Role of endogenous retroviruses in murine SLE

Autoimmunity Reviews 10 (2010) 27–34

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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev

Review

Role of endogenous retroviruses in murine SLE Lucie Baudino, Kumiko Yoshinobu, Naoki Morito, Marie-Laure Santiago-Raber, Shozo Izui ⁎ Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland

a r t i c l e

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Article history: Received 15 July 2010 Accepted 21 July 2010 Available online 24 July 2010

a b s t r a c t Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by B cell hyperactivity leading to the production of various autoantibodies and subsequent development of glomerulonephritis, i.e. lupus nephritis. Among the principal targets of the autoantibodies produced in murine SLE are nucleic acid–protein complexes and the envelope glycoprotein gp70 of endogenous retroviruses. Recent studies have revealed that the innate receptor TLR7 plays a pivotal role in the development of a wide variety of autoimmune responses against DNA- and RNA-containing nuclear antigens, while TLR9 rather plays a protective role. In addition, the regulation of autoimmune responses against endogenous retroviral gp70 by TLR7 suggests the implication of endogenous retroviruses in this autoimmune response. Moreover, the demonstration that TLR7 is involved in the acute phase expression of serum gp70 uncovers an additional pathogenic role of TLR7 in murine lupus nephritis by promoting the expression of nephritogenic gp70 autoantigen. Clearly, the eventual identification of endogenous retroviruses implicated in murine SLE and of mouse genes regulating their production could provide a clue for the potential role of endogenous retroviruses in human SLE. © 2010 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 2. Polygenic control of the expression of serum retroviral gp70 . 3. Sgp-mediated increase of gp70 production during acute phase 4. TLR7 and endogenous retroviruses in murine SLE . . . . . . 5. Endogenous retroviruses in human SLE . . . . . . . . . . . 6. Concluding remarks . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Systemic lupus erythematosus (SLE) is a disorder of systemic autoimmunity characterized by the production of a variety of autoantibodies and subsequent development of glomerulonephritis, i.e. lupus nephritis [1]. Mice of the (NZB × NZW)F1 hybrid, MRL and BXSB strain have been extensively used as experimental models of human SLE [2]. They are characterized by a wide spectrum of autoimmune manifestations culminating in the development of immune complex (IC)-mediated lupus nephritis. The severity of kidney lesions is closely associated with the increase in serum titers of IgG autoantibodies directed against various ⁎ Corresponding author. Tel.: +41 22 379 5741; fax: +41 22 379 5746. E-mail address: [email protected] (S. Izui). 1568-9972/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2010.07.012

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27 28 29 29 31 32 32 32 32

nuclear antigens. In addition, lupus-prone mice spontaneously develop autoantibodies against serum glycoprotein gp70 derived from endogenous retroviruses [2–5]. The availability of several murine strains with distinct genetic backgrounds has offered an invaluable opportunity for elucidating the genetic and immunological basis underlying the etiopathogenesis of SLE. Genome-wide linkage analyses in mice obtained through intercrosses or backcrosses of different lupus-prone and nonautoimmune strains have shown that (1) major histocompatibility complex (MHC)-linked and multiple non-MHC-linked genetic factors independently or additively contribute to the overall susceptibility and progression of the disease; (2) heterogeneous combinations of multiple disease-promoting genes operate in a threshold-dependent manner to achieve full expression of the disease; and (3) contributions are unlikely to be linked to “true” genetic mutations, but are

