What do mouse models teach us about human SLE?

Commonly used murine lupus models

The earliest murine lupus models comprise of various inbred strains, which develop systemic autoimmunity and lupus nephritis spontaneously. These spontaneous lupus nephritis models, including NZB/W F1, BXSB/y aa , BXD2/TyJ, and MRL/ lpr strains, demonstrate many characteristic features of human SLE and mirror human SLE pathogenesis remarkably well. When the NZB strain of mice are bred with either the relatively normal New Zealand White (NZW) or Swiss-Webster mice, the F1 progeny, designated as NZB/W F1, and SNF1, respectively, develop early onset immune complex-mediated glomerulonephritis (GN), and enhanced autoantibody production. Both the NZB/W F1 and SNF1 strains exhibit a female predominance, closely resembling that of lupus patients. The BXSB/ yaa mouse model is a recombinant inbred strain derived from the backcross of (B6x SB/Le) F1 to SB/Le, where the ensuing male-predominant disease is dependent upon the duplication of Toll-like receptor 7 ( TLR7 ) gene.

Another mouse strain that develops lupus spontaneously is the Murphy's recombinant large (MRL)/ lpr strain, which carries the lymphoproliferation ( lpr ) mutation in the Fas gene on the lupus-prone MRL background. This strain also exhibits more widespread systemic involvement and some unique phenotypes rarely observed in NZB/W F1 and BXSB/ yaa , such as sialoadenitis, inflammatory arthritis, skin damage, as well as neuropsychiatric symptoms, not unlike the disease seen in humans. Similar to the MRL/lpr model, BXD2 mice derived from the C57BL/6J and DBA/2J progenitor strains also develop spontaneous arthritis, GN, splenomegaly, and autoantibody production. Besides these strains, accidental brother–sister mating between NZB/W F1 and NZW led to the generation of the New Zealand Mixed (NZM) strains. The widely used NZM2410 and NZM2328 strains have also provided researchers additional insights into lupus pathogenesis.

Another category of lupus mouse models include congenic strains developed by several groups, including the B6. Sle 1, B6. Sle 2, B6. Sle 3 strains generated by Wakeland and coworkers. These congenic lupus models are commonly generated by introgressing a susceptibility locus from a disease-prone strain onto a disease-free or resistant strain (e.g., B6), through a process called “selective breeding.” Congenic strains are unique in that each mouse harbors a particular susceptibility locus on a common immune-disease free genetic background. This characteristic renders the congenic mouse model a powerful tool for genetic dissection analysis, because one can readily compare the congenic strain bearing the disease locus with a genomically matched background strain to decipher the function of each disease locus very precisely, something that can never be done with humans.

Besides these spontaneous mouse models of lupus, many investigators have been utilizing experimentally induced lupus models that can be triggered using pristane or by inducing chronic graft versus host disease. Both models facilitate the rapid analysis of how various cells and molecules impact lupus development, and the testing of immunomodulatory agents on disease, relatively quickly. Finally, genetically engineered murine models represent a very powerful tool for studying the role of individual genes in the development of lupus. Indeed, a large number of transgenic mouse models (where a particular gene is hyper-expressed) and gene-knockout models (where the function of a gene is ablated) have taught us how various genes could potentially impact lupus development, as reviewed.

Conditional knockout system of lupus models helps delineate the cell-intrinsic mechanisms of autoimmunity and lupus development

The availability of conditional knockout LoxP (or floxed) mice and various tissue-specific Cre systems have become instrumental in identifying the role and mechanism by which a particular gene or the pathway may promote lupus development in a cell-type specific manner. However, most floxed and Cre mice are available on a B6 background. As a result, in case of the polygenic models of lupus such as MRL/lpr, BXSB or BXD2 mice, floxed and Cre mice need to be backcrossed for many generations to the lupus background to establish cell-type specific conditional knockout mice on the lupus model. Although time-consuming, this system is invaluable in delineating the cell-intrinsic mechanisms of autoimmunity and disease development. Using this system on the MRL/lpr background the Shlomchik group previously identified the B cell- and DC-specific roles of MyD88 signaling in autoimmunity and LN development. The generation of floxed and cell-type specific Cre mice directly on the autoimmune background using the CRISPR/Cas system may facilitate analysis of the cell-intrinsic mechanisms of autoimmunity and lupus pathogenesis.

Besides the polygenic models of lupus in which dissecting the cell-intrinsic mechanisms requires a time-consuming and laborious process of backcrossing, many researchers prefer lupus models that are already on the B6 background. One widely-used model in the lupus field is B6 mice deficient in the inhibitory IgG Fc receptor FcγRIIB. FcγRIIB deficiency, and polymorphisms in tightly-linked 129 strain-derived SLAM (signaling lymphocyte activation molecule) family genes promote autoimmunity and lupus-like disease in FcγRIIB-deficient mice generated using 129 ES cells and backcrossed to B6 mice. Other B6-based models that have been used for the analysis of cell-intrinsic mechanisms are B6.Lyn ^−/− mice and TLR7 transgenic B6 mice both of which develop high titers of autoantibodies, splenomegaly and severe LN. Although the lupus-prone NZM2410 strain-derived B6.Sle1/Sle1b congenic model has been commonly used for the investigation of the cell-intrinsic role in autoimmune B and T cell responses, and autoantibody production, B6.Sle1/Sle1b mice expressing the Yaa locus, or B6.Sle1/Sle1b mice treated epicutaneoulsy with TLR7 ligand (S.B.C. et al. submitted) develop full-blown lupus disease, and these newer mouse models can also be used to delineate the cell-intrinsic mechanisms in autoimmune responses and lupus pathogenesis.

