Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease

Abstract

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation1. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4–5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive2 and still associated with inflammation3,4, increased risk of cancer5 and Parkinson disease6, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

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Figure 1: Strong local and systemic generation of C5a in Gba19V/− mice drives activation of antigen-presenting cells and T cells and production of proinflammatory cytokines.
Figure 2: C5aR1-deficient mice are protected from the development of pharmacologically induced Gaucher disease.
Figure 3: C5aR-targeting in Gaucher-disease-prone Gba19V/− mice protects from GC accumulation and inflammation.
Figure 4: Formation of GC-specific IgG autoantibodies in Gba19V/− mice and patients with Gaucher drive local and systemic C5a generation.

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Acknowledgements

We thank S. L. Tinch, V. Inskeep, B. Quinn and D. N. Magnusen for technical assistance. We thank L. Bailey for help with submission of the IRB protocol to use healthy human control and Gaucher disease patients’ sera and C. Loftice for office and laboratory support. We also thank Cincinnati Children’s Hospital Medical Center FACS, pathology and animal cores for their efforts and C. M. Karsten for his help with the model detailing the role of C5a in Gaucher disease. This study was supported by the Alexion Rare Disease Innovation (31-91010-584000-137015) and Division of Human Genetics funds to M.K.P. as well as the German Research Foundation (EXC 306/2, CL XII; and IRTG 1911) to J.K.

Author information

M.K.P., G.A.G. and J.K. contributed to study design, data analysis and writing the paper. J.K. also provided the C5aR A8(Δ71−73). M.K.P. generated background matched Gba19V/−C5ar1+/+, Gba19V/−C5ar1−/−, wild-type and C5ar1−/− mice and performed in vivo and ex vivo functional immunological assays, lipid extraction, and IgG2a purification work. T.A.B. contributed to data interpretation and helped in preparation of the IRB protocol to use healthy human control and Gaucher disease patients’ sera. R.R. performed immune cell characterization and cytokines assays. L.J.M. executed and interpreted the statistical data. D.W. performed and analysed haematoxylin and eosin and CD68 tissue staining. K.D.S. and W.Z. performed GC and GS analyses. M.A.M determined protein expression of GCS. A.F.M. determined mRNA expression of UGCG/Ugcg. B.L. assessed GCase activity in mouse tissues.

Correspondence to Manoj K. Pandey or Jörg Köhl.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks P. A. Ward and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Surface expression of C5aR1 on tissue DCs and macrophages, and costimulatory molecule expression in pulmonary DCs and CD4+ T cells from Gba19V/− mice.

af, C5aR1 expresssion in FACS-sorted CD11c+CD11b+ DCs (ac) and CD11b+F4/80+ macrophages (df) from spleen and lung of strain-matched Gba19V/− and wild-type mice (n = 15 per each group). gl, Expression of CD40, CD80 and CD86 on pulmonary CD11c+CD11b+ DCs (gi) and CD40L and CD69 on CD3+CD4+ T cells (jl) from wild-type and Gba19V/− mice (n = 6 per group); ΔMFI: C5aR1 MFI − isotype MFI. In the histograms (b, e, h, k), the dark blue lines correspond to wild-type and the orange lines to Gba19V/− cells. Yellow- and black-lined histograms depict corresponding isotype controls. Values in c, f, i and l are mean ± s.d. Asterisks show significant differences between wild-type and Gba19V/− mice (***P < 0.001). Groups were compared using Student’s t-tests.

Extended Data Figure 2 C5a drives dose-dependent increases of co-stimulatory molecule expression in DCs and CD4+ T cells from Gba19V/− mice.

DCs and CD4+ T cells purified from wild-type and Gba19V/− mice (n = 15 per each group) were stimulated with the indicated concentrations of C5a for 24 h. af, DCs (a) or CD4+ T cells (d) were identified as CD11c+CD11b+ or CD3+CD4+ cells. Depicted is the CD40, CD80 and CD86 expression in DCs (b, c) and the CD40L and CD69 expression in T cells (e, f). The dark blue and orange lines correspond to wild-type and Gba19V/− cells, respectively. The light blue and pink histograms depict the corresponding isotype controls. Values in c and f are mean ± s.d. Asterisks show significant differences between wild-type and Gba19V/− mice (**P < 0.01; ***P < 0.001). Wild type were compared to Gba19V/− mice at the indicated C5a concentrations using ANOVA. Four separate a priori comparisons were performed for each experimental condition, BST = 0.0125 (0.05 / 4).

