Background/Purpose: Mouse models of SLE have been indispensable tools for studying disease pathogenesis; however, each model only recapitulates limited aspects of lupus. SLE1.2.3. (TC) mice are mice homozygous for three NZM2410-derive lupus susceptibility loci on the C57Bl/6J background. These mice develop systemic autoimmunity with fatal glomerulonephritis and die typically by 12 months of age. The polymorphic Rab4a locus has been associated with genetic susceptibility and protection from lung disease in SLE patients and has been found to be overexpressed in T cells of patients and mice with SLE. To determine the impact of this gene on disease development, the TC mice has been backcrossed with C57Bl/6 wild-type (WT) mice that carry floxed Rab4A^Q72L (TC-FL) alleles or lack Rab4A in T cells (TC-KO).

Methods: Proteinuria was assessed by the Bradford assay. ANA production was measured by ELISA. Mice were sacrificed around 50 weeks of age for analysis via flow cytometry, Seahorse assay and mass spectrometry. Histological scoring for glomerulonephritis (GN) was done blindly by Dr. Mark Haas.

Results: TC mice had increased proteinuria (males 160%, p=0.035, females 230%, p=0.008) over matched controls. Deletion of Rab4A in T cells reduced proteinuria (-50%, p=0.015) in female TC-KO mice relative to TC-FL mice at 21 and 40 weeks of age (TC-FL: 0.85±0.2 mg/ml, n=22; TC-KO: 0.39±0.1 mg/ml; n=12; p=0.041). Similar trends were noted in male mice (TC-FL: 1.01±0.1 mg/ml, TC-KO: 0.79±0.1 mg/ml; p=0.048). ANA production was reduced in male (-70.3%, p=0.009) and female (-24%, p=0.003) TC-KO mice relative to TC mice at 23 weeks of age. GN scores were significantly elevated (425%, p=0.009) in female TC mice compared to WT control at 53 weeks of age. Importantly, TC-KO mice showed a reduction (-40%; p=0.0342) in GN severity over TC-FL mice.  

Immunophenotyping unveiled a 45% depletion of CD4⁺ T cells (p=0.003) and 50% expansion of CD8⁺ T cells (p=0.016) in TC-KO female mice relative to TC-FL controls. Interestingly, mTORC1 activity in CD4⁺ was elevated by 100% in TC and by 290% (p=3E-05) in TC-FL mice, an effect that is diminished in TC-KO mice by 47% compared to TL-FL mice (p=0.0395). The same effect was not observed in CD8⁺ T cells or B cells of TC-KO mice. Additionally, IFNy-producing CD4⁺ T cells were expanded by 370% in TC (p=0.042) and by 310% in TC-FL mice (p=0.025). These proinflammatory cells were depleted by 47% in the TC-KO mice relative to lupus-prone TC mice.  

Relative to WT controls, flow cytometry revealed elevated mitochondrial mass (+38%, p=0.0416) and that occurred with a reduction in mitochondrial potential in CD4⁺ T cells of TC mice (53%, p=0.009). Seahorse metabolic analysis unveiled increased mitochondrial basal respiration (+80%, p=0.024), maximal respiration (+48%, p=0.02), ATP production (+84%, p=0.023) and spare respiratory capacity (+174%, p< 0.001) in CD8⁺ T cells of TC-WT mice relative to WT controls.

Conclusion: These findings establish how Rab4A promotes mTOR pathway activation, pro-inflammatory T cell lineage development, and disease pathogenesis through its influence on mitochondrial metabolism in SLE. Therefore, inactivation of Rab4A may have therapeutic potential in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rab4a-controls-mtor-pathway-activation-pro-inflammatory-lineage-development-and-disease-pathogenesis-in-lupus-prone-mice/