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. 2022 Jan 13;11(2):262.
doi: 10.3390/cells11020262.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus

Tania Colasanti  1 , Francesca Romana Spinelli  1 , Cristiana Barbati  1 , Fulvia Ceccarelli  1 , Susanna Scarpa  2 , Marta Vomero  1 , Cristiano Alessandri  1 , Guido Valesini  1 , Fabrizio Conti  1
Affiliations

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus

Tania Colasanti et al. Cells. .

Abstract

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients' PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors' PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14- (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients' serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.

Keywords: B lymphocyte stimulator (BLyS); BLyS receptors; IL-18; autophagy; belimumab; citrullination; peptidylarginine deiminases (PADs); systemic lupus erythematosus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Belimumab decreases autophagy levels in PBMCs from SLE patients. (A) Western blot analysis of LC3-II, p62-SQSTM, and α-synuclein in lysates from SLE patients’ PBMCs at basal level (t0) and after 2, 4, and 12 weeks (t2, t4 and t12) of belimumab administration, and (B) from healthy donors (HDs). Results from a blot in a representative experiment are shown. (C) Densitometric analysis of SLE patients’ and HDs’ LC3-II (left panel), p62-SQSTM (middle panel), and α-synuclein (right panel) levels relative to β-actin, represented as the mean ± standard deviation (SD). In SLE: for LC3-II, **** p < 0.0001 t0 vs. t12, *** p = 0.0003 t2 vs. t12, *** p = 0.0005 t4 vs. t12; for p62-SQSTM, *** p = 0.0002 t0 vs. t4, **** p < 0.0001 t0 vs. t12, ** p = 0.0026 t2 vs. t12; for α-synuclein, ** p = 0.0098 t0 vs. t2, **** p < 0.0001 t0 vs. t4, t2 vs. t4, and t4 vs. t12. HDs’ mean LC3-II and p62-SQSTM expression levels are comparable to those of SLE patients at t12, and opposite (lower or higher, depending on the single marker trend) to SLE at baseline (t0) (** p = 0.002 for LC3-II, *** p = 0.0001 for p62-SQSTM, **** p < 0.0001 for α-synuclein). Only for α-synuclein, HDs’ expression levels are comparable to those of SLE patients at t4, given the dramatic decrease in levels observed at t12 (*** p = 0.0002 SLE t12 vs. HDs). (D) Correlation and linear regression analysis of α-synuclein and LC3-II levels relative to β-actin in HDs (* p = 0.0298, r= −0.55, 95% confidence interval (CI) = −0.8251 to −0.053).
Figure 2
Figure 2
Correlation between LC3-II autophagy marker levels determined by western blotting and clinical parameters. Correlation and linear regression analysis of LC3-II expression levels in lysates from 26 patients with SLE and SLE Disease Activity Index 2000 (SLEDAI-2K) scores after 12 weeks (t12) of belimumab administration:** p = 0.0087, r = 0.5, 95% confidence interval (CI) = 0.133−0.751.
Figure 3
Figure 3
Immunofluorescence analysis of LC3B and LAMP-1 localization in PBMCs from SLE patients and HDs. A representative image of LC3B expression as LC3 puncta (red fluorescence) and LAMP-1 expression (green fluorescence) in Triton-X-100-permeated cells. PBMCs from SLE patients at (A) baseline (t0) and after 2, 4, and 12 weeks (t2, t4 and t12) of belimumab administration, and from HDs. Results from a representative experiment are shown. Cells were stained with Hoechst dye to reveal nuclei (blue staining). The yellow spots indicate colocalization (merge) of the two markers in autophagic cells. A diffuse cytoplasm staining with virtually no puncta is visible at t0 and t2, while at t4 and t12, a typical punctate staining is observable, more similar to that of the HDs, in which rare yellow spots (autophagic vacuoles) are present. Magnification: 50X. (B) Quantification by densitometric evaluation of LC3B and LAMP-1 colocalization (merge yellow spots) from positive cells, as resulting from immunofluorescent staining for each condition in 3 independent experiments. In PBMCs from SLE patients, reductions at all of the timepoints and significant differences with respect to HDs were observed (* p = 0.029 t0 vs. t12, ** p = 0.008 t0 vs. HDs; * p = 0.011 t2 vs. HDs). Results are represented as the mean ± standard deviation (SD) of densitometric units.