Extended Data Figure 3 Similar GCase activity but low GC accumulation and reduced costimulatory molecule expression in DCs and CD4+ T cells, and low cytokine and chemokine production in CBE-treated C5ar1−/− mice.

a, b, Expression levels of GC species in FACS-sorted pulmonary DCs (a) and CD4+ T cells (b) isolated from vehicle-treated wild-type (black) and C5ar1−/− (white), and from CBE-treated wild type (red) and CBE-treated C5ar1−/− mice (blue) (n = 10 per group). The total GCs were normalized to 1 × 106 of each cell type. cf, DCs (c, d) were assessed for CD40, CD80 and CD86 expression; CD4+ T cells were stained for CD40L and CD69 (e, f). In the histograms, the orange and dark blue lines correspond to CBE-treated wild-type and C5ar1−/− cells, respectively. The light blue and the pink lines correspond to vehicle-treated wild-type and C5ar1−/− cells, respectively. g, Cytokine production of co-cultured pulmonary DCs and CD4+ T cells. Values are mean ± s.d. Statistical differences between groups were determined by ANOVA with the a priori comparison being wild-type to C5ar1−/− mice. For each experiment, two conditions were evaluated (PBS and CBE), BST = 0.025. h, Serum cytokines and chemokines from the indicated CBE-treated and untreated mice as determined by proteome array. For each experiment, 40 cytokines or chemokines were evaluated. Given the high degree of correlation between cytokines/chemokines, a modified Bonferroni corrects (adjusting for correlation between cytokines) would result in a P value threshold of 0.021. Further, as two conditions were evaluated (PBS and CBE), the final multiple testing corrected significance threshold is 0.0105. i, GCase activity in extracts from liver, lung, and spleen of vehicle-treated wild-type or C5ar1−/− mice as well as CBE-treated wild-type or C5ar1−/− mice (n = 4 per group). All values are mean ± s.d. Group comparisons were done by ANOVA with the a priori comparison being wild-type to C5ar1−/− mice (*P < 0.05; **P < 0.01; ***P < 0.001); ns, not significant.

Extended Data Figure 4 CBE-treated C5ar1−/− mice show decreased cellularity, tissue disruption, and lower numbers of antigen-presenting cells and T cells.

a, Histological examination (haematoxylin and eosin) of liver, spleen and bone marrow of CBE-treated and untreated wild-type and C5ar1−/− mice (n = 15 per group). be, Total cell numbers in liver, spleen and lung (b), and DC and CD4+ T cell numbers in liver (c), spleen (d) and lung (e) (n = 15 per group) of vehicle-treated wild-type and C5ar1−/− mice as well as CBE-treated wild-type and C5ar1−/− mice. Values are mean ± s.d. Group comparisons were done by ANOVA with the a priori comparison being wild-type to C5ar1−/− mice. For each experiment, two conditions were evaluated (PBS and CBE), BST = 0.025 (***P < 0.001); ns, not significant.

Extended Data Figure 5 Decreased GC accumulation, reduced Ugcg expression and low expression of costimulatory molecules in pulmonary DCs and CD4+ T cells of Gba19V/−C5ar1/− mice.

a, b, GC accumulation in pulmonary DCs (a) and CD4+ T cells (b) of wild-type (black) and C5ar1−/− mice (white) as well as Gba19V/−, (red) and Gba19V/−C5ar1−/− mice (blue) (n = 15 per group). The total GC species in each cell type were normalized to 1 × 106 cells. c, Relative Ugcg mRNA levels in lung, liver and spleen of wild-type, C5ar1−/−, Gba19V/− and Gba19V/−C5ar1−/− mice (n = 8 per group). dg, CD40, CD80 and CD86 expression in pulmonary CD11c+CD11b+ cells (d, e) as well as CD40L and CD69 expression in CD4+ T cells (f, g). In the histograms, the orange and dark blue lines correspond to cells from Gba19V/− and Gba19V/−C5ar1−/− mice, respectively. The light blue and pink lines correspond to cells from wild-type and C5ar1−/− mice, respectively. Data are mean ± s.d. Group comparisons were done by ANOVA with the a priori comparison being C5aR1-deficient to non-deficient mice. For each experiment, two different mouse strains were evaluated (wild-type or Gba19V/−), thus the BST = 0.025 (**P < 0.01, ***P < 0.001); ns, not significant.

