLE, where locally produced IFN␣ can directly act on cells in the immune system and promote the ongoing immune response. Gene signature. Several investigators have used the microarray technique to study the gene expression profile in cells from patients with SLE (1,2,48). A consistent finding in these studies has been a clear up-regulation of IFN-stimulated genes in the majority of patients, which is known as the IFN signature. It has been established that type I IFN, not IFN␥, is responsible for this IFN signature (49). However, it remains to be elucidated to what extent type III IFNs (IL-28/29) also contribute, because these cytokines activate Stat-1 and Stat-2 like the type I IFNs, despite acting on a receptor other than the IFNAR (50). The IFN signature 411 was observed in PBMCs from almost all pediatric SLE patients with relatively active disease of recent onset (48). This is in contrast to studies in adults with more long-lasting and less active disease, in which approximately half of the patients displayed the IFN signature (1,2). In adult patients, the IFN signature is associated with severe disease manifestations, such as central nervous system involvement or nephritis (1). The IFN signature was also seen in the majority of laser-captured glomeruli in patients with lupus nephritis, although some patients demonstrated other gene expression profiles (51). These findings support previous observations (36–38,52,53) that activation of the type I IFN system is most prominent in early and active disease. An important observation in the gene expression studies is the finding that several lupus autoantigens, such as Ro 52 and laminin-1b, are up-regulated by IFN␣ (48). An increased expression of these autoantigens may influence the autoantibody repertoire (see below). At the moment, it is not known how the IFN signature is expressed and varies in SLE patients during long-term followup, but longitudinal studies are under way (1). Such investigations will provide important information on the value of analyzing the gene signature in SLE patients in order to predict and monitor flares and organ manifestations, as well as treatment response and outcome. Polymorphisms of IFN-related genes. Several genes are associated with increased susceptibility for the development of SLE (54), but genes with major functions in the type I IFN system (type I IFN–related genes) have not until recently been investigated in humans. We analyzed single-nucleotide polymorphisms (SNPs) in 11 type I IFN–related genes in SLE patients, 4 of which were linked to the production and 7 of which were linked to the action of type I IFN (55). This investigation revealed SNPs in 2 of the studied genes, Tyk-2 and IRF-5, with strong signals in joint analysis of linkage and association with SLE. Tyk-2 binds to the IFNAR-1 and is responsible both for maintaining the expression of the complete IFNAR on the cell surface and for signaling via the receptor after IFN␣/␤ ligation. Interestingly, Tyk-2 is also involved in the function of several other cytokines that have been reported to be elevated in SLE patients, such as IL-6 and IL-10 (56). The importance of Tyk-2 in autoimmunity is further supported by the observation that Tyk-2–deficient mice are resistant to experimental arthritis (57). IRF-5, in contrast, is important for induction of IFN␣ production by viral infections and TLR-7/8 agonists and is constitutively expressed in 412 NIPCs/PDCs (58,59). In addition, IRF-5 increases the expression of several other genes involved in cell signaling, apoptosis, cell cycle regulation, and immune activation (60). Therefore, polymorphisms altering the function of IRF-5 may, in many ways, facilitate the development of an autoimmune disease such as SLE. The association of TYK-2 and IRF-5 genes to SLE further strengthens the proposed important role of the type I IFN system in the etiopathogenesis of this disease. Because many hundreds of genes are involved in the activation, regulation, and action of the type I IFN system, more type I IFN–related genes should be analyzed for a possible connection to SLE. Several genes already reported to be associated to SLE may actually have their most important effect on the disease by affecting the type I IFN system (e.g., the genes encoding Fc␥RIIa, which are important in the activation of NIPCs/PDCs by interferogenic immune complexes (see below), and the genes for complement factors C1q and C-reactive