Genes and (2009) 10, 390–396 & 2009 Macmillan Publishers Limited All rights reserved 1466-4879/09 $32.00 www.nature.com/gene

REVIEW Three checkpoints in lupus development: central tolerance in adaptive immunity, peripheral amplification by innate immunity and end-organ

H Kanta1,2 and C Mohan1,2 1Division of Rheumatology, Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX, USA and 2Department of , University of Texas Southwestern Medical School, Dallas, TX, USA

Although the etiology of systemic lupus erythematosus (SLE) remains to be fully elucidated, it is now apparent that multiple genetic and environmental factors are at play. Over the past decade, several studies have helped uncover genetic associations and susceptibility loci in human and murine lupus. In particular, recent genome-wide association studies have uncovered a large number of associated genes in human SLE. Given this plethora of candidate genes, the next challenge for lupus biologists is to fathom how these different genes operate to engender lupus. In this context, recent genetic studies in mouse models of lupus have been particularly informative. The purpose of this review is to overview three key genetically determined checkpoints in lupus development that have emerged from studies of NZM2410-derived congenic strains bearing individual lupus susceptibility loci. These three events include a breach in central tolerance in the adaptive arm of the , peripheral amplification of the autoimmune response by the innate immune system and local processes in the target organ that facilitate end-organ disease. Collectively, murine congenic dissection studies provide a framework for understanding and analyzing the steady stream of gene candidates that are currently emerging from human lupus studies. Genes and Immunity (2009) 10, 390–396; doi:10.1038/gene.2009.6; published online 5 March 2009

