Basophil-derived exosomes exacerbate systemic lupus erythematosus by regulating B-cell proliferation via miR-24550

Abstract

Background: Systemic lupus erythematosus (SLE) is a complex autoimmune disease where B-cell proliferation and activation play a pivotal role in pathogenesis. While the role of basophils in SLE is recognized, the impact of basophil-derived exosomes on B-cell proliferation and activation has not been thoroughly investigated.

Methods: Exosomes from human basophils in both resting and activated states were isolated and characterized. These exosomes were then co-cultured with B cells to assess their effects on B-cell survival and proliferation. To investigate the in vivo roles, a Pristane-induced lupus model in Mcpt8^{flox/flox CAGGCre-ERTM} mice was utilized. The Pristane-Mcpt8^{flox/flox, CAGGCre-ERTM} mice were analyzed for basophil-derived exosome accumulation in the spleen and kidneys, and the effects on immune cell proliferation and plasma cell-plasmablast balance were assessed. Transcriptomic analysis was conducted on basophil-derived exosomes to identify key non-coding RNAs. Lupus mice were humanized by transplanting peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice to evaluate the effects of intervening miR-24550 in B cells.

Results: Activated basophil-derived exosomes were found to enhance B-cell survival and proliferation in patients with SLE. In the lupus mouse model, basophil-derived exosomes accumulated primarily in the spleen and kidneys, inducing excessive immune cell proliferation and disrupting the plasma cell-plasmablast balance, which worsened kidney damage. Transcriptomic analysis revealed key non-coding RNAs within basophil-derived exosomes. Activated basophil-derived exosomes were internalized by B cells, releasing miR-24550, which promoted B-cell proliferation. In humanized SLE mice, inhibiting miR-24550 in B cells reduced immune hyperactivation and improved renal function, similar to the effects of inhibiting basophil-derived exosomes release in Pristane-Mcpt8^{flox/flox, CAGGCre-ERTM} mice. Ultimately, basophil-derived exosomal miR-24550 promotes B-cell proliferation and activation by targeting Krüppel-like factor 5 (KLF5), which exacerbates SLE progression.

Conclusions: Basophil-derived exosomal miR-24550 promotes B-cell proliferation and activation by targeting KLF5, thereby exacerbating SLE progression. This study presents a novel strategy for SLE prevention and treatment.

Keywords: B cells; Basophils; Exosomes; Systemic lupus erythematosus; miR-24550.

Fig. 1

Basophil-derived exosomes enhance the survival and proliferation of B cells in SLE patients. A Flow cytometric analysis of CD123⁺ CD203c⁺ basophil purity. B Flow cytometric analysis of CD203c expression in human basophils. C Statistical analysis of CD203c expression in human basophils (n = 6). D Transmission electron microscopy images of human basophil morphology. E Western blotting analysis of human basophils and their exosomes. F Transmission electron microscopy images of human basophil-derived exosome morphology. G Nanoparticle tracking analysis of basophil-derived exosome size. H Flow cytometric analysis of CD19⁺ B-cell purity. I Immunofluorescence analysis of basophil-derived exosomes and B-cell colocalization. J Flow cytometric analysis of B-cell survival. K Flow cytometric analysis of B-cell proliferation. L Statistical analysis of B-cell survival (n = 4). M Statistical analysis of B-cell proliferation. Results are shown as mean ± SEM (n = 4). C, L, and M were analyzed by independent-samples t-tests. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Basophil-derived exosomes migrate to spleen and kidneys in Pristane-Mcpt8^{flox/flox, CAGGCre−ERTM} lupus mice. A Timeline of basophil and exosome transfer in Mcpt8^{flox/flox, CAGGCre−ERTM} mice. B Schematic of Mcpt8^{flox/flox, CAGGCre−ERTM} mouse construction. C Flow cytometric analysis of basophil proportion in peripheral blood of Mcpt8^{flox/flox, CAGGCre−ERTM} mice. D Flow cytometric gating for sorting induced basophils from mouse bone marrow. E Flow cytometric analysis of mouse basophil purity after sorting. F Nanoparticle tracking analysis of basophil exosome size in mice. G Western blotting analysis of mouse basophils and their exosomes. H Transmission electron microscopy of mouse basophil exosome morphology. I Immunofluorescence analysis of distribution of basophil-derived exosomes in Pristane-Mcpt8^{flox/flox, CAGGCre−ERTM} lupus mice