protein, which contribute to clearance of apoptotic cells) (54). Murine models. A role for type I IFN in the development of experimental SLE was suggested early on by the finding that administration of exogenous IFN␣/␤ to NZB and NZB/W mice accelerates the progression and severity of the disease (61,62). In addition, when agents that induce type I IFN production were injected into lupus-prone mice, a more severe disease was observed (63–65). The importance of IFN␣ in the disease process is also supported by the observation that in vivo expression of IFN␣ by an adenovirus vector induced early lethal lupus in susceptible animals (66). The potential role of the type I IFN system as a therapeutic target in SLE is suggested by the finding that crosses between lupus-prone NZB or C57BL/6-lpr/lpr mice and type I IFNAR–knock-out mice have a dramatically reduced SLE disease (67,68). In contrast, the absence of a functional IFNAR in lupus-prone MRL/lpr mice causes enhanced production of autoantibodies, increased lymphadenopathy, and a more severe renal disease (69). The reason for the contradicting results is unclear, but the results indicate important differences between experimental murine lupus models. Activation and regulation of NIPCs/PDCs in SLE Activation. The ongoing production of IFN␣ in many patients with SLE suggests the presence of inducers that trigger expression of IFN␣ genes. Therefore, an important finding was that DNA-containing immune complexes in patients with SLE can induce IFN␣ pro- RÖNNBLOM ET AL duction by NIPCs/PDCs, and that the occurrence of such interferogenic immune complexes is associated with active disease (5,6,70). When formed, these immune complexes are internalized via Fc␥RIIa and reach the endosomes of NIPCs/PDCs, with subsequent activation of the relevant TLR and induction of IFN␣/␤ production (20,70). The nature of the cellular nucleic acids that trigger IFN production by these immune complexes is unknown, but in vitro studies show that apoptotic cells generate DNA-containing material that can form interferogenic immune complexes (71,72). Cells dying by apoptosis or necrosis also release RNA-containing material with interferogenic properties (72), and it was recently shown that ssRNA with guanosine- and uridinerich sequences can activate IFN␣ production via interaction with TLR-7 (73,74). Such RNA sequences are commonly observed in normal RNA, and obvious candidate IFN␣ inducers include the U-series small nuclear RNA that are associated with several RNPs and are part of the spliceosomes. Autoantibodies to the small nuclear RNPs are common in SLE and may be responsible for the formation of interferogenic immune complexes. Accordingly, the same molecules that are released at the time of cell death and constitute major autoantigens in SLE and several other systemic autoimmune diseases are also potent IFN␣ inducers and can act as endogenous adjuvants in the autoimmune process (see below). Because patients with SLE have reduced clearance and increased numbers of apoptotic cells (75), apoptotic cell material should be readily available in vivo to generate interferogenic immune complexes. Regulation. The in vitro production of IFN␣ triggered by interferogenic immune complexes in NIPCs/PDCs is markedly increased in the presence of IFN␣/␤ and, in some instances, also GM-CSF (6,76). One possible reason for this phenomenon, which is termed priming, is that type I IFNs increase the levels of transcription factors IRF-5 and IRF-7 necessary for induction of major IFN␣ genes (13). During viral infection, this positive feedback mechanism for enhancement of IFN␣ production is essential and gives rise to an early and strong antiviral response. In contrast, the disease flares that are frequently seen during infection in patients with SLE (77) may be attributable to priming effects of type I IFN induced by virus or bacteria. Most immune complexes do not induce IFN production in NIPCs/PDCs. In fact, immune complexes that lack interferogenic DNA or RNA cause a general inhibition of the response of NIPCs/PDCs to a wide variety of IFN inducers, even those that are independent TYPE I INTERFERON IN SLE of Fc␥RIIa (20). One possible explanation for such inhibition is that immune complex delivers negative signals to NIPCs/PDCs via crosslinking of the Fc␥RIIa. This Fc receptor is regarded as an activating receptor because of its content of immunoreceptor tyrosinebased activation