The Long (and Sometimes Endless) Road to Murine Lupus Genes

Bielschowsky, Helyer, and Howie described the very first murine models for lupus in the late 1950s (1, 2). These “New Zealand” strains of mice displayed different types of autoimmunity. Whereas the New Zealand Black (NZB) strain exhibited hemolytic anemia, the F₁ hybrids of this strain with the New Zealand White (NZW) strain developed highly penetrant lupus nephritis. The latter strain, termed BWF1, rapidly became one of the most researched animal models of lupus, even to this day. This strain has been very useful in delineating the contributions of various lymphocyte subsets toward the loss of immune tolerance and the emergence of end-organ inflammation affecting primarily the kidneys.

Although early researchers quickly learned that murine lupus had a strong genetic basis, the road to gene identification has been long and arduous. Early studies explored the genetic role of key molecules in the immune system, such as the H-2 locus, Ig loci, and TCR loci, as reviewed elsewhere (3). Most of these studies revealed no disease association, with the exception that H-2, the murine equivalent of the human MHC or HLA locus, was implicated in some studies as being associated with murine lupus (reviewed in Ref. 3). Only a few years prior to this, Benacerraf and Katz (4) had defined the importance of the H-2 locus in immunity. Hence, perhaps not surprisingly, the ensuing decade of lupus genetics research was focused on elucidating how various H-2 alleles and H-2 heterozygosity impacted murine lupus development (5–9) (Fig. 1).

Whether additional genetic loci were needed for lupus to manifest became apparent in 1978, when Knight and Adams (11) generated offspring by crossing the NZB and NZW strains, in different ways, and concluded that lupus susceptibility was polygenic, and that two to three genetic loci were likely to be responsible. This initial report was followed by additional breeding studies by other investigators (12) and analyses of recombinant inbred lines (13, 14), all of which came to the same conclusion: that a couple of genes were likely to be responsible for murine lupus.

Although there was consensus that lupus in the BWF1 strain was oligogenic, the chromosomal locations of these genetic loci remained unknown for a decade and a half. In the early 1990s, the availability of polymorphic DNA microsatellite markers distributed across all murine chromosomes and the accessibility of powerful linkage analysis software facilitated linkage analysis of various traits in murine models (15, 16). The year 1994 was a landmark in the history of murine lupus genetics (Fig. 1). The first report applying these novel technologies to murine lupus emerged from someone who was new to the field of lupus. Edward K. Wakeland was an MHC geneticist at the University of Florida who was researching factors shaping the evolution of the MHC locus, including the role of sexual mating preferences (17, 18). In 1993, his group used polymorphic microsatellite markers to map susceptibility loci for murine diabetes (19). In 1994, Wakeland and colleagues (10) published the first report of the chromosomal locations of genetic loci linked to murine lupus, having studied a mouse strain that was totally new to the murine lupus community, the New Zealand mixed (NZM)2410 strain (20). It is this study that is the featured Pillars of Immunology article (10).

Unlike the BWF1 strain, which is an F₁ hybrid between the NZB and NZW inbred strains, the NZM2410 strain is a recombinant inbred strain derived from the same two parental strains, NZB and NZW (20). Whereas each locus in BWF1 is a hybrid of NZB and NZW alleles, each locus in the NZM2410 strain is homozygous, with either an NZB/NZB or NZW/NZW genotype. Using the C57BL/6 (B6) strain as a “disease-resistant” control strain, Wakeland and colleagues generated (NZM2410 × B6)F₁ hybrids and backcrossed them to the NZM2410 mice to derive a panel of 158 “backcross” mice, which exhibited varying aspects of lupus and differing degrees of disease. In effect, these offspring represented 158 unique mouse models, each with a unique assortment of NZM2410- and B6-derived genetic intervals, each exhibiting varying degrees of lupus. When linkage analysis was performed to identify the genetic intervals associated with disease development, Wakeland and colleagues noted that there were four genetic loci associated with lupus nephritis in this backcross cohort, consistent with earlier reports indicating that murine lupus is oligogenic. Also consistent with earlier studies, the H-2 locus on chromosome 17 was one of the four loci mapped by Wakeland and colleagues. Three additional novel loci were identified and named Sle1, Sle2, and Sle3 on chromosomes 1, 4, and 7, respectively (10).