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rather due to polymorphic alleles with subtle functional differences, except for the Fas and Yaa (Y-linked autoimmune acceleration) mutations observed in MRL and BXSB mice, respectively [6,7]. The Yaa mutation was recently identified to be a translocation from the telomeric end of the X chromosome, containing the gene encoding Toll-like receptor 7 (TLR7), onto the Y chromosome in BXSB male mice [8–10]. Accordingly, the Tlr7 gene duplication has been proposed to be the disease-accelerating mechanism conferred by the Yaa mutation. Current efforts are focused on evaluation of candidate genes located within lupus susceptibility loci by analyzing autoimmune phenotypes in mice congenic for different susceptibility intervals. The involvement of endogenous retroviruses in SLE is a longstanding theory. This relationship was first suggested when murine leukemia viral antigens were found in immune deposits of diseased glomeruli in lupus-prone NZB and (NZB × NZW)F1 hybrid mice [11,12]. Subsequently, it was demonstrated that relatively large amounts of the envelope glycoprotein gp70, derived from endogenous retroviruses, are present in the sera of lupus-prone (NZB × NZW)F1, MRL and BXSB mice, and that only lupus-prone mice spontaneously develop autoantibodies against serum retroviral gp70 [2–4]. Indeed, gp70-anti-gp70 IC (gp70 IC) were detected close to the onset of renal disease in the circulation and found within diseased glomeruli of lupus mice [3,4]. Several genetic studies have revealed a remarkable correlation of serum levels of gp70 IC with the development of severe lupus nephritis [13–16], thus further supporting the pathogenic role of gp70 IC in murine SLE. 2. Polygenic control of the expression of serum retroviral gp70 Retroviruses are RNA viruses that employ the virus enzyme reverse transcriptase to transcribe their RNA genome into DNA, which is integrated into the host cell genome, called provirus. Two identical long terminal repeats (LTR), which flank the coding sequences of retroviruses, contain sequences termed U3 (3′ unique region), R (repeat) and U5 (5′ unique region). Transcription of the provirus initiates at the U3-R boundary in the upstream LTR and RNA cleavage/polyadenylation occurs at the R-U5 boundary in the downstream LTR. The U3 sequence located on the 5′-LTR serves as a viral promoter. The murine retroviral env (envelope) gene encodes a precursor polyprotein which is cleaved to produce two subunits; an envelope glycoprotein, gp70, and a transmembrane protein, p15E. Both subunits remain associated on the intact virions through disulfide linkage. The p15E protein contains an amino-terminal hydrophobic peptide that is thought to mediate membrane fusion, whereas gp70 bears the receptor binding function. All inbred strains of mice carry numerous endogenous retroviruses as chromosomal genes. Certain viral gene segments are expressed selectively, depending on their site of integration into chromosomal loci, in which induction is linked to the differentiation state of the cells [17]. Indeed, gp70 of endogenous retroviruses is a constituent of the surface of various epithelia and lymphocytes, and shares immunological and biochemical properties with the thymocyte differentiation antigen GIX [17–20]. In addition, gp70 circulates in the blood of virtually all strains of mice, but is not associated with viral particles. It has previously been demonstrated that lymphoid cells are not a major source for serum retroviral gp70 because neither thymectomy nor splenectomy affected serum levels of gp70 [21]. Rather, serum gp70 behaves like an acute phase protein (APP) and is secreted by hepatocytes into the circulating blood [22–24]. Endogenous retroviruses are classified as ecotropic (Eco), xenotropic (Xeno) or polytropic according to their host range, which is dictated by their respective gp70 proteins [25]. Eco viruses utilize the cationic acid transporter (CAT-1) as an entry receptor [26] and can infect murine, but not non-murine cells. Xeno and polytropic viruses share a common entry receptor, XPR1 (xenotropic and polytropic retrovirus receptor 1) [27,28]. However, the polymorphic form of

XPR1 in laboratory mouse strains renders these strains resistant to infection by Xeno viruses [29]. Thus, Xeno viruses can infect nonmurine cells but not cells derived from laboratory mouse strains, while polytropic viruses can infect both murine and non-murine cells. Based on differences in their gp70 nucleotide sequences, the polytropic viruses have been further divided into two subgroups, termed PT and modified PT (mPT) [25]. It has long been believed, based on serological and tryptic peptide mapping analysis, that gp70 derived from Xeno viruses is the predominant source of gp70 present in the serum [5,30,31]. However, our recent analysis of the abundance of different hepatic gp70 RNAs and of the genomic composition of corresponding proviruses in various strains of mice revealed a heterogeneous origin of serum gp70 with a substantial contribution of PT and mPT gp70s, in addition to Xeno gp70 [23]. Serum concentrations of gp70 are highly variable among different strains of mice [4]. All SLE-prone strains have relatively high concentrations of gp70 in their sera (N15 μg/ml), whereas C57BL/6 (B6), C57BL/10 (B10) and BALB/c mice produce low serum levels of gp70 (b5 μg/ml). Interval mapping of backcross progeny between lupus-prone mice and B6 or B10 mice identified a major locus controlling gp70 production on mid-chromosome 13, designated Sgp3 (serum gp70 production 3) or Bxs6 (the Bxs6 locus identified in BXSB mice is likely to be identical to Sgp3) [16,32,33]. Indeed, B6 or B10 mice congenic for the Sgp3 or Bxs6 locus derived from either the NZB, NZW or BXSB strain display approximately 10-fold higher levels of gp70, as compared with wild-type B6 or B10 mice [32–34]. Furthermore, analysis of gp70 RNA revealed that Sgp3 controls in trans the expression of Xeno, PT and mPT retroviruses in the liver [23]. This is reminiscent of the way the Gv1 (Gross virus antigen 1) gene regulates the expression of thymic GIX gp70 antigen [35]. Since the expression of the GIX gp70 antigen is closely correlated to serum levels of gp70 [36] and since Gv1 directly overlaps with the Sgp3 locus [37], it is reasonable to assume that Gv1 and Sgp3 are identical or related genes regulating the transcription of retroviral sequences. To explore the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE, we have recently conducted an extensive analysis of the abundance of four different classes of retroviral gp70 RNAs in different strains of mice (Table 1). This analysis revealed a more than 15-fold increased expression of mPT gp70 RNA in all four lupus-prone mice, as compared with that in nine non-autoimmune strains of mice [38]. Notably, this was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT

Table 1 Expression of different classes of endogenous retroviral gp70 RNAs in livers of various strains of mice. Strains

Eco

Xeno

PT

mPTa

mPT D1b

mPT D2c

NZB NZW BXSB MRL NFS 129 AKR DBA/2 B6 B10 BALB/c CBA C3H

− + − − − − +++ + − − − − ++

+++ +++ ++ ++ ++ − + ++ + + − ++ ++

++ ++ ++ ++ ++ ++ ++ ++ + − + ++ +

+++ +++ +++ +++ + + + + + + − − +

+ + + + + + + + + + + + +

− − + − + − + − + + + − −

Adapted from Yoshinobu, K. et al. [38]. a mPT virus bearing the intact env gene. b mPT D1 virus carrying a deletion in the 3′ portion of the gp70 surface protein. c mPT D2 virus carrying a deletion in the 5′ portion of the p15E transmembrane protein.

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transcripts, many strains of mice expressed two defective (D1 and D2) mPT env transcripts which carry a deletion in the 3′ portion of the gp70 surface protein and the 5′ portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to non-autoimmune strains of mice, all four lupus-prone mice predominantly and abundantly expressed intact mPT env transcripts at the near exclusion of defective env transcripts, and their levels were more than 100-fold higher than those in non-autoimmune strains of mice. The analysis of Sgp congenic mice revealed that the Sgp3 locus was responsible for the selective up-regulation of the intact mPT env RNA [38]. Since BXSB is a recombinant strain derived from a cross of B6 and SB/Le mice and since the Sgp3 allele of BXSB mice is inherited from the SB/Le strain, it was somehow unexpected that SB/Le mice failed to display the predominant expression of the intact mPT env transcripts [38]. This was also the case for the GIX+ 129 strain of mice, which is expected to share the Sgp3 allele with lupus-prone mice, because the 129 strain carries the Gv1 locus promoting an up-regulated expression of the GIX gp70 antigen [35]. Indeed, our recent studies on B6 congenic mice bearing the 129-Sgp3 allele revealed that the 129-Sgp3 allele is able to promote the predominant and abundant expression of intact mPT env transcripts in B6 mice (Baudino, L. et al., J. Autoimmun. in press.). Thus, the lack of predominant expression of intact mPT env transcripts in 129 as well as SB/Le mice could be rather due to the absence of a particular fraction of mPT provirus, the expression of which can be selectively up-regulated by Sgp3. Notably, other GIX+ strains of mice, such as AKR, DBA/2 and C3H/He [39], also displayed the expression pattern of the three species (WT, D1 and D2) of mPT env RNAs similar to that of 129 and SB/Le mice [38]. If we assume that all the GIX+ strains of mice carry the same Sgp3 allele, the copy number of the unique mPT provirus responsive to Sgp3 and its strain distribution may be very limited. In view of the potential association of mPT retroviruses with murine SLE, it is of interest to identify the genetic origin of the mPT provirus that is highly expressed by Sgp3 in lupus-prone mice, since this would help determine its pathogenic role in the development of murine SLE. Our ongoing analysis of Sgp3 subcongenic lines has narrowed down the Sgp3 region to a 5.42 Mb between 64.54 and 69.96 Mb of chromosome 13. Among 30 genes mapped to this region in the NCBI database, 21 of them encode Krüppel-associated box zinc-finger proteins (KRAB-ZFP), although the precise function of most of these Zfp genes has not yet been identified [40,41]. KRAB-ZFP are composed of a KRAB domain, which represses transcription by recruiting the KAP1 (KRAB-associated protein 1) corepressor acting as a scaffold for chromatin-condensing protein, and of a zinc-finger domain, which selectively recognizes target genes through recognition of specific regulatory sites within the DNA [42]. It has recently been shown that KRAB suppressed lentivirus proviral transcription by inducing heterochromatization in the lentiviral integration sites [43] and that ZFP809 silences integrated retroviral DNAs through the recruitment of KAP1 in embryonic stem cells [44]. Thus, Zfps are the primary candidate genes for Sgp3. The second genetic locus linked to serum gp70 in the NZB strain is the Sgp4 locus on chromosome 4. The contribution of Sgp4 to the production of serum gp70 is more modest than that of Sgp3, which is consistent with the finding that Sgp4 controls only the expression of Xeno gp70 [23]. Double congenic mice bearing both NZB-derived Sgp3 and Sgp4 revealed a synergic effect on the production of serum gp70 [24]. Although basal levels of serum gp70 in B6.NZB-Sgp3/4 congenic mice are comparable to those of lupus-prone BXSB and MRL mice, they are still lower than those in NZB and NZW mice, suggesting that an additional locus can contribute to serum gp70. Indeed, the presence of an additional Sgp locus on proximal chromosome 12 from NZB and NZW mice has been identified [45]. B6 mice bearing this locus derived from NZB mice displayed modest, but significant increases of serum gp70, in association with a selectively up-regulated