motifs, but it can also be inhibitory by activating SH2-containing inositol phosphatase 1 and the protein tyrosine phosphatase src homology phosphatase 1 in a manner similar to that of the inhibitory Fc␥RIIb (78,79). Several other receptors on NIPCs/PDCs can also modulate IFN␣ production. Thus, ligation of not only BDCA-2, but also BDCA-4, inhibits the IFN␣ response (19,42). In addition, certain cytokines have a negative impact on NIPCs/PDCs. In particular, IL-10 is a potent inhibitor of the IFN␣ production triggered by both viruses and immune complexes, but TNF␣ also reduces IFN␣ production by NIPCs/PDCs (76,80). Induction of autoimmunity by type I IFN The possible role of IFN␣ as an inducer of autoimmunity was originally suggested by the observation that in patients with malignant disease treated with IFN␣, autoantibodies and sometimes also overt autoimmune disease frequently developed (4). For instance, as many as 19% of patients with malignant carcinoid tumors who were receiving long-term treatment with IFN␣ eventually manifested an autoimmune disease, including SLE (81). Preexisting autoantibodies are not necessary for the development of autoimmunity, although the presence of autoantibodies before IFN␣ therapy considerably increases the risk of autoimmune disease developing. The conclusions from these observations are that type I IFN can both break tolerance and promote ongoing autoimmune reactions in humans. What are the mechanisms behind this propensity of type I IFNs to cause autoimmune reactions? As outlined above, type I IFNs have several effects on key immune cells that are likely to promote the activation of potential autoimmune cells (Table 1). Furthermore, type I IFN promotes the production of IFN␥ and other cytokines such as IL-6, IL-10, and IL-15 that are important for sustained activation of the immune system (27). Type I IFN also potentiates the effects of IFN␥ and IL-6 (82,83) and shifts the actions of IL-10 from an antiinflammatory to a more proinflammatory profile (84). The increased expression of all of these molecules decreases the threshold for cellular activation and increases the likelihood for the initiation of an autoimmune reaction. Type I IFN can also have direct effects on target tissues 413 in autoimmune disease by causing expression of autoantigens to which autoantibodies are directed, a phenomenon commonly seen in SLE (48,85). This action of type I IFNs may be as important as their direct effects on the immune system in the initial phase of the development of autoimmunity. In addition, it has been proposed that class I MHC up-regulation caused by IFN␣ in tissues makes them a target for autoimmune attack by cytotoxic T cells (86). A special case is the human endogenous retrovirus (HERV)-K18 superantigen, which is induced by IFN␣ and may have a pathogenic role in type I diabetes (87). A role for HERV in SLE has also been postulated, because sex hormones, especially estrogens, appear to increase the expression of autoantigens such as HERV (88). This could contribute to the fact that the prevalence of SLE is higher in females than in males. Taken together, results of many clinical and experimental studies have established that type I IFN can push the immune system toward an autoimmune state. Several of the alterations seen in the immune system after type I IFN exposure have also been observed in SLE and may be highly relevant for the understanding of the pathogenesis of this disease. The type I IFN system in the etiopathogenesis of SLE We have integrated the many findings concerning the type I IFN system in SLE in an etiopathogenic model of SLE, which also includes other observations in this disease (Figure 2). This model was first described in 1999 (6) and has subsequently been updated (89,90). It is envisioned that infections by viruses or bacteria can provoke an initial autoimmune response by activating dendritic cells (in particular NIPCs/PDCs). The production and action of type I IFN are important in this context. Indeed, autoantibodies are common during viral infections (91), either due to loss of tolerance to ubiquitous cellular autoantigens or due to molecular mimicry. The latter possibility is supported by data that link Epstein-Barr virus to SLE, because the major autoantigens SmB⬘ and SmD1 cross-react with sequences from Epstein-Barr nuclear antigen 1 (EBNA-1), and immunization of mice with cross-reactive EBNA-1 sequences precipitates