Reduced late endosome/lysosome function promotes SLE through chronic PI3K activity and SHP-1/SHIP-1 defects
SunAh Kang, Andrew J. Monteith, Liubov Arbeeva, Karissa Grier, Shruti Saxena Beem, Anthony C. Trujillo, Xinyun Bi, Kai Sun, Rebecca E. Sadun, Mithu Maheswaranathan, Megan E.B. Clowse, Saira Z. Sheikh, Jennifer L. Rogers, Barbara J. Vilen
SunAh Kang, Andrew J. Monteith, Liubov Arbeeva, Karissa Grier, Shruti Saxena Beem, Anthony C. Trujillo, Xinyun Bi, Kai Sun, Rebecca E. Sadun, Mithu Maheswaranathan, Megan E.B. Clowse, Saira Z. Sheikh, Jennifer L. Rogers, Barbara J. Vilen
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Research Article Immunology

Reduced late endosome/lysosome function promotes SLE through chronic PI3K activity and SHP-1/SHIP-1 defects

Abstract

Degradation of cellular waste from phagocytosis, endocytosis, and autophagy occurs through hydrolases that become activated during acidification of late endosomes and lysosomes (LELs). In our cross-sectional study, we showed diminished LEL acidification and the accumulation of surface-bound nucleosome on monocytes, dendritic cells, B cells, neutrophils, and T cells from patients with systemic lupus erythematosus (SLE). Diminished acidification and exocytosis of undegraded IgG-immune complexes were evident in active, but not inactive, disease. This was supported by our murine study in which LEL acidification was diminished, promoting exocytosis and the accumulation of cell surface IgG-immune complexes. Mechanistically, LEL dysfunction was induced by chronic PI3K activation in lupus-prone MRL/lpr mice. We also showed that on a non-autoimmune C57BL/6 background, deficiency in SHP-1 and inhibition of SHIP-1 activity were sufficient to recapitulate LEL dysfunction found in MRL/lpr mice. Non-acidic LELs were evident in the majority of patients and associated with SLEDAI arthritis, rash, and nephritis. The high frequency of LEL dysfunction in SLE suggests that it could serve as a biomarker identifying a specific disease endotype.

Authors

SunAh Kang, Andrew J. Monteith, Liubov Arbeeva, Karissa Grier, Shruti Saxena Beem, Anthony C. Trujillo, Xinyun Bi, Kai Sun, Rebecca E. Sadun, Mithu Maheswaranathan, Megan E.B. Clowse, Saira Z. Sheikh, Jennifer L. Rogers, Barbara J. Vilen

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Figure 6

Deficiency in SHP-1 and inhibition of SHIP-1 in B6 mice phenocopy the LEL dysfunction seen in MRL/lpr.

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Deficiency in SHP-1 and inhibition of SHIP-1 in B6 mice phenocopy the LE...
To assess the effects of SHIP-1 and/or SHP-1 on LEL defects, BMMφs from B6, B6.SHIP-1–/–, and B6.SHP-1–/– mice were treated or not treated with inhibitors of SHP-1 (10 μM NSC-87877, 3 hours before IgG-IC treatment) or SHIP-1 (50 nM 3AC, 48 hours before IgG-IC treatment). The effects of single deficiency of SHIP-1 (A–C) or SHP-1 (D–F) or double deficiency (G and H) were analyzed. BMMφs were stimulated with IgG-ICs (25 μL IgG-ICs per 0.25 × 106 cells) with or without inhibitors. At designated times, LEL pH (A, D, and G), ROS (B, E, and H), and exocytosis (C, F, and I) were measured by flow cytometry. Absolute pH was calculated using a standard curve (A, D, and G). vATPase activity in unstimulated samples [t0(CMA)] was inhibited with concanamycin A (CMA; 2 ng/mL). ROS levels (B, E, and H) were measured using CellROX including t0 samples untreated with IgG-ICs, and fold of B6 t0 was graphed. BMMφs were preloaded (t0) with Alexa Fluor 488–labeled IgG-ICs, and exocytosis was measured at designated times (C, F, and I). Surface-bound fluorescence was assessed by subtraction of internalized fluorescence (surface quenched) from total (unquenched) and normalized to individual t0. Statistical analysis used Kruskal-Wallis test with multiple comparisons. Adjusted P values with significance are shown. N = 4–12; 3–4 experiments. Bars, median; boxes, 25th–75th percentiles; whiskers, minimum and maximum values.