Figure 4
Figure 4
Belimumab decreases protein citrullination in PBMCs from SLE patients. (A) Western blot analysis of citrullinated proteins and PADI4/PAD4 enzyme levels in lysates from SLE patients’ PBMCs at baseline (t0) and after 2, 4, and 12 weeks (t2, t4 and t12) of belimumab administration, and (B) from healthy donors (HDs). Results from a blot in a representative experiment are shown. (C) Densitometric analysis of SLE patients’ and HDs’ PADI4/PAD4 expression levels relative to β-actin, represented as the mean ± standard deviation (SD). In SLE: * p = 0.0144 t0 vs. t2, ** p = 0.0047 t0 vs. t4, *** p = 0.0003 t0 vs. t12. HDs’ PADI4/PAD4 mean expression levels are comparable to those of SLE patients at t12, and lower than those of SLE patients at baseline (*** p = 0.0003 SLE t0 vs. HDs). (D) Western blot analysis of citrullinated vimentin immunoprecipitated from SLE patients’ PBMCs lysates at t0, t2, t4, and t12, and (E) from HDs. An appropriate IgG isotypic control (irrelevant IgG; Irr. IgG) was employed to verify that immunoprecipitations were correctly performed. Results from a blot in a representative experiment are shown. (F) Densitometric analysis of SLE patients’ and HDs’ citrullinated vimentin expression levels, represented as the mean ± standard deviation (SD). In SLE: ** p = 0.0044 t0 vs. t2, *** p = 0.0005 t0 vs. t4, * p = 0.0403 t0 vs. t12, * p = 0.0122 t4 vs. t12). HDs’ citrullinated vimentin mean expression levels are comparable to those of SLE patients at t12, and lower than those of SLE patients at baseline (** p = 0.005 SLE t0 vs. HDs).
Figure 5
Figure 5
Belimumab reduces circulating IL-18 concentrations evaluated in sera from SLE patients. IL-18 levels in sera and lysates from 26 SLE patients were assessed using a commercially available ELISA kit. Sera and lysates were collected at baseline (t0) and after 2, 4, and 12 weeks (t2, t4, and t12) of belimumab therapy. All of the samples were run in triplicate in three different assays, and the results are shown as the mean ± standard deviation (SD). (A) For systemic IL-18 concentrations in sera, * p = 0.0191 t0 vs. t2; ** p = 0.0046 t0 vs. t4; * p = 0.0116 t0 vs. t12. (B) For IL-18 expression levels analyzed in lysates, p > 0.05 for all the conditions.
Figure 6
Figure 6
Surface expression of BAFF-R, BCMA, and TACI (BLyS receptors) on CD4+, CD8+ T cells and CD14+ monocytic cells of SLE patients. (A) Phenotypic characterization of PBMCs from 26 SLE patients at baseline (before treatment with belimumab), showing the expression of CD4, CD8, CD14, and CD19. Cytofluorimetric images show PBMC populations (R1 gate, upper panel) and CD4+, CD8+, CD14+, and CD19+ cells (R2 gate, lower panels). Results from a representative experiment are shown. (B) Flow cytometric analysis after staining of PBMCs with anti-BAFF-R, anti-BCMA, and anti-TACI mAbs. CD19+ B cells were used as positive controls for the surface expression of the receptors. Isotypic control staining is represented by the solid black line, while anti-BLyS receptors are represented by the broken black line. Results obtained in a representative experiment are shown. (C) Box and whisker plots indicate the levels of BLyS receptors observed in CD4+, CD8+, and CD14+ cells, expressed as mean fluorescence intensity (MFI). CD19+ cells are displayed as positive controls for the expression of BLyS receptors. Results are represented as the mean ± standard deviation (SD) of the fold increase.
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