lupus-like autoimmunity (92). Furthermore, a peptide from the EBNA-1 antigen induces production of antibodies against 60-kd Ro in mice, and autoantibodies to this EBNA-1–related Ro 60 epitope were detected in patients an average of 3.9 years before the development of SLE. In fact, low levels of different autoantibodies directed to nucleic acid– 414 RÖNNBLOM ET AL Figure 2. The role of interferogenic DNA/RNA-containing immune complexes and type I IFN produced by immature plasmacytoid dendritic cells (PDCs; also termed natural IFN␣-producing cells [NIPCs]) in the induction and maintenance of autoimmune disease. Production of autoantibodies occurs during viral or bacterial infection, in part because of adjuvant effects of microbial components and molecular mimicries. Cells dying by apoptosis or necrosis release DNA- or RNA-containing autoantigens that form interferogenic immune complexes with autoantibodies. Such immune complexes act as endogenous inducers of type I IFN production in NIPCs/PDCs (see Figure 1) and can directly activate autoantibody-producing B cells via the B cell receptor (BCR) and TLRs 7–9. Type I IFN, especially IFN␣, has multiple immunostimulatory actions that include induced maturation of monocytederived dendritic cells (moDC), development of Th1 cells and cytotoxic T (Tc) cells, as well as facilitation of B cell activation. All of this results in further autoantibody production, formation of more IFN-inducing immune complexes, IFN␣ production, and IFN-mediated immunostimulation. The autoimmune process is consequently sustained by a mechanism with the features of a vicious circle. IL-12R ⫽ interleukin-12 receptor (see Figure 1 for other definitions). associated proteins or nucleic acids can be detected for long periods of time in apparently healthy individuals in whom SLE eventually develops (93). Once autoantibodies are available, they can form immune complexes by combining with apoptotic or necrotic cell material that contains interferogenic DNA or RNA. Such interferogenic immune complexes stimulate long-term IFN␣ production by NIPCs/PDCs in the absence of exogenous inducers such as virus or bacteria, thereby explaining the IFN signature that is present in patients with SLE. The produced IFN␣ will increase autoantibody production by promoting dendritic cell maturation, T cell activation, and stimulation of B cells, as outlined above. DNA-containing immune complexes can, in addition, directly activate autoreactive B cells (94), which will further increase the production of autoantibodies necessary for formation of interferogenic immune complexes. The increased apoptosis and decreased clearance of apoptotic cell material seen in SLE will provide autoantigens that will be both immunogenic and IFN␣ inducing. Exposure to ultraviolet light or infections will generate more apoptotic or necrotic material, and the resulting inflammatory process will promote the extracellular release of nucleic acid (95). In addition, IFN␣ can increase both apoptosis (96) and expression of autoantigens (85), thereby adding fuel to the autoimmune process. A process that has the features of a vicious circle is thus established and maintains autoimmunity by continuously exposing the immune system to endogenous IFN␣ inducers and to the IFN␣ produced by NIPCs/ PDCs. The activity of this vicious circle is increased by recruitment of new NIPCs/PDCs to tissues by chemokines and priming of these cells by, for example, IFN␣. TYPE I INTERFERON IN SLE The induction of production of such cytokines by microorganisms may reactivate the vicious circle, explaining the disease flares sometimes observed in SLE patients during infection. Finally, cytokines that limit the activity of NIPCs/PDCs, especially IL-10 and TNF␣, may constitute a beneficial negative feedback mechanism in SLE (76). The described disease process is obviously dependent on several environmental and genetic factors. Genes that influence the function of the type I IFN system, including the capacity of the immune system to be activated by type I IFN and establish a vicious circle, are expected to confer increased or decreased risk for SLE. It is, however, important to recognize the many different hormonal, inflammatory, and vascular factors that determine the final clinical phenotype of SLE disease in individual patients. The type I IFN system in other autoimmune diseases Given the fact that the type I IFN system acts as a sensor of environmental