Aberrant signaling in the TNFα/TNF receptor 1 pathway of the NZM2410 lupus-prone mouse
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high autoantibody production against nuclear proteins. The most severe form is caused by deposition of immune complexes on the basement membrane of the kidney leading to glomerulonephritis, and ultimately kidney failure. It has been well documented that apoptotic defects in the lymphoid system that result in failure to remove autoreactive cells culminate in autoimmune diseases [1]. In particular, defects in FAS and FASL members of the TNF/TNFR family, which result in severe lymphoproliferation, result in autoimmunity [1]. Deletion of the p55 TNF-receptor (TNFR1) in the B6-lpr/lpr mouse resulted in acceleration of lymphoproliferation and autoimmune disease [2]. This suggested that the p55 TNF-R pathway may play a compensatory role in lymphocyte apoptosis and that TNFα can regulate the survival of autoreactive lymphocytes [2]. Although several studies have associated Tnfa sequence polymorphisms and SLE [3], [4], the precise contribution of TNFα on systemic autoimmunity is controversial [5].
The NZM2410 mouse, derived from the NZB and NZW strains, is a spontaneous lupus model that closely resembles the severe form of human SLE [6], [7]. The NZM2410 mouse and its derived B6.NZM congenic strains have been characterized genetically and phenotypically over the last decade [8], [9]. QTL analysis identified four lupus susceptibility loci, Sle1-4, in NZM2410 [7]. Congenic strains were produced with genomic intervals corresponding to each of the Sle loci on a C57BL/6 (B6) background [10]. Characterization of the NZM2410 mouse has revealed that the MHC region, including Tnfa, was derived from NZW [7], implying that NZM2410 carries the TNFα low producer NZW Tnfa allele (Tnfaw) [11], [12], [13], [14]. The Sle4 interval, which includes the H2z haplotype and Tnfaw, has been associated with recessive resistance to SLE [7]. Specific immunological abnormalities have been associated with each of the three other Sle loci [10]. Sle1 results in the production of antichromatin antibodies and increased B and T cell activation [15], Sle2 is associated with a decreased threshold of activation in B cells [16], and Sle3 results is an accumulation of CD4 T cells due to an impaired activation-induced cell death [17]. The combination of these three loci in the triple congenic B6.Sle1.Sle2.Sle3 strain was necessary and sufficient to reconstitute a fully penetrant lupus nephritis comparable to that of the NZM2410 parental strain [18].
The purpose of this study was to evaluate the ability to induce TNFα-dependent apoptosis in vivo in young predisease NZM2410 mice. A murine endotoxicosis model that utilizes LPS with d-galactosamine, a hepatocyte-sensitizing agent, was used to induce hepatocyte apoptosis leading to fulminate hepatic failure and eventually death [19], [20], [21]. This process is mediated by TNFα signaling through its p55 receptor (TNFR1) [22] and is caspase dependent [23]. This system allows for the assessment of TNFα production, TNFα apoptotic signaling, and TNFα effects on the production of IL-6 and IL-10. The well-documented overlap between apoptosis defects and autoimmunity prompted us to assess whether a genetic overlap also existed in the NZM2410 mouse as a model of both systemic autoimmunity and inflammatory/apoptosis defects. Although the congenic mapping approach does not replace genome-wide genetic mapping, we used the B6.Sle4 strain to assess whether the LPS-induced endotoxin resistance in NZM2410 could be accounted for by its Tnfaw allele. We also used the existing B6.Sle1, B6.Sle2, B6.Sle3, and B6.Sle1.Sle2.Sle3 congenic strains to assess whether the LPS-induced endotoxin resistance loci segregated with SLE-susceptibility loci.
In this study, we show that young NZM2410 mice were fully resistant to LPS with d-galactosamine induced endotoxicosis. Multiple functional differences in several biological markers associated with this model were found in NZM2410 as compared to B6. Since all of these defects occurred in young mice, they are likely to represent intrinsic genetic defects rather than a consequence of SLE pathogenesis. We also propose an initial genetic mapping of the resistance loci relative to the Tnfa gene and the Sle-susceptibility loci, and an initial functional mapping relative to the TNFα signaling pathway.
Section snippets
Mice
Six- to eight-week-old C57BL/6J (B6), NZM2410, B6.Sle1.Sle2.Sle3, B6.Sle1, B6.Sle2, B6.Sle3, and B6.Sle4 age- and sex-matched mice were used. They were either maintained in specific pathogen free (subset of B6 and NZM2410 for the rhTNFα study only) or conventional housing at the University of Florida Department of Animal Resources, as indicated in the text. The production of the B6.NZM congenic strains was previously described [10], [18]. All animal protocols were approved by the Institutional
Establishment of the LPS with d-galactosamine model in the NZM2410 mouse and mapping of resistance loci using B6.NZM congenic strains
The NZM2410 strain was completely resistant to LPS with d-galactosamine induced mortality, as compared to the B6 strain (0% vs. 70–75% mortality, respectively) up to 100 μg of LPS (Fig. 1). Similar results were obtained with SPF NZM2410 and B6 mice with LPS doses of up to 10 μg, where some mortality was observed in NZM2410, suggesting that an increased pathogen load was associated with a higher resistance. As expected, administration of the same doses of LPS alone induced high levels of TNFα
Discussion
The role of apoptosis in the establishment or maintenance of tolerance has been demonstrated in numerous studies [1]. It has been shown that the p55 TNF-R pathway can compensate for the FAS apoptotic pathway, suggesting that TNFα can regulate the survival of autoreactive cells [2]. To assess the integrity of the TNFα signaling pathway in the NZM2410 lupus-prone mouse and its derived B6.NZM congenic strains, we used a TNFα-dependent LPS-induced endotoxicosis model of hepatocyte apoptosis and
Acknowledgements
We would like to thank Tadahiko Kohno from Amgen, Inc. (Thousand Oaks, CA) for the generous gift of rhTNFα and B.P. Croker for helpful suggestions. This work was supported by grants from the NIH (ES-10277-01 to L. Morel, GM-40586-13 and GM-61807-01 to L.L. Moldawer).
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