Keywords: SLE; genetics; ; kidney disease

Introduction review is to overview three key genetically determined checkpoints in lupus development: breach in central Systemic lupus erythematosus (SLE) is a chronic auto- tolerance in the adaptive arm of the immune system, immune disease of complex etiology in both humans and peripheral amplification of the autoimmune response by animal models, characterized by the presence of wide- the innate immune system and local processes in the spread immunological abnormalities and multiorgan target organ that facilitate end-organ disease. injury. The hallmark of SLE is the production of high titers of autoantibodies directed against nuclear such as double-strand DNA and chromatin, which result Genetic dissection of murine lupus ultimately in -mediated end-organ damage. Although the etiology of SLE remains to be fully The NZM2410 mouse strain is a New Zealand black/ elucidated, it is now apparent that multiple genetic and white (NZB/NZW)-derived inbred strain that sponta- environmental factors are at play. Over the past decade, neously develops lupus nephritis that is very similar to human SLE.5 Through linkage analysis, several non-H2 several studies have helped uncover genetic associations z 1–4 chromosomal intervals, notably, Sle1 on chromosome 1, and susceptibility loci in human and murine lupus. In z z particular, recent genome-wide association studies have Sle2 on chromosome 4 and Sle3 on chromosome 7 have been found to confer lupus susceptibility in this mouse uncovered a large number of associated genes in human 6 SLE. Given this plethora of candidate genes, the next model. By introgressing these different chromosomal challenge for lupus geneticists is to fathom how these intervals onto the healthy C57BL/6 (B6) background, congenic strains bearing Sle1z, Sle2z and Sle3z have been different genes operate to engender lupus. In this 7,8 context, recent genetic studies in mouse models of lupus generated for functional analysis. Hence, for the first have been particularly informative. The purpose of this time, researchers have been able to study ‘monogenic’ models of lupus as opposed to studying ‘polygenic’ strains. This genetic simplification through ‘congenic Correspondence: Dr C Mohan, Division of Rheumatology, Depart- dissection’ has been instrumental in demonstrating that ment of Internal Medicine, University of Texas Southwestern each lupus susceptibility locus in this model infringes a Medical Center, 5323 Harry Hines Boulevard, Mail Code 8884, different checkpoint in disease development. Y8.204, Dallas, TX 75390-8884, USA. z E-mail: [email protected] The B6.Sle1 congenic strain exhibited a breach of Received 15 December 2008; revised and accepted 21 January 2009; to nuclear antigens, resulting in the published online 5 March 2009 production of autoantibodies to chromatin, autoreactive Checkpoints in murine lupus H Kanta and C Mohan 391 T cells responding to histone , with increased Checkpoint I: aberrant adaptive immunity expression of activation markers on T and B cells.8–10 in lupus B6.Sle2z mice exhibited B-cell hyperactivity and elevated B1 cell numbers, leading to polyclonal and/or polyreactive B cells that are generated in the (BM) and T hypergammaglobulinemia.11–13 B6 mice bearing the Sle3z cells that are generated in the populate and disease interval exhibited phenotypes affecting primarily constitute the adaptive arm of the immune system. The the T-cell compartment, as well as modest levels of anti- produces and T cells nuclear IgG antibodies and nephritis.14,15 Of particular note that are highly specific for a particular pathogen (or is the observation that Sle1z Sle2z or Sle3z in isolation was ). The relative specificity of SLE sera to a select not sufficient for the development of fatal lupus but only subset of nuclear antigens (as opposed to reacting