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expression of Xeno gp70 RNA, as in the case of the Sgp4 locus (unpublished observations). Collectively, all these data indicate that serum levels of gp70 are under the control of multiple regulatory genes. Thus, diverse levels of serum gp70 in various murine strains can be explained by the presence of a different assortment of multiple structural and regulatory genes implicated in the production of serum gp70 in liver. 3. Sgp-mediated increase of gp70 production during acute phase responses The expression of serum retroviral gp70 in lupus-prone mice is enhanced by different inducers of APP such as LPS, turpentine oil or polyriboinosinic-polyribocytidylic acid, indicating that serum gp70 behaves like an APP [22]. This notion has been confirmed by recent studies showing that IL-1, IL-6 and TNF, which are well known inducers for APP, increase similarly serum levels of gp70 in NZB mice [24]. Strikingly, unlike conventional APPs, the serum gp70 response is strain-dependent, as only mice with high basal levels of serum gp70, such as lupus-prone mice, display an up-regulated production of serum gp70 in response to LPS. Studies on Sgp3, Sgp4 and Sgp3/4 congenic mice revealed that the Sgp loci act synergistically and play a major role in the acute phase expression of serum gp70 [24]. This indicates that Sgp3 and Sgp4 control the expression of serum gp70 under not only steady-state but also inflammatory conditions. In contrast to the selective effect of Sgp3 on the steady-state expression level of the intact mPT env gene, it was unexpected that NZB, BXSB and B6.Sgp3 mice injected with LPS also displayed marked increases in D1 mPT env RNA, as compared with intact mPT env RNA, while levels of D2 mPT env RNA were not modulated [24]. Since increases in D1 mPT env RNA were not observed in control B6 mice, the LPS-induced up-regulated expression of D1 mPT env RNA is regulated by Sgp3. These results indicate that the expression of serum gp70 under steady-state and inflammatory conditions is controlled by distinct genes in the Sgp3 locus, further underlining the complexity of the genetic control of the expression of different classes of endogenous retroviruses in mice. At present, we cannot offer a straightforward explanation for the strong up-regulated expression of the D1 mPT env RNA in response to LPS, as compared with other mPT env RNAs. However, the D1 mutant has two unique mutations in the U3 region: a substitution of guanine with adenine in a SV40 core-like motif (GTGATCA instead of GTGGTCA) and an insertion of thymine in the UCR (upstream conserved region), which negatively regulates the expression of endogenous retroviruses [46]. It remains to be determined whether these two mutations contribute to the upregulated transcription of the D1 mPT provirus in presence of inflammatory stimuli. 4. TLR7 and endogenous retroviruses in murine SLE TLRs are a family of germ-line encoded receptors which recognize a diverse range of conserved molecular motifs commonly found in microbial pathogens. Recognition of microbial components by TLR is critical in host responses against pathogens [47]. At least three TLRs act as receptors for nucleic acids in mice: TLR3 for double-stranded RNA, TLR7 for single-stranded RNA and TLR9 for unmethylated CpG (cytosine–phosphate–guanine) DNA. Notably, unlike other TLRs expressed on the cell surface, these three nucleic acid-specific TLRs are localized in the endosome, which is a mechanism to restrict TLR responses to nucleic acids from microbial pathogens and to prevent their activation by host nucleic acids. The possible activation of autoreactive B cells through recognition of endogenous nucleic acids by TLR7 and TLR9 prompted a number of investigators to define more precisely the role of both receptors in autoimmune responses against distinct nuclear antigens and the subsequent development of lupus nephritis. It was found that the

