Background/Purpose: Polymorphic haplotypes of the HRES-1 endogenous retrovirus at the 1q42 chromosomal locus have been associated with lupus susceptibility, in particular, with predisposition to antiphospholipid syndrome (APS) and protection from lung disease in patients with SLE (Arthritis Rheum. 58:532-540). Expression of the HRES-1/Rab4 protein of this genomic locus, which has been recently designated as Rab4A, is increased in T cells of SLE patients (J Immunol 182: 2063–2073) and lupus-prone mice (Ann Rheum Dis 73:1888–1897). Intra-alveolar hemorrhage is a rare but fatal complication of pulmonary involvement SLE. To determine the impact of this gene on disease development, mice with constitutively active Rab4A^Q72L alleles or lacking expression of Rab4A in T cells (Rab4A-KO^CD4Cre) have been generated in the C57Bl/6 strain.

Methods: Female mice were injected intraperitoneally with 0.5 ml of pristane and evaluated for pulmonary capillaritis (Rheumatology 46:1405-10). Mice were treated with 3 mg/kg rapamycin or solvent control for 7 days prior and 14 days post pristane injection. 14 days after pristane injection, spleen and lung tissues were examined for lineage specification within the adaptive and innate arms of the immune system by flow cytometry. Expression of Rab4A and activation of the mechanistic target of rapamycin complexes 1 (mTORC1) and 2 (mTORC2) were also examined by western blot. Production of antinuclear antibodies (ANA) and antiphospholipid antibodies (aPL), such as anti-cardiolipin (ACLA) and anti-β₂ glycoprotein I autoantibodies (anti-β₂GPI) were measured by ELISA. Statistical analyses were performed using 2-tailed t-test and stated changes were significant at p < 0.05.

Results:  Relative to wild-type (WT) mice (2.4±0.4), vasculitis scores were reduced in Rab4A^Q72L (1.1±0.1; p=0.021) and increased in Rab4A-KO^CD4Cre mice (3.6±0.2; p=0.042). ANA production was increased in Rab4A-KO^CD4Cre mice relative to age-matched WT (p=0.038) and Rab4A^Q72L controls (p=0.048). ACLA and anti-β₂GPI production was significantly reduced in Rab4A^Q72L mice relative to WT and Rab4A-KO^CD4Cre mice. mTORC1⁺/mTORC2^– IL-17-producing Th17 cells were increased 1.9-fold in the CD4⁺ T cell compartment of Rab4A-KO^CD4Cre mice, and these cells were reduced by 48% in Rab4A^Q72L mice. In turn, mTORC1⁺/mTORC2⁺ CD4⁺CD25⁺ regulatory T cells (Tregs) were expanded 2.4-fold in Rab4A^Q72L mice, and these Tregs were reduced by 42% in Rab4A-KO^CD4Cre mice. Thymus-derived FoxP3⁺Helios⁺ Tregs were also expanded Rab4A^Q72L mice and depleted in Rab4A-KO^CD4Cre mice. IL-17-producing CD11b⁺ pro-inflammatory macrophages were expanded 2.1-fold in the spleen and Gr1⁺ neutrophils were expanded 8.4-fold in the lung of Rab4A-KO^CD4Cre mice. Rapamycin treatment did not block lung injury in pristane-treated WT or Rab4A-KO^CD4Cremice.

Conclusion:  Rab4A is required for mTORC1- and mTORC2-dependent development of Tregs, which restrict aPL production and pro-inflammatory expansion of macrophages and neutrophils and block pristane-induced intra-alveolar hemorrhage in a mouse model of SLE. Thus, constitutive activation of Rab4A blocks organ-specific disease development and offers a novel target for diagnosis and treatment of SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rab4a-is-required-for-development-of-tregs-restricts-antiphospholipid-antibody-production-and-pro-inflammatory-expansion-of-macrophages-and-neutrophils-and-blocks-pristane-induced-intra-alveolar-hem/