to the elicited modest serological and cellular features of auto- whole universe of antigens) suggests that lupus genes reactivity. In contrast, the epistatic interaction of these loci must be impacting adaptive immunity, at some level. with each other and other loci such as Faslpr and Yaa led to Our recent genetic dissection studies have indicated that highly penetrant glomerulonephritis (GN).16–20 Sle1z may be one such locus/gene (Figure 1). The above ‘congenic dissection’ studies illustrate that The Sle1z interval, located on distal chromosome 1, is the genesis of fatal lupus is the end result of multiple perhaps one of the most extensively studied chromoso- genes and pathways acting in concert. Though the above mal intervals in murine lupus, because it confers disease studies had originated with the NZM2410 model of susceptibility in multiple spontaneous lupus models lupus, parallel findings have also been reported in other including the BWF1, SNF1, BXSB and NZM2410 strains mouse models of lupus, as reviewed.21 Collectively, these of mice. The Sle1z interval is home to three subloci: Sle1az, studies have revealed that both the innate and adaptive Sle1bz and Sle1cz22 Among these, the NZM2410/NZW- arms of the immune systems have to be dysregulated for derived ‘z’ allele of Sle1bz leads to the highest levels and full-blown lupus to ensue. Additional studies have also penetrance of antinuclear autoantibodies (ANAs).23,24 indicated that further checkpoints may be operative Studies employing crosses to HEL-reactive B-cell recep- within the end organs in lupus. This review discusses tor (BCR) transgenic models have demonstrated that recent evidence indicating that murine lupus suscept- Sle1z could breach tolerance among B cells with low- ibility genes may infringe all of the above checkpoints in avidity but not high-avidity reactivity to self-antigens.24 lupus development, as captioned in Figure 1. Though the mature B cells from these mice were

Figure 1 Three key steps in lupus pathogenesis, as indicated by congenic dissection studies in mice. Checkpoint I patrols central immune tolerance in the adaptive arm of the immune system, ensuring that anti-self B cells and T cells are censored in the bone marrow (BM) and thymus, respectively. Ly108 (the candidate gene within the Sle1/Sle1b lupus susceptibility interval on chromosome 1) is an example of a gene that can breach the first checkpoint. Checkpoint II patrols the innate arm of the immune system. When this checkpoint is breached, peripheral amplification of the autoimmune response results in the generation of potentially pathogenic autoantibodies and effector . The Sle3 lupus susceptibility locus on murine chromosome 7 is an example of a locus that can breach checkpoint II. Yaa/Tlr7 is a second example of a locus/gene that profoundly impacts checkpoint II. The consequence of this breach is the emergence of hyperactive, pro-inflammatory myeloid cells, which can secondarily impact the activation of autoreactive lymphocytes. It is envisioned that a final checkpoint might be operative in the end organs, where autoantibodies, T cells and myeloid cells mediate pathology. Congenic dissection of lupus nephritis in mouse models has recently suggested that kallikreins may be renoprotective in immune-mediated nephritis and may constitute candidate genes for the disease. The coordinate activation of disease susceptibility genes at all three checkpoints appears to be necessary for full-blown disease in murine models. Indicated in the right margin are candidate disease genes that have recently been implicated in human systemic lupus erythematosus (SLE), largely through genome-wide association studies. On the basis of the known properties of these human genes (as briefly referenced in the text), the disease checkpoints they are most likely to influence are also highlighted.