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development of SLE was markedly suppressed in (NZB × NZW)F1 mice treated with a dual inhibitor of TLR7 and TLR9 [48] as well as in B6Faslpr and BXSB mice bearing the Unc93b1 mutation which impairs translocation of TLR7 and TLR9 from the endoplasmic reticulum to the endolysosomes where they encounter and respond to their respective ligands [49,50]. The duplication of the Tlr7 gene in mice bearing the Yaa mutation and transgenic overexpression of TLR7 in B6 mice deficient in the inhibitory IgG Fc receptor, FcγRIIB, resulted in an enhanced production of RNA-specific autoantibodies [8–10,51]. Moreover, recent studies in TLR7-deficient B6 mice congenic for the Nba2 (NZB autoimmunity 2) locus (B6.Nba2) demonstrated that TLR7 also promotes the production of anti-DNA autoantibodies [52]. In contrast, MRL-Faslpr, B6-Faslpr and B6.Nba2 mice deficient in TLR9 displayed increased production of autoantibodies against nuclear antigens, including anti-DNA autoantibodies, and developed an accelerated form of lupus nephritis [52–54]. Collectively, these results underline the pivotal role of TLR7 in autoimmune responses against DNA- and RNA-containing nuclear antigens, and the rather protective role of TLR9 against the development of murine SLE. Although the underlying mechanism responsible for the opposing effects of TLR7 and TLR9 on the development of murine SLE has remained elusive, it has been shown that TLR9 may exert an inhibitory effect on TLR7. Indeed, in vitro studies in HEK293 cells transfected with human TLR cDNAs revealed that the expression of TLR9 inhibited the activation of TLR7, but not vice versa [55]. This could be due to the fact that TLR7 and TLR9 compete for association with UNC93B1, which preferentially binds and translocates TLR9 to the endolysosomes [56]. Thus, a deletion of TLR9 could result in more robust signaling through TLR7. Notably, we have recently observed a functionally upregulated expression of TLR7 in B cells and plasmacytoid dendritic cells (pDC) of TLR9-deficient mice [52], which could promote the production of nephritogenic anti-nuclear autoantibodies, thereby accelerating the progression of lupus nephritis. This idea is consistent with findings that disease exacerbation in TLR9-deficient MRL-Faslpr and B6.Nba2 mice was completely suppressed by the deletion of TLR7 [52,57]. It is significant that the production of gp70 IC was also completely suppressed in B6.Nba2 mice deficient in TLR7 [38,52], indicating an essential role of TLR7 in the development of the anti-gp70 autoimmune response. Since it is unlikely that virion-free serum gp70 is able to trigger TLR7, an attractive hypothesis would be that Sgp3 enhances the production of endogenous retroviral virions carrying single-stranded RNA, which would then promote the development of autoimmune responses against serum retroviral gp70 through the activation of TLR7. This hypothesis is also supported by the finding that the Sgp3 locus contributes to the production of anti-gp70 autoantibodies [34]. As mentioned above, lupus-prone mice express more than 100fold increased levels of mPT env RNA, as compared with nonautoimmune strains of mice [38]. Consistent with our findings, it has been reported that an 8.4-kb transcript corresponding to the fulllength mPT retroviruses was expressed uniquely in thymi of NZB, BXSB and MRL mice, while the expression of full-length transcripts of Xeno and PT viruses was not limited to lupus-prone mice [58]. Notably, NZB mice spontaneously produce a very high titer of replication-competent Xeno viruses from birth [59]. Although endogenous mPT viruses are likely to be replication defective, one can speculate that abundant and preferential expression of mPT proviruses possessing an intact env gene could facilitate the generation of replication-competent mPT-derived infectious viruses through recombination with Xeno viruses in lupus-prone mice. These recombinant infectious viruses could then act as a triggering factor for the development of murine SLE. It has been well established that DC play a pivotal role in the induction and regulation of the immune response because immature, non-activated DC that capture autoantigens induce self tolerance,

