Fig. 3: Rab4A-mediated pro-inflammatory T and B cell development during lupus pathogenesis is responsive to treatment by rapamycin and N-acetylcysteine (NAC) in vivo.

Treatment with rapamycin (Rapa) and NAC was implemented in female B6.TC/Rab4A^Q72L mice beginning at 27 ± 1.4 weeks of age. 3 mg/kg rapamycin was dissolved in phosphate-buffered saline (PBS) with 0.2% carboxymethylcellulose (CMC) solvent vehicle (Veh) and administered intraperitoneally (ip) three times weekly, while 10 g/l of NAC was provided in drinking water for 12 weeks. Control mice were treated ip three times weekly with 0.2% CMC solvent control alone. Age-matched female B6.TC and B6.TC/Rab4A^Q72L.CD4Cre-KO mice were also treated with rapamycin or solvent control. A Rapamycin, NAC, and inactivation of Rab4A block GN, GS, and glomerular hyalinosis. Kidneys were scored by an experienced renal pathologist blinded to mouse genotypes and treatments. Overall one-way ANOVA p values are shown in the header of each figure panel, while Sidak’s post-hoc test p values < 0.05 over brackets reflect comparison between experimental groups. The numbers (n) of mice in each experimental group were as follows: B6.TC Veh (n = 8), B6.TC Rapa (n = 5), B6.TC/Rab4A^Q72L Veh (n = 15), B6.TC/Rab4A^Q72L NAC (n = 5), B6.TC/Rab4A^Q72L Rapa (n = 6), B6.TC/Rab4A^Q72L-KO Veh (n = 8), B6.TC/Rab4A^Q72L-KO Rapa (n = 2); B Effect of rapamycin and inactivation of Rab4A on renal infiltration by CD3⁺ T cells and B220⁺ B cells and expression of pS6RP were assessed by immunohistochemistry. C Rapamycin and NAC, and inactivation of Rab4A block splenomegaly and cardiomegaly in lupus-prone mice. Overall one-way ANOVA p values are shown in the header of each figure panel, while Sidak’s post-hoc test p values < 0.05 over brackets reflect comparison between experimental groups. The numbers (n) of mice in each experimental group were as follows: B6.TC (Veh) (n = 5), B6.TC Rapa (n = 5), B6.TC/Rab4A^Q72L (Veh) (n = 4), B6.TC/Rab4A^Q72L (NAC) (n = 5), B6.TC/Rab4A^Q72L (Rapa) (n = 6), B6.TC/Rab4A^Q72L-KO (Veh) (n = 3), B6.TC/Rab4A^Q72L-KO (Rapa) (n = 2). Spleen weights one-way ANOVA p = 0.0002, Sidak’s post-hoc test p values corrected for multiple comparisons: B6.TC (Veh) vs B6.TC/Rab4A^Q72L (Veh) p = 0.0007, B6.TC/Rab4A^Q72L (Veh) vs B6.TC/Rab4A^Q72L-KO (Veh) p = 0.0007, B6.TC/Rab4A^Q72L (Veh) vs B6.TC/Rab4A^Q72L (NAC) p = 0.0062, B6.TC/Rab4A^Q72L (Veh) vs B6.TC/Rab4A^Q72L (Rapa) p < 0.0001; heart weights one-way ANOVA p = 0.1282, body weights one-way ANOVA p = 0.2232. D Effect of rapamycin and NAC and inactivation of Rab4A on anemia, leukopenia, and thrombocytopenia of lupus-prone mice. Overall one-way ANOVA p values are shown in the header of each figure panel, while Sidak’s post-hoc test p values < 0.05 over brackets reflect comparison between experimental groups. The numbers (n) of mice in each experimental group were as follows: B6.TC Veh (n = 5), B6.TC Rapa (n = 5), B6.TC/Rab4A^Q72L Veh (n = 3), B6.TC/Rab4A^Q72L NAC (n = 5), B6.TC/Rab4A^Q72L Rapa (n = 6), B6.TC/Rab4A^Q72L-KO Veh (n = 3), B6.TC/Rab4A^Q72L-KO Rapa (n = 2); E Effect of rapamycin and NAC, and inactivation of Rab4A on the abundance and mTOR activation of CD4⁺, CD8⁺, and DN T cells of lupus-prone mice. Overall one-way ANOVA p values are shown in the header of each figure panel, while Sidak’s post-hoc test p values < 0.05 over brackets reflect comparison between experimental groups. The numbers (n) of mice in each experimental group were as follows: B6.TC Veh (n = 5), B6.TC Rapa (n = 4), B6.TC/Rab4A^Q72L Veh (n = 3), B6.TC/Rab4A^Q72L NAC (n = 5), B6.TC/Rab4A^Q72L Rapa (n = 6), B6.TC/Rab4A^Q72L-KO Veh (n = 3), B6.TC/Rab4A^Q72L-KO Rapa (n = 2); F Effect of rapamycin and NAC, and inactivation of Rab4A on the activation of mTORC1 and mTORC2 in CD19⁺ and CD19⁺CD38⁺ B cells and abundance of CD19⁺CD11c⁺ B cells. Overall one-way ANOVA p values are shown in the header of each figure panel, while Sidak’s post-hoc test p values < 0.05 over brackets reflect comparison between experimental groups.