Genes and Immunity Checkpoints in murine lupus H Kanta and C Mohan 392 functionally normal, immature B cells from the Sle1z- and Genetic dissection studies in murine models of lupus Sle1bz-bearing BM exhibited a profound reduction in have uncovered a lupus susceptibility locus on mid- calcium flux, RAG expression and cell death following chromosome 7 similarly positioned in several strains of BCR cross-linking, revealing that the Sle1bz lupus mice, including the NZM2410, NZB/NZW and MRL/ susceptibility locus significantly dampened central B-cell lpr.6,14,32–34 This locus has been termed Sle3z in the tolerance.24 NZM2410 model. B6.Sle3z congenics exhibit low levels Through a meticulous positional cloning approach, of ANAs and several phenotypes.14 Impor- Wakeland and co-workers23 demonstrated the SLAM tantly, Sle3z-bearing T cells were spontaneously activated family of costimulatory molecules to be the candidate and exhibited elevated CD4/CD8 ratios and impaired genes for Sle1bz. Among this family of molecules, one activation-induced cell death.1,14,35 To explore the cellular member, Ly108, exhibited interesting expression differ- origin of the Sle3z-associated phenotypes, Sobel et al.15 ences associated with functional consequences when transferred BM from -marked B6 and B6.Sle3 immature B cells from B6 and B6.Sle1bz mice were congenic mice into B6 hosts. In these chimeras, T cells of compared.24 The normal ‘b’ allele of Ly108 encodes both origins (that is, with or without Sle3z) exhibited predominantly the Ly108.2 isoform (bearing three in- elevated CD4/CD8 ratios and spontaneous T-cell activa- tracellular immunotyrosine switch motif (ITSM) signal- tion, phenotypes that have been attributed to Sle3z These ing motifs); in contrast, the lupus-associated ‘z’ allele of studies demonstrated that the Sle3z-associated pheno- Ly108 encodes predominantly the Ly108.1 isoform (bear- types may not be encoded in a T-cell-intrinsic fashion, ing two intracellular ITSM motifs) due to splice-site although they were BM transferable. Likewise, the same polymorphisms. Importantly, immature B cells trans- study also revealed that autoantibody production in the fected with the lupus-associated Ly108.1 isoform showed chimeras was also not contingent upon the intrinsic impaired calcium flux, apoptotic cell loss and BCR expression of Sle3z within B cells.15 editing, compared to transfectants bearing the normal Consistent with the above findings, the Sle3z-asso- Ly108.2 isoform. The normal Ly108.2 isoform appears to ciated phenotypes arise from the intrinsic impact of render immature B cells sensitive to BCR cross-linking, this locus on myeloid cells.35 Dendritic cells (DCs) and effectively facilitating the operation of several tolerance isolated from B6.Sle3z spleens, lymph mechanisms including receptor editing and deletion, nodes and BM exhibited increased surface levels of whereas the lupus-associated isoform, Ly108.1, may be CD40, CD80, CD86, CD54, CD106 (VCAM-1) and FcR functioning by blocking these processes.24 (CD16/32) and increased expression levels of pro- Collectively, the above studies reveal that polymorph- inflammatory such as interleukin (IL)-12, isms in the SLAM family gene Ly108 can infringe key IL-1b and tumor necrosis factor-a (TNF-a).35 Also, when checkpoints in central B-cell tolerance, hence leading to Sle3z-bearing DCs were ovalbumin (OVA)-pulsed and the emergence of self-reactive antibodies. Recent work cocultured with OVA-specific T-cell receptor Tg T cells, from the Wakeland laboratory indicates that the same the T cells demonstrated greater expansion and reduced polymorphisms in Ly108 may also breach thymic , compared to T cells cocultured with B6 DCs.35 tolerance in B6.Sle1bz mice (submitted). Hence, Ly108 is Most importantly, after adoptive transfer into young B6 the prototype of a class of genes that can infringe central hosts, Sle3z-bearing DCs led to elevated splenic CD4/ tolerance in both the B- and T-cell compartments that CD8 ratios and serum autoantibody levels. These constitute the adaptive arm of the immune system. findings indicated that Sle3z-bearing DCs were sufficient Whether similar genes may also be operative in human to recreate the Sle3z-associated lupus phenotypes. More SLE is discussed further below. In addition to Sle1bz it is recently, the subinterval within Sle3z that may be worth noting that the more distal locus on murine responsible for these phenotypes has been narrowed to chromosome 1, Sle1cz, may also harbor genes that breach a sublocus termed Sle3b (Wakeland EK et al., unpub- B-cell tolerance either centrally or peripherally, including lished). The candidate gene(s) within this locus that may Cr2.25–28 be responsible for these myeloid cell phenotypes remain to be decoded. It is of interest to note that similar myeloid cell abnormalities have also been described in Checkpoint II: aberrant innate immunity human SLE.36,37 A more recent breakthrough in murine lupus genetics in lupus that has highlighted the importance of the innate The innate immune system is a universal and ancient immune system revolves around the Yaa lupus suscept- form of host defense presumably designed to fight ibility locus derived from the BXSB lupus strain, which infection. Before launching an effective adaptive immune was originally derived from the SB/Le and C57BL/6 (B6) response, the host must deal with acute assaults, sense parental strains.38–41 The male bias in BXSB lupus has the presence of pathogens, distinguish infectious nonself clearly been shown to be encoded by the BXSB Y from noninfectious self and mount an effective immune chromosome, and the putative locus on Y chromosome response against invading organisms rapidly. Myeloid has been termed Yaa or Y chromosome cells, interferons (IFN) and Toll-like receptors (TLRs) all accelerating locus. Interestingly, the Yaa locus leads to are important in innate immune responsiveness.29–31 different degrees of autoimmunity on different genetic Unfortunately, when innate immune responses are backgrounds, in epistasis with other lupus susceptibility misdirected to components of self, autoimmunity can loci.39–42 Defined about 30 years ago, it was only recently ensue. Recent genetic studies in mice have yielded at that the identity of Yaa was elucidated—two indepen- least two examples of loci/genes that could potentially dent groups identified Tlr7 as the candidate gene for contribute to lupus by dysregulating innate immunity, as Yaa.19,43 Finally, another lupus susceptibility gene identi- diagrammed in Figure 1, and discussed further below. fied in mice with spontaneous lupus is Ifi202, a gene that