danger and has potent and pleiotropic effects on the immune system, one could anticipate that type I IFN is involved in other autoimmune diseases besides SLE. In fact, a large number of different autoimmune conditions develop in patients during IFN␣ treatment (4). Furthermore, studies of the type I IFN system in several autoimmune diseases indicate that NIPC/PDC activation and IFN␣ production may be common events in autoimmunity. Increased serum levels of IFN␣ and expression of IFN␣ mRNA in the pancreatic islets have been observed in patients with insulin-dependent diabetes mellitus (97). In addition, expression of IFN␣ genes in islets precedes the lymphocyte infiltration and islet cell destruction observed in experimental diabetes, which moreover can be prevented by neutralizing anti-IFN␣ antibodies. Recently, increased IFN␣ mRNA expression was also noted in liver biopsy specimens obtained from patients with primary biliary cirrhosis (98). Among patients seen in rheumatology clinics, those with rheumatoid arthritis, dermatomyositis, primary Sjögren’s syndrome (pSS), and psoriasis all have signs of type I IFN production. Thus, infiltration of NIPCs/PDCs and expression of IFN␤ and the type I IFN–inducible protein MxA can be detected in the synovia of patients with rheumatoid arthritis (99–101). Patients with dermatomyositis have a typical IFN gene signature and IFN␣–containing BDCA-2–positive cells in muscle tissue (102,103). In a recent study, we found IFN␣-producing cells in the salivary glands of patients with pSS (104). In addition, necrotic or apoptotic cell 415 material in combination with autoantibodies to RNAbinding proteins from pSS patients specifically triggered IFN␣ production by NIPCs/PDCs. This IFN␣-inducing capacity of pSS sera was associated with both lymphocytic foci in the labial salivary glands and several extraglandular disease manifestations (e.g., dermatologic, hematologic, and pulmonary involvement). Gene expression profiles of minor salivary gland biopsy specimens also show an up-regulation of type I IFN– regulated genes (105). Skin biopsy specimens obtained from patients with psoriasis display increased expression of mRNA for both IFN␣ and IFN-induced proteins as well as an infiltration of NIPCs/PDCs (46,106). Furthermore, an essential role for NIPCs/PDCs and IFN␣ in the development of psoriatic lesions was recently demonstrated in a xenograft model of human psoriasis (107). Taken together, emerging data suggest that the type I IFN system could be an important etiopathogenic factor in several systemic and organ-specific autoimmune diseases. The genetic background of the individual, the nature of the initial trigger of the IFN␣ production, as well as the target organ affected by NIPCs/PDCs and IFN␣, could all be factors determining the type of disease that eventually develops. Therapeutic consequences Although the precise role of the type I IFN system in the various autoimmune disorders remains to be established, the observed connection between IFN␣ and autoimmune disease suggests that down-regulation of this system should have beneficial effects in patients. In fact, 2 of the best-documented therapeutic agents in SLE, chloroquine and glucocorticoids, inhibit IFN␣ production by NIPCs/PDCs (108) and down-regulate the IFN signature (48), respectively. These drugs, however, have multiple cellular effects, and more specific therapies directed to the type I IFN system should be explored (Table 2). The most obvious therapeutic target in SLE is IFN␣, and a humanized monoclonal anti-IFN␣ antibody that neutralizes all IFN␣ subtypes has been developed (109). Soluble IFNAR is another therapeutic option, although such a molecule might also act as a ligand-dependent agonist (110). Furthermore, the NIPCs/PDCs could be directly targeted by human monoclonal antibodies to BDCA-2, because ligation of this molecule turns off the IFN␣ production by NIPCs/PDCs (19,42). Several approaches can be used to reduce the amount of endogenous IFN␣ inducers or the expression of type I IFN genes in response to these inducers. DNase 416 RÖNNBLOM ET AL Table 2. Therapeutic targets of potential importance for reducing the action of type I interferon in systemic lupus erythematosus* Target Type of drug Interferogenic DNA and RNA Autoantibodies Nucleases Fc␥ receptor IIa Antagonist Toll-like receptors Type I IFN Type I IFN receptor Type I IFN signaling pathway IFN-inducible genes NIPC/PDC