This landmark study by Wakeland and colleagues in 1994 was important in many respects. First, it reaffirmed the idea that murine lupus development was oligogenic, and possibly polygenic, and that these genetic factors included the well-researched H-2 locus. Second, it was the first demonstration that lupus nephritis can be inherited in a gene dose threshold–dependent manner, because backcross offspring with increasing numbers of disease genes exhibited increased penetrance of the disease (10). Third, it provided the first indication that some, but not all, of the genetic loci linked with lupus nephritis may be linked with anti-DNA Ab production. Fourth, it was also the first illustration that healthy controls can bear potential disease-causing genes, because the B6 allele of the H-2 locus conferred disease susceptibility. Nevertheless, the B6 strain remains disease-free because this strain’s lupus susceptibility genetic load (with just one locus, i.e., H-2) is insufficient to surpass the threshold needed for lupus development. Finally, this seminal work proved to be the wellspring of a series of congenic dissection studies that have contributed to our understanding of possible disease mechanisms in murine lupus.

It is important to point out two additional publications in the same year that reported similar findings, beginning with the BWF1 strain rather than the NZM2410 strain. By studying the genotypes and phenotypes of (NZB × NZW)F₁ × NZW backcross mice, as well as an additional backcross panel, Kotzin and colleagues (21, 22) mapped lupus susceptibility loci on chromosomes 1, 4, 7, 10, 13, 17, and 19. Using an (NZB × NZW)F₂ intercross panel of mice, Theofilopoulos and colleagues (23) identified eight lupus susceptibility loci on chromosomes 1, 4, 7, 8, 11, and 17. Given that the NZM2410 strain studied by Wakeland and colleagues was originally derived from the NZB and NZW parental strains, it was not a surprise that shared or overlapping quantitative trait loci (QTLs) were identified in the different studies, such as the loci on chromosomes 1, 4, 7, and 17. Following the pioneering publications by these three research groups, many additional genetic mapping studies have been carried out in related and independent murine lupus strains, as reviewed elsewhere (24–28). Thus, compared with the lethargic pace of progress prior to 1994, murine lupus genetics witnessed an avalanche of interesting studies in the ensuing decades (Fig. 1).

Following the initial mapping study, Wakeland and colleagues embarked on a series of “congenic dissection” studies that helped greatly simplify the study of murine lupus. Wakeland and colleagues adapted a technology that had been pioneered by another H-2 geneticist several decades earlier. George Snell (29) had described how H-2 alleles can be selectively bred onto other genetic backgrounds as “congenic” intervals. Likewise, by selective breeding, a single genetic interval of interest can be “extracted” from the genome of one strain (from a disease-prone strain, for example) and bred or “introgressed” onto a new strain background (onto a disease-free or disease-resistant strain, for example) to generate a congenic strain. This powerful approach also allows one to “position” a QTL of interest onto a totally “new” background to determine how that locus functions in the context of different background genomes.

Wakeland and colleagues devised a strategy to derive congenic strains more rapidly by using a marker-assisted breeding protocol for the production of “speed congenics” (30). This modification was very useful because it almost halved the number of generations required to produce congenic strains. Essentially, in this modified approach one institutes marker-assisted screening at the earliest breeding generations so that offspring with excessive irrelevant donor gene content can be selected against while offspring bearing the target genetic interval can be positively selected. Wakeland and colleagues successfully bred the Sle1, Sle2, Sle3, and H-2 QTL intervals from the NZM2410 strain onto the disease-resistant B6 strain to derive the B6.Sle1, B6.Sle2, B6.Sle3, and B6.H2^z congenic strains, each of which shared >99% of their genome with the B6 control strain (31, 32).

When compared with each other, the phenotype of each B6.Sle congenic strain was distinctive (31–35). B6.Sle1 mice were noted to suffer a breach in tolerance to chromatin, but minimal glomerulonephritis or further impairment in lymphocyte function was observed (33). In contrast, the phenotypes of B6.Sle2 and B6.Sle3 mice appeared to have resulted from changes in the B cell compartment and myeloid/T cell compartments, respectively, accompanied by different types of serum antinuclear autoantibodies (34, 35). Interestingly, the phenotype of each B6.Sle congenic strain represented a component of full-blown lupus, but none of these congenic loci was sufficient to drive the entire disease process acting in isolation. These findings shore up the hypothesis that lupus pathogenesis is the consequence of multiple susceptibility genes acting in a synergistic fashion to breach critical thresholds in autoimmune regulation.