Extended Data Figure 6 Decreased GC accumulation, reduced Ugcg expression and decreased costimulatory molecule and and proinflammatory cytokine production in C5aRA-treated Gba19V/− mice.

a, GC accumulation in liver and spleen of vehicle (PBS; black) and C5aRA-treated (white) wild-type as well as vehicle-treated (PBS; red) and C5aRA-treated (blue) Gba19V/− mice (n = 15 per group). b, Relative Ugcg mRNA levels from lung, liver and spleen of vehicle-treated or C5aRA-treated (n = 4 per group) mouse strains. ch, FACS-sorted DCs and CD4+ T cells prepared from lung of vehicle-treated (PBS; light blue line/black column) or C5aRA-treated wild-type (pink line/white column) as well as vehicle-treated (orange line/red column) or C5aRA-treated Gba19V/− mice (dark blue line/blue column) (n = 15 per group) were co-cultured. Such cells and supernatants were used to determine the expression of the indicated co-stimulatory molecules (cg) and proinflammatory cytokines (h) by flow cytometry and ELISA. Values shown in b and eh are mean ± s.d. Group comparisons were done by ANOVA with the a priori comparison being treatment with vehicle or C5aRA. For each experiment, two different mouse strains were evaluated (wild-type or Gba19V/−), BST = 0.025. **P < 0.01, ***P < 0.001; ns, not significant.

Extended Data Figure 7 Increased IgG autoantibodies to GC and C5a, and strong tissue deposition of C3b in CBE-induced GCase deficiency.

ac, IgG1, IgG2a/c, IgG2b and IgG3 antibodies to GC (a), C5a serum concentrations (b) (n = 10 per group) and C3b deposition in liver, spleen and lung sections from 9 individual wild-type mice (c) that were injected i.p. with PBS (black columns in a) or CBE (100 mg per kg per day; white columns in a). Values in a, b are mean ± s.d. Group comparisons were done by ANOVA (a) or Student’s t-test (b) with the a priori comparison being wild-type to C5ar1−/− mice. For each experiment, two conditions were evaluated (PBS and CBE), BST = 0.025. ***P < 0.001.

Extended Data Figure 8 Genetic deficiency of activating or inhibitory FcγRs has no effect on survival after CBE-induced GCase deficiency.

a, b, Survival plots of CBE-treated wild-type (a and b, red lines), Fcer1g−/− (a, grey line), and Fcgr2b−/− (b, green line) mice. Wild-type, Fcer1g−/− and Fcgr2b−/− mice (n = 10 per group) were injected i.p. with CBE (100 mg per kg) or vehicle (PBS) daily for up to 30 days.

Extended Data Figure 9 Pharmacological blockade of C5aR reduces proinflammatory cytokine production in response to GC immune complexes in naive and GCase-inhibited U937 cells.

TNF, IL-1β, IL-6 and IL-23 production from untreated (−CBE, black columns) and CBE-treated (white columns) macrophage-like U937 cells that were stimulated with vehicle (PBS), GC, anti-GC IgG, GC immune complexes or C5aRA + GC immune complexes. Three independent experiments were performed for each group. Values are mean ± s.d. Statistical differences between groups were determined by ANOVA. For each experiment, vehicle-treated was compared to other conditions. We also considered post hoc comparisons for the comparison with C5aR-targeting. As there would be 10 different pairwise comparisons, significance for conditions other than to vehicle would require a P value ≤ 0.005. *P < 0.05, ***P < 0.001; ns, not significant.

Extended Data Figure 10 Model detailing the role of the C5a–C5aR1 axis in Gaucher disease.

(1) Mutations in GBA1, encoding defective GCase, result in the accumulation of GC preferentially in visceral macrophages. (2) Continuous release of GC from macrophages, uptake and processing by B cells and Cd1d-restricted activation of T lymphocytes results in the differentiation of B lymphocytes into plasma cells and the production of GC-specific IgG autoantibodies. (3) Such IgG autoantibodies form GC immune complexes that activate the classical pathway of complement, eventually leading to systemic C5 cleavage and C5a generation. (4) GC immune complex also bind to activating FcγRs present on macrophages and induce local C5 production by an LAT-dependent mechanism. Further, FcγR-activated macrophages cleave C5 into C5a by a cell-specific protease. This local C5 production and C5a may also occur in circulating, inflammatory monocytes, which express IgG2a/c-binding FcγRIV. (5) The binding of systemically or locally generated C5a to C5aR1 enhances the accumulation of GC within macrophages through increased expression of GCS, driving a vicious cycle that fuels the autoimmune response against GC. Importantly, the abrogation of this cycle at the level of C5a–C5aR1 interaction is sufficient to greatly, but not completely, reduce cellular GC accumulation and protect from death in genetic and pharmacologically-induced Gaucher disease models. (6) Activation of the C5a–C5aR1 axis in DCs upregulates costimulatory molecules (CD80/CD86/CD40) and drives the activation of T cells (CD40L/CD69) eventually resulting in the induction of a proinflammatory environment (IFNγ and IL-17A/F) that promotes the tissue destruction in Gaucher disease.

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Pandey, M., Burrow, T., Rani, R. et al. Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease. Nature 543, 108–112 (2017). https://doi.org/10.1038/nature21368

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