Background/Purpose: Systemic Lupus Erythematosus (SLE) is an autoimmune disease with an incompletely understood etiology. Previous work has identified Rab4A, a small GTPase responsible for endosomal recycling, to be a potential pathogenic driver of disease [Caza, et al. Ann Rheum Dis. 2014], which involves activation of mTOR that is responsive to accumulation of kynurenine. Here, we investigated whether Rab4A activity controls kynurenine metabolism through regulating surface expression of CD98 via recycling.

Methods: Using the wild-type parental line Sle1.2.3. (Sle Rab4a^WT), constitutively active (Sle Rab4A^Q72L), or T-cell deleted Rab4A (Sle Rab4A^CD4-KO) were generated using a Lox-Cre system. Splenocytes were harvested from the spleens of mice (n=12) and analyzed by flow cytometry using antibodies against CD3, CD4, CD8,  CD19 and tryptophan/kynurenine transporter, CD98. Metabolomic data was acquired using a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher). CD4+ T cells were isolated using a positive selection kit (ThermoFisher), then stimulated for 72hrs with anti-CD3 and anti-CD28 antibodies. The metabolomic profile of Sle Rab4^Q72L (n=4) and SLERab4^CD4-KO (n=4) were compared using Metaboanalyst 3.0. Disease progression was assessed by glomerulonephritis (GN) scoring [1]. Statistical analysis was carried with ANOVA and Student’s t-test using GraphPad.

Results: Mean GN score was significantly reduced in Sle Rab4a^CD4-KO (0.3) compared to Sle Rab4A^WT (1.1, p=0.039) and Sle Rab4A^Q72L mice (1.5, p=0.001). In pathogenic CD3+CD4-CD8- double-negative (DN) T cells, CD98 expression was significantly increased in Sle Rab4A^Q72L compared to Sle Rab4a^WT (p=0.029) and Sle Rab4A^CD4-KO mice (p=0.019) (Figure 2). This trend was also seen in CD4+ T cells without reaching significance (Figure 2). CD98^hi cells were depleted in Sle Rab4A^CD4-KO compared to Sle Rab4A^WT (p=0.044) and Sle Rab4A^Q72L mice (p=0.040). The tryptophan metabolomic pathway was measured by mass spectrometry to determine downstream effects of CD98 recycling by Rab4A. In Sle Rab4A^Q72L mice, accumulation of several tryptophan metabolites, including kynurenine, anthranilate, and serotonin was seen compared to Sle Rab4A^CD4-KO mice (Figure 3). A global impact on tryptophan metabolism was supported by pathway analysis effectively discriminating between Sle Rab4A^Q72L and Sle Rab4A^CD4-KO mice.

Conclusion: Rab4A activity controls the onset and severity of GN Sle1.2.3. lupus-prone mice. Constitutively active Rab4A has increased glomerulonephritis, while deleting Rab4A in T cells ameliorates disease activity. Activation of Rab4A promotes the expression the CD98 and the accumulation of kynurenine and other tryptophan metabolites, suggesting that Rab4A-dependent tryptophan and kynurenine uptake may control mTOR activation seen in human subjects with SLE.

Figure 1. Glomerulonephritis scoring. Kidney sections from Sle Rab4AWT, Sle Rab4AQ72L, and Sle Rab4ACD4-KO mice were scored blind on a 0-4 scale. Left, scores of n=31 mice. Right, representative sections in Sle Rab4AWT, Sle Rab4AQ72L, and Sle Rab4ACD4-KO mice.

Figure 2. CD98 surface expression in CD4+ T cells and DN T cells. Surface expression was measured by flow cytometry. Left, mean fluorescent intensity and prevalence of CD98hi cells. Right, representative dotplots showing CD98 expression in DN T cells in Sle Rab4AWT, Sle Rab4AQ72L, and Sle Rab4ACD4-KO mice.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rab4a-regulates-glomerulonephritis-and-tryptophan-metabolism-in-sle1-2-3-lupus-prone-mice-via-recycling-of-cd98/