In addition to the above monocongenic strains, the study by Wakeland and colleagues of polycongenic strains has yielded additional insights. Despite the modest autoimmune phenotypes noted in B6.Sle1, B6.Sle2, and B6.Sle3 monocongenic mice, bicongenic and tricongenic offspring derived from these mice exhibited extremely high penetrance of lupus nephritis and early mortality, resembling the disease seen in the original NZM2410 strain (36, 37). These studies have demonstrated that interaction between loci cannot only lead to stronger or novel phenotypes, but may also facilitate the progression to full-blown lupus nephritis. Thanks to these efforts, a new mouse model of lupus, the B6.Sle1.Sle2.Sle3 triple congenic strain, has been deposited into The Jackson Laboratory and is rapidly gaining popularity among the murine lupus research community as a mouse model of choice because of its B6 genetic background. Several other investigators have subsequently reported various phenotypes in other independently bred congenic strains bearing other lupus susceptibility loci or protective loci, as reviewed elsewhere (24–28). Regardless, the series of congenic dissection studies that were carried out by Wakeland and colleagues, in which a polygenic disease model is parsed into its component monogenic elements, and the subsequent “genetic reconstitution” studies, in which the individual genetic components are recombined back into a polygenic model, remain the earliest and most elaborate of all such studies in the field.

Having an isolated disease-associated QTL on a control genetic background facilitates subsequent gene identification, provided the isolated interval in the congenic strain is associated with robust disease-relevant phenotypes. Fortunately, this was the case with several of the genetic loci that Wakeland and colleagues had isolated as congenic intervals. Using these reporter phenotypes, a series of recombinants across the congenic interval were bred and phenotyped to track the disease locus. Performed recursively, this allows the location of the causative allele to be “fine mapped” to a small interval (generally <1 Mb). The causative disease variant can then be identified by a combination of DNA sequencing and functional studies, followed by confirmation with genetic knockins or other approaches. To date, a few NZB, NZW, or NZM2410 genes have been reported as being disease causative, including Ly108, Cr2, and Ifi202 on chromosome 1, and Klk and Coro1a on chromosome 7, by Wakeland and colleagues as well as by others (38–42). This represents only the tip of the iceberg, as several more lupus susceptibility loci have been documented, and their underlying causative genes remain to be uncovered or reported.

Compared with the pace with which researchers were able to map disease loci and generate congenic strains, the process of gene identification has proven to be far more complex and challenging than anticipated. Most of the disease loci pursued by Wakeland turned out to harbor two or more subloci, each with weaker disease contributions, as illustrated by the exploration of the Sle1 and Sle2 QTL intervals (43, 44). Indeed, this appears to be the experience with disease QTLs associated with other disease models as well (45, 46). These weaker gene–phenotype associations complicate fine mapping and may sometimes preclude the identification of the responsible disease genes. Even when trackable phenotypes were present, pinpointing the real gene dispersed among a large number of positional candidates (with sequence polymorphisms) has proven to be anything but simple. Despite being tackled by an army of scientists and huge funding investments, many lupus-susceptibility QTLs have remained recalcitrant to gene discovery. For instance, the H-2 locus has been intensively studied for more than three decades, but the causative sequence variants still remain unclear. Nevertheless, the few success stories that have been published serve to highlight how these new genes (and gene families) influence immune tolerance, inflammation, and end-organ disease processes.

Although murine lupus genetics has been useful in teasing apart the component pathogenic mechanisms or checkpoints that lead to lupus development (which appear to be similar in human systemic lupus erythematosus [SLE] as well), the uncovered murine lupus genes do not appear to be major genes for human SLE. Indeed, in the recent past, Wakeland and colleagues have turned their attention to humans and have been expending substantial efforts in identifying and fine mapping the disease genes for human SLE (47–51). The outcome of this new direction of research is likely to be as illustrious as Wakeland’s contribution to murine lupus genetics. For having orchestrated the very first and most elaborate and productive gene mapping and genetic dissection program in the nearly 60-y history of murine lupus research (Fig. 1), and for having systematically evaluated arguably the largest number of potential genetic loci in murine lupus, Wakeland could well be considered to be the “father of murine lupus genetics.”