In summary, regardless of the use of polygenic or B6 models of lupus the Cre/LoxP system is powerful in delineating the cell-intrinsic mechanisms by which a GWAS-identified gene or implicated pathway may contribute to autoimmune B and T cell responses and lupus pathogenesis. The knowledge gained from conditional knockout lupus models will also assist in delving into cell-type specific mechanistic studies in human SLE.

Murine lupus strains constitute excellent models for defining the genetic architecture of SLE

Since the 1980s, more than 100 genomic regions have been identified to be associated with increased susceptibility to lupus in mice. Though linkage and association studies have been carried out in murine and human lupus, murine strains have facilitated research in a very important way—murine lupus geneticists were able to establish “gene-function” relationships through the construction of congenic strains. Over the past decade, an increasing number of double congenic and triple congenic strains have demonstrated that lupus can not only be genetically dissected into its functional components, it can also be reconstituted back to full blown disease, as illustrated by the B6. Sle1.Sle2.Sle3 triple congenic strain.

Congenic strains contribute in another important fashion. By progressively shortening the chromosomal interval of interest, scientists can reduce the pool of potential candidate genes encompassed within the genetic interval, and then eventually identify the causative disease genes. Importantly, several such murine lupus genes identified by congenic studies have been validated by genetic association studies in human SLE. For example, the Sle1c locus is linked to increased circulating IgG antibodies and immune alterations in T cells. Cr2 , a gene encoding complement receptor type 2 that functions as a B-cell co-receptor, has been considered as a candidate gene within this locus. A point mutation in Cr2 leads to alterations in B-cell responses and germinal center formation. Similarly, in human SLE, a common three single nucleotide polymorphism (SNP) Cr2 haplotype has been associated with lupus susceptibility. Several functional variants in this gene also alter gene transcription, splicing efficiency, or the formation of multiple protein-DNA complexes. Thus besides underscoring the fact that the same gene can be causative in murine and human lupus, murine studies may also offer potential mechanisms by which Cr2 could contribute to the development of lupus. Additional murine lupus genes identified using a variety of congenic strains include Ifi202 , Ly108, FAAH, Coronin-1A, and EAT-2 . . In general, there is limited overlap between the lupus genes identified in mice versus those described in humans, although the functional pathways implicated may be similar in both species. More importantly, mouse models can be used to validate GWAS-identified SLE risk alleles, as described below.

Mouse models help validate GWAS-identified lupus risk alleles

To date, GWAS and fine mapping studies have identified nearly 400 SNP risk variants in more than 100 genetic loci that confer increased risk of developing SLE. More than 80% of these SLE risk variants are located in noncoding regions, whereas a small percentage of these SNPs is located in coding regions of genes. Since many risk variants are in intronic regions and are not homologous to mouse, they cannot be easily characterized using mouse models. However, researchers have found ways to validate the causality of coding region risk variants using mouse models. One such well-characterized coding region SNP is rs2476601 (R620W) within the PTPN22 gene that encodes the protein Lyp and also impacts BCR signaling. A knock-in mouse expressing an analogous mutation (R619W) introduced by site-directed mutagenesis of 129 ES cells and injected into B6 blastocysts, developed autoantibodies, and systemic lupus-like disease due to increased antigen receptor (BCR and TCR) hyperactivity. R619W mice generated on a pure B6 background, however, do not develop similar magnitude of autoimmune responses and disease, indicating a potential interaction of the R619W allele with 129 derived genes in accelerating SLE pathogenesis. Another coding region risk variant rs1990760 has recently been characterized, located within the IFIH1 gene (A946T). IFIH1 (MDA5) is a cytosolic sensor which induces a rapid Type I IFN (INF-I) response following stimulation by double-stranded RNA and branched, high-molecular weight RNA. Mice generated through homologous knock-in containing the polymorphism rs1990760 within the IFIH1 gene (A946T) demonstrate increased recognition of self-ligands and IFN-I production. Although lupus phenotypes have not been examined in A946T mice other than the IFN-I response, these mice further demonstrate increased IFN-I production following encephalomyocarditis virus infection or in the streptozocin-induced Type 1 diabetes (T1D) model. These reports suggest that the causality of other coding region risk variants can be validated similarly using mouse models which will help gain insights into how various risk alleles in human SLE may contribute to autoimmunity and subsequent development of end-organ manifestations.