Genes and Immunity Checkpoints in murine lupus H Kanta and C Mohan 393 may not only facilitate IFN-I-driven peripheral amplifi- nephritis, proteinuria, azotemia, glomerular and tubu- cation of the autoimmune response, but may also impact lointerstitial disease all ensue with a rapid and pre- cellular apoptosis in a variety of cell types.44–46 dictable time course in the experimentally induced Collectively, the above studies suggest that dysregu- models. Over the past decade, researchers have assessed lated expression or function of DCs, the TLRs they bear the functions of 425 different molecules (including (for example, TLR7) and cytokines they express (for various complement proteins and TLR ligands, FcR, example, IFN-I) can profoundly impact lupus pathogen- B7/CD28/CTLA4, LFA1/ICAM1, P-selectin, TNF-a, IL- esis. Though the breach in checkpoint I in the adaptive 1, IL-6, IL-12, IL-18, IFN-g, M-CSF, PDGF, MCP-1 and arm of the immune system may suffice for autoreactive B NO) in the pathogenesis of spontaneous lupus nephritis and T cells to emerge in the periphery, these may not be as well as experimental anti-GBM disease. Importantly, sufficient to induce disease. A breach in the innate arm of the molecules that have been studied thus far have the immune system (that is, checkpoint II) may be shown excellent concordance in how they affect both necessary for the ‘amplification’ of the autoimmune disease settings, as recently reviewed.67 In other words, response and the generation of pathogenic effector molecules known to influence the progression of experi- molecules, including pathogenic antibodies, effector T mental anti-GBM disease also impacted the development cells and pro-inflammatory myeloid cells, all of which of spontaneous lupus nephritis in the same direction. may be responsible for the ensuing end-organ pathology Thus, although experimental anti-GBM nephritis and (Figure 1). spontaneous lupus nephritis may differ in the nature of Although checkpoint II has been described as being the inciting antibodies and the localization of the focused on the innate immune system, genes that immune deposits, a shared network of downstream amplify T-cell/B-cell collaboration such as Roquin47 molecular pathways is likely to be mediating disease in and major histocompatibility complex (MHC) can also both settings. be expected to have critical functions in amplifying the Indeed, when different strains were challenged with autoimmune response in the periphery. Indeed, the anti-GBM serum, it became evident that B6 mice bearing MHC locus has consistently emerged as the strongest the Sle3 congenic interval at chromosome 7 exhibited susceptibility factor for murine lupus in multiple murine significantly increased renal disease.58 Mapping using models.48,49 Though this locus (referred to Sle4 or Sles1 in recombinant congenics revealed that a sublocus harbor- NZM2410 mice) has been phenotypically very well ing the kallikrein gene complex on chromosome 7 was characterized, definitive evidence indicating that the H2 responsible for this phenotype.68 Functional studies genes are indeed the disease genes within this locus is indicated that increased renal kallikrein had a protective still elusive. function in immune-mediated nephritis.68 Collectively these studies allude to a third checkpoint in the development of lupus nephritis that could potentially Checkpoint III: genetically determined modulate the degree of end-organ disease, in the face of events in the end organs that dictate potentially pathogenic autoantibodies and autoaggressor pathology lymphocytes (Figure 1). The consequence of breaching checkpoints I and II in the adaptive and innate arms of the immune system, Relevance of the three-checkpoint model respectively, is the generation of potentially pathogenic to human SLE genetics mediators, including autoantibodies, immune com- plexes, T cells and myeloid cells. However, leads from Though the three-checkpoint model depicted in Figure 1 the literature indicate that an additional set of genes/ may be an oversimplification of the molecular events that molecules may have the potential to modulate the lead to murine lupus, it provides us with a working severity of the end-organ disease that ensues. Discor- framework for classifying and understanding the large dance between serum ANAs and GN have been number of likely human lupus genes that have recently documented in murine as well as in human lupus, as been reported, as reviewed in.69 On the basis of the reviewed.50,51 Strongly nephrophilic seropositivity can be known function of these molecules one can readily uncoupled from renal disease, in experimental models identify gene candidates that may potentially infringe where key molecular mediators (for example, FcR, MCP- each of the three checkpoints, as indicated on the right 1, complement, TNF-a, ICAM-1) are deficient.52–56 In margin of Figure 1. Genes such as Bank1, Blk and the addition, genetic mapping studies have uncovered loci complement molecules are candidates that could be that are strongly linked to nephritis, but not autoanti- envisioned to breach checkpoint I, based on the bodies.49,57,58 Reports of familial clustering of primary/ published properties of these molecules.70–74 In fact, idiopathic GN59–61 and of GN following lupus, diabetes there is already functional evidence from repertoire and hypertension62–65 further support the potential monitoring studies indicating that early B-cell tolerance importance of Genetics in determining intrinsic suscept- checkpoints may also be infringed in human SLE.75,76 A ibility to renal disease in lupus as well as in other relatively extensive subset of the genes implicated in diseases. human SLE could be envisioned to upregulate myeloid A valuable tool for uncovering key molecular deter- cell activity or enhance T-cell/B-cell or DC/T-cell minants in lupus nephritis is the anti-glomerular base- interactions, leading in essence to a breach of checkpoint ment membrane (GBM) experimentally induced model II, as captioned in Figure 1. Once again, these proposed of nephritis. The ability of heterologous ‘anti-kidney’ (or assignments to the respective checkpoints are based on anti-GBM) sera to inflict nephritis was first recognized by the published properties of these molecules.77–82 Finally, Masugi in 1934.66 In contrast to spontaneous lupus genes such as ITGAM, complement, and the various FcR

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