Inhibitory ODN or ORN Antibody/soluble IFN receptor Antagonist Kinase inhibitors, etc Effect Tolerogen/B cell depletion Histone deacetylase inhibitors Anti–BDCA-2 antibodies Reduce amount of IFN inducer in immune complexes Reduce amount of interferogenic immune complexes Prevent uptake by NIPC/PDC of interferogenic immune complexes Block induction of IFN production Neutralize biologically active IFN Prevent binding of IFN to the receptor Reduce the IFN signal downstream of the receptor Inhibit IFN-stimulated gene transcription Inhibit IFN production * ODN ⫽ oligodeoxynucleotide; ORN ⫽ oligoribonucleotide; BDCA-2 ⫽ blood dendritic cell antigen 2 (see Table 1 for other definitions). treatment has been tried in SLE patients, with the rationale to eliminate pathogenic dsDNA/anti-dsDNA antibody complexes (111). No clear therapeutic effect was noted, but this could theoretically be due to the importance of RNA-containing immune complexes in the induction of IFN␣ in SLE in vivo. The tolerogen LPJ-394 that reduces the level of anti-dsDNA antibodies in SLE has only a moderate effect on disease activity (112), perhaps because antibodies with other specificities are important in the generation of interferogenic immune complexes. There are several possible ways to prevent the action of interferogenic immune complexes on NIPCs/PDCs. Besides blocking Fc␥RIIa by specific antibodies (20), the TLRs involved in IFN␣ gene activation by such immune complexes could be inhibited by TLR antagonists in the form of oligodeoxynucleotides (ODNs) or oligoribonucleotides. In fact, treatment of lupus-prone mice with suppressive ODNs prolonged their survival (113), but the effects on the type I IFN system was not extensively investigated. Several of the signaling molecules downstream of the TLRs, such as MyD88, IL-1 receptor–associated kinase 1, IRF-5, and IRF-7, remain to be investigated as therapeutic targets in SLE (55,114,115). Drugs that interfere with the post-IFNAR events could be used to reduce the effects of IFN␣. Several possible target molecules exist, with inhibitors of Tyk-2 being one example. In addition, the transcription of IFN-stimulated genes could be blocked by histone deacetylase inhibitors (116,117). In fact, such drugs can reduce glomerulonephritis in the MLR-lpr/lpr mouse (118). In conclusion, a large number of molecules within the type I IFN system have the potential to serve as therapeutic targets in SLE. Several of these molecules have pleiotropic effects in the immune system, and inhibition of their function can have additional beneficial effects besides those on the type I IFN system. Unresolved questions There are several questions that should be addressed before embarking on clinical trials with the objective to inhibit the type I IFN system in patients with SLE. The first question is, will the therapy work as well in humans as in some experimental lupus models? One word of caution is warranted, because MLR/lpr mice lacking the IFNAR developed a more severe disease compared with wild-type animals (69). This is in contrast to the NZB and C57BL/6-lpr/lpr strains (67,68). Which of these 2 experimental SLE disease models is most relevant to human SLE? This issue can be resolved only by clinical studies in human patients. Second, why do not all patients with SLE have an activated type I IFN system? As discussed above, there may be several reasons for this observation, but patients without an activated type I IFN system cannot be expected to benefit from type I IFN inhibitors. Alternatively, some patients with SLE may display an activation of the type I IFN system due to ongoing viral infection, and not because of increased SLE disease activity. In this situation, interference with the type I IFN system may be dangerous and even cause mortality in treated patients. How is it possible to discriminate between active lupus disease and an infection, since these 2 conditions often resemble each other and may even exist together in the same individual? The answer to this question is complex and illustrates the fact that clinical skill and TYPE I INTERFERON IN SLE judgment, as well as advanced laboratory techniques, are necessary in order to optimize therapy for each patient. In addition, even if therapy that targets type I IFN is administrated based on the correct indication, this treatment may increase susceptibility to viral infections. 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