Fig. 3

Basophil-derived exosomes exacerbate disease progression in Pristane-Mcpt8^{flox/flox, CAGGCre−ERTM} lupus mice. A Statistical analysis of plasma antinuclear antibodies in mice (n = 4). B Statistical analysis of plasma anti-dsDNA antibodies in mice (n = 4). C Morphological analysis of mouse spleen. D Hematoxylin and eosin (H&E) staining analysis of mouse spleen pathology. E Immunofluorescence staining analysis of plasma cell and plasmablast proportions in mouse spleen. F Statistical analysis of 24-h urinary protein levels in mice (n = 4). G Statistical analysis of urea levels in mice (n = 4). H Statistical analysis of creatinine levels in mice (n = 4). I H&E, PAS, and MASSON staining analysis of mice kidney pathology (n = 4). J Immunofluorescence staining analysis of IgG and C3 deposition in mouse kidneys. Results are shown as mean ± SEM. A, B, F, G, and H were analyzed by one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

MiR-24550 in basophil-derived exosomes is crucial for their function. A miRNA differential expression heatmap (red indicates upregulation; green indicates downregulation; higher expression is indicated by brighter colors). B miRNA differential expression volcano plot (horizontal axis: fold change; vertical axis: P-value). C Relative expression levels of miRNAs in basophil-derived exosomes before and after activation (n = 3). D Relative expression levels of miRNAs in exosomes derived from activated basophils. E Flow cytometric gating scheme for detecting CD19⁺ B cells. F Flow cytometric analysis of CD19⁺ B cell purity before and after negative selection. G miRNA expression levels in B cells from healthy volunteers and patients with SLE (n = 3). H Changes in miR-24550 expression levels after basophil activation (n = 3). I Relative expression levels of miR-24550 in B cells after co-culturing of activated basophil-derived exosomes with B cells from patients with SLE (n = 3). Results are shown as mean ± SEM. C and G were analyzed by independent-samples t-tests. I was analyzed by paired t-tests. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

MiR-24550 promotes B-cell differentiation and activation. A Flow cytometric analysis of CD19⁺ B cell purity before and after negative selection. B qPCR analysis of miR-24550 overexpression efficiency in B cells (n = 5). C TUNEL assay to detect the effect of miR-24550 overexpression on B-cell apoptosis. D Statistical analysis of the effect of miR-24550 overexpression on B-cell apoptosis (n = 5). E Flow cytometric analysis of the effect of miR-24550 overexpression on the expression of the B-cell activation markers CD80 and CD86. (F) Statistical analysis of the effect of miR-24550 overexpression on the expression of the B-cell activation markers CD80 and CD86 (n = 5). G Flow cytometric analysis of the effect of miR-24550 overexpression on the expression of the B-cell proliferation marker Ki-67. H Statistical analysis of the effect of miR-24550 overexpression on the expression of the B cell proliferation marker Ki-67 (n = 5). I Flow cytometric analysis of the effect of miR-24550 overexpression on memory B-cell subsets. J Statistical analysis of the effect of miR-24550 overexpression on memory B-cell subsets (n = 5). K Flow cytometric analysis of the effect of miR-24550 overexpression on plasma cells and plasmablasts. L Statistical analysis of the effect of miR-24550 overexpression on plasma cells (n = 5). M Statistical analysis of the effect of miR-24550 overexpression on plasmablasts (n = 5). Results are shown as mean ± SEM. B was analyzed by one-way ANOVA. D, F, H, J, L, and M were analyzed by independent-samples t-tests. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

Construction of humanized SLE mouse models. A Procedure for construction of humanized SLE mouse models. B qRT-PCR analysis of miR-24550 overexpression and knockdown efficiency in B cells from patients with SLE (n = 3). C Flow cytometric analysis of CD19⁺ B cells from the bone marrow of humanized SLE mice. D Flow cytometric analysis of plasmablasts (PB) from the bone marrow of humanized SLE mice. E Flow cytometric analysis of memory B cells from the bone marrow of humanized SLE mice. F Statistical analysis of PB proportion in bone marrow of humanized SLE mice (n = 4). G Statistical analysis of switched memory (SM) B-cell proportion in bone marrow of humanized SLE mice (n = 4). H Statistical analysis of unswitched memory (UM) B-cell proportion in bone marrow of humanized SLE mice (n = 4). I Statistical analysis of naïve B cell proportion in bone marrow of humanized SLE mice (n = 4). Results are shown as mean ± SEM. B, F, G, H, and I were analyzed by one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

B-cell miR-24550 knockdown ameliorates disease progression in humanized SLE mice. A Statistical analysis of plasma antinuclear antibodies in humanized SLE mice (n = 4). B Statistical analysis of plasma anti-dsDNA antibodies in humanized SLE mice (n = 4). C Statistical analysis of plasma IFN-γ in humanized SLE mice (n = 4). D Statistical analysis of plasma IL-17 in humanized SLE mice (n = 4). E Statistical analysis of plasma TNF-α in humanized SLE mice (n = 4). F Statistical analysis of plasma IL-4 in humanized SLE mice (n = 4). G) Statistical analysis of plasma IL-12P70 in humanized SLE mice (n = 4). H Statistical analysis of plasma IL-10 in humanized SLE mice (n = 4). I