while the activation of antigen-loaded DC triggers their maturation and enables them to induce antigen-specific immunity. A particular role for pDC, a subset of DC which highly expresses TLR7, in SLE has been proposed since this DC subset is the major source of IFNα [60,61], a cytokine that plays a substantial role in the development of SLE [62,63]. Thus, one attractive hypothesis would be that endogenous retroviruses could enter pDC through receptor-mediated endocytosis and gain access to TLR7, leading to the activation of pDC. Activated pDC then rapidly secrete robust amounts of IFNα and, to a lesser extent, other proinflammatory cytokines such as IL-6 or TNF [64]. Notably, IFNα and TNF promote the differentiation of monocytes into myeloid DC which are highly efficient at antigen presentation, express high levels of costimulatory molecules, and produce B-cell survival factors. In addition, IL-6 and IFNα promote differentiation of plasma cells. Thus, excessive activation of pDC by endogenous retroviruses could play an important role in the accelerated development of SLE. The activation of pDC by endogenous retroviruses might be dependent on the XPR1 entry receptor, which allows their internalization and subsequent interaction with TLR7 in endosomes. XPR1 expressed in laboratory strains of mice confers susceptibility to PT and mPT retroviruses, but not to Xeno viruses due to the Xpr1 polymorphism [29]. However, one cannot exclude the possibility that non-infectious Xeno viruses can be internalized through other receptors and activate pDC via interacting with TLR7 in endosomes. For example, Xeno viruses in the form of IC with IgG anti-gp70 autoantibodies could be internalized through FcγR and subsequently interact with TLR7 in pDC (Fig. 1). Furthermore, analogous to a model proposed for the activation of anti-nuclear autoreactive B cells through BCR/TLR engagement [65], gp70 on endogenous retroviruses could bind to the BCR of anti-gp70 B cells causing BCR-mediated signaling and endocytosis of retroviruses (Fig. 1). TLR7 translocates from the endoplasmic reticulum to endolysosomes where it engages single-stranded RNA motifs contained in retroviruses and initiates a signaling cascade leading to anti-gp70 B-cell activation. In this regard, it should be stressed that the activation of anti-gp70 autoreactive B cells does not necessarily require infectious retroviruses, since endogenous retroviruses can be internalized through BCR but not through XPR1. Thus, if high titers of endogenous retroviruses are produced by the presence of the Sgp loci, this might be sufficient to trigger anti-gp70 autoimmune responses in mice predisposed to SLE. These autoimmune responses can be further accentuated during the course of SLE as a result of activation of pDC in response to IgG IC containing nuclear antigens and endogenous retroviruses. This mechanism can sustain the production of IFN-α through IC-mediated activation of pDC, thereby establishing a vicious cycle not only aggravating the autoimmune process but also promoting the development of autoimmune responses against a wide array of autoantigens that do not engage TLR7. In addition to the contribution of TLR7 to the development of autoimmune responses against nuclear antigens as well as retroviral gp70, we observed that the stimulation of TLR7 and TLR9 induced high levels of serum gp70 in NZB mice with kinetics identical to those induced by LPS or inflammatory cytokines [24]. Notably, activation of TLR7 and TLR9 in monocytes/macrophages induced the secretion of IL-6 and TNF [66,67], both of which are a good inducer of APP. These data suggest that TLR7 and TLR9 are involved in the acute phase expression of serum gp70. Thus, we can speculate that DNA- and RNAcontaining IgG IC activate macrophages through interaction with FcγR and then TLR7 and TLR9, which induce secretion of cytokines such as IL-6 and TNF, acting as a positive feedback on the production of serum gp70 and endogenous retroviruses. Thus, TLR7 displays dual effects on the development of SLE. On the one hand, it promotes autoimmune responses against nuclear and retroviral antigens through the activation of autoreactive B cells as well as pDC, and on the other hand, it enhances the production of serum gp70 and endogenous

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Fig. 1. Model for activation of pDC and anti-gp70 autoreactive B cells by endogenous retroviruses through TLR7. In pDC, endogenous retrovirus complexed with IgG anti-gp70 autoantibodies can be internalized through FcγR and subsequently interacts with endolysosomal TLR translocated from endoplasmic reticulum (ER). Endogenous retroviruses bearing PT or mPT gp70 can also be internalized through the XPR1 entry receptor, potentially leading to the activation of endolysosomal TLR7. In anti-gp70 autoreactive B cells, specific recognition of gp70 on endogenous retroviruses by anti-gp70 BCR can lead to their internalization and to the activation of TLR7.

retroviruses, thereby providing an additional source for antigenic stimulation and nephritogenic IC formation. It has been shown that neonatal infection with a murine leukemia virus isolated from NZB mice induced a lupus-like autoimmune syndrome in (BALB/c × NZB)F1 mice [68]. In addition, the possible importance of endogenous retroviruses as a triggering factor for autoimmune responses in SLE has also been suggested due to the production of anti-nuclear autoantibodies in Sgp3 and GIX congenic mice [32,34,69]. Furthermore, a more recent study has shown that raltegravir, a retroviral integrase inhibitor, induced accumulation of pre-integration cDNA of endogenous retroviruses, which may increase type I IFN responses, thereby accelerating the development of kidney disease in lupus-prone (NZB × NZW)F1 mice [70]. This finding is consistent with the demonstration that the absence of 3′ repair exonuclease 1 (Trex1) contributes to the development of lupus-like autoimmune syndrome [71].

All these results allow us to propose the following model (Fig. 2). The expression of endogenous retroviruses depends on their site of integration or transcriptional regulation. Sgp3 and Sgp4 are the major genetic loci controlling the expression of serum gp70 and endogenous retroviruses. Endogenous retroviruses can be internalized through XPR1 receptor and/or FcγR and stimulate the TLR7 signaling cascade in pDC. Activated pDC aggravate the autoimmune process, leading to increases of various autoantigen–autoantibody IC, such as DNA-antiDNA IC, implicated in lupus nephritis. Furthermore, endogenous retroviruses can be recognized by anti-gp70 BCR resulting in the activation of gp70-specific autoreactive B cells through TLR7 signaling. The production of IgG anti-gp70 autoantibodies and subsequent formation of gp70 IC further contributes to the development and progression of lupus nephritis. In addition, IgG IC containing nucleic acids, such as nuclear antigens and endogenous retroviruses, activate macrophages via TLR7 and TLR9 signaling, resulting in an increased secretion of inflammatory cytokines, which further enhance the production of serum gp70 and endogenous retroviruses.

5. Endogenous retroviruses in human SLE

Fig. 2. Model of the implication of endogenous retroviruses in murine SLE.

A possible contribution of endogenous retroviruses to the development of human SLE has long been suspected. With the use of polyclonal antibodies raised against murine and feline leukemia viruses, the presence of an antigen related to mammalian retroviral core protein, p30, was reported in immune deposits of glomerular lesions in human SLE patients [72]. In addition, increased titers of antibodies reactive with peptides derived from the env and gag genes of human endogenous retroviruses have also been detected in sera from human SLE patients [73–76]. However, the search for serum retroviral gp70 and gp70 IC in human SLE has not been successful. One possible explanation for this failure may be a lack of appropriate antibodies to specifically detect retroviral gp70 antigens implicated in human SLE. Nevertheless, a member of human endogenous retroviruses, called multiple sclerosis-associated retroviral agent (MSRV), was isolated from patients with multiple sclerosis [77]. The MSRV Env protein was

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shown to stimulate activation of T lymphocytes and the production of inflammatory cytokines [78,79], suggesting a possible role for MSRV in the pathogenesis of MS. Moreover, a possible involvement of xenotropic murine leukemia virus-related virus (XMRV) in the pathogenesis of human prostate cancer and chronic fatigue syndrome (CFS) has recently been claimed [80–82], although there have been conflicting reports describing the association of XMRV with prostate cancer and CFS [83–85]. The XMRV sequence is not found in the human genome, suggesting that XMRV must have been acquired exogenously from rodents. However, transfer of XMRV from rodents to humans would require unlikely high levels of exposure to rodents for our society. Therefore, it has been suggested that XMRV may have been resident in the human population for some time. Clearly, further studies are awaited to define whether XMRV is indeed a contributing factor in the pathogenesis of prostate cancer, CFS and possibly other human diseases. Nevertheless, the suggested roles for MSRV and XMRV in the pathogenesis of different human diseases argue in favor of a possible contribution of either human or murine retroviruses to the pathogenesis of human SLE.

unsolved question: the contribution of endogenous retroviruses to the development of human SLE. Take-home messages • Lupus-prone mice spontaneously develop autoantibodies against retroviral gp70 derived from endogenous retroviruses. • The development of murine lupus nephritis is strongly correlated with the presence of circulating gp70-anti-gp70 immune complexes. • Retroviral gp70 behaves as an acute phase protein and its expression is under a polygenic control. • TLR7 plays a critical role in the development of anti-gp70 autoimmune responses and is involved in the acute phase expression of serum retroviral gp70. • Lupus-prone mice preferentially and abundantly express a particular class of endogenous retrovirus, modified polytropic (mPT) virus.

6. Concluding remarks

Acknowledgments

Genome-wide linkage analyses of test-crosses between lupusprone and non-autoimmune mouse strains have shown that lupuslike disease is controlled by sets of susceptibility loci that contribute through epistatic interactions and in a threshold manner to full-blown development of the disease. Although the nature of these genetic components has not been completely defined, it is becoming clear that abnormalities of proteins which regulate the thresholds for activation of autoreactive B cells, such as FcγRIIB and SLAM (signaling lymphocytic activation molecule), play an essential role in the development of autoimmune responses characteristic of SLE. Moreover, recent studies revealed that the innate receptor TLR7 is critically involved in the activation of pDC and autoreactive B cells through the recognition of endogenous nuclear autoantigens and the subsequent development of autoimmune responses against DNA- and RNAcontaining nuclear antigens. In addition, TLR9 deficiency in lupusprone mice resulted in accelerated development of SLE, which was completely suppressed by deletion of TLR7, indicating that TLR7 and TLR9 play a pathogenic and protective role, respectively, in the development of murine SLE. Clearly, further elucidation of the precise molecular mechanisms responsible for the opposing roles of TLR7 and TLR9 in the development of autoimmune responses will help to identify novel therapeutic targets for SLE. It is striking that TLR7 not only regulates autoimmune responses against the envelope glycoprotein gp70 of endogenous retroviruses but also up-regulates the expression of serum gp70 during acute phase responses in lupus-prone mice. Thus, the activation of TLR7 by apoptotic cells accumulating in lupus-prone mice or by IgG IC containing nucleic acids likely boosts the production of serum gp70 during the course of the disease, thereby further accelerating the progression of lupus nephritis. Furthermore, gp70-specific autoreactive B cells could also recognize and internalize endogenous retroviruses by BCR and subsequently be activated by TLR7. This hypothesis is supported by the finding that the abundance of gp70 RNA of one of the endogenous retroviruses, mPT, in lupus-prone mice is more than 100 times higher than that in non-autoimmune strains of mice. This indicates that lupus-prone mice possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor for the development of autoimmune responses via TLR7. Clearly, it is of importance to define the possible role of endogenous retroviruses for autoimmune responses in SLE and to identify the molecular basis responsible for the increased expression of endogenous retroviruses implicated in murine SLE. These experiments will enable us to address the relevance of their human counterparts, thus providing clues to answer a long-standing

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Th17 related cytokines in systemic lupus erythematosus: smoking bullets Systemic lupus erythematosus (SLE) is an autoimmune condition sustained by a cytokine driven autoimmune response. For many years the Th1/Th2 imbalance paradigm has served to explain many immunological phenomena occurring in the course of the disease, and SLE has been considered a Th2 disease, at least during the early stages. The discovery of a new CD4+ T cells subphenotype has raised interest on the role exerted by these lymphocytic population in autoimmunity. These are the IL-17 producing T-cells, termed as ‘Th17’, which not only can produce IL-17, but also a burden of inflammatory Th17-related cytokines. Although Th17 cells seem to contribute to several autoimmune diseases, their role in SLE is far less clear. Recently, Kwan and colleagues (Rheumatology 2009; doi:10.1093/rheumatology/kep255) showed in a cohort of 25 subjects with a history of lupus nephritis in remission, in 30 SLE patients with no history of renal involvement and in 23 subjects with active lupus nephritis, compared with 8 healthy subjects, that urinary expression of IL-17 and IL-27 was increased in SLE patients. However, the degree of up-regulation was reduced with active disease, inversely correlating with the SLEDAI score. They also observed a significant rise in the urinary IL-27 expression in patients with complete response, while no changes in patients with partial or no response. This dichotomous behavior raises several questions. First of all, the role of the CD4+ Th17 cells in the kidney has not been studied in detail. Secondarily, the production of Th17 is strictly dependent on the presence of TGF-b and IL-6. However, we have to remind that in case of absence of IL-6, and in the presence of TGF-b, T lymphocytes rather differentiate towards the more anti-inflammatory Treg cells. A further confounding factor is that also the Treg cells may favor IL-17 production and produce IL-17-related cytokines. It is thus possible that the Th17 pathway may be a marker of control of the intra-renal inflammation in SLE. However, we cannot talk of the Th17 pathway if flow cytometry is not performed and the cellular source of IL-17 is not specified. Nonetheless, evaluation of IL-6 and TGF-b goes within the Th17 pathways, thus, further studies are needed to clarify this issue.

Cutting Edge: Novel function of B cell-activating function in the induction of IL-10-producing regulatory B cells Although B cells have been shown to possess a regulatory function, micro-environmental factors or cytokines involved in the induction of regulatory B cells remain largely uncharacterized. B cell-activating factor (BAFF), a member of TNF family cytokines, is a key regulator for B cell maturation and function. In this study, Yang M. et al. (J Immunol 2010; 184: 3321-25) detected significantly increased numbers of IL10-producing B cells in BAFF-treated B cell cultures, an effect specifically abrogated by neutralization of BAFF with TACI-Fc. BAFF-induced IL10-producing B cells showed a distinct CD1dhighCD5+ phenotype, which were mainly derived from marginal zone B cells. Moreover, BAFF activated transcription factor AP-1 for binding to IL-10 promoter. Notably, BAFF treatment in vivo increased the number of IL-10-producing B cells in marginal zone regions. Furthermore, BAFF-induced IL-10-producing B cells possess a regulatory function both in vitro and in vivo. Taken together, these findings identify a novel function of BAFF in the induction of IL-10-producing regulatory B cells.

Recurrent lupus nephritis after kidney transplantation: a surveillance biopsy study To determine the incidence of recurrent lupus nephritis (LN) in renal transplant recipients with systemic lupus erythematosus (SLE). All patients with SLE that had undergone transplant with a functioning graft were asked in 2009 to participate in a cross-sectional study Norby GE. et al. (Ann Rheum Dis 2010; 69: 1484-7). The study included a standardized clinical examination, laboratory tests and biopsy of the transplanted kidney. A total of 41 (93%) of a cohort of 44 patients with SLE with renal transplants participated. Of the biopsies, 3 were indication biopsies and 38 were surveillance biopsies. In all, 22 patients (54%) had biopsy-proven recurrence of LN. The majority of the cases were subclinical and characterized as class I/class II LN. Proteinuria (mg protein/mmol creatinine) was significantly increased in patients with recurrence, 70.6 (104.9) mg/mmol vs 11.9 (6.7) mg/mmol in patients without recurrence (p= 0.038). Lupus anticoagulant was found more frequent in the patients with recurrence, nine vs two patients (p = 0.033). Recurrence of LN was associated with receiving a kidney from a living donor (p = 0.049). In all, 83% (34 of 41) had chronic allograft nephropathy in the transplanted kidneys with no difference between patients with recurrence or without. Thus, subclinical recurrence of LN is common in patients with renal transplants with SLE. The majority of the patients have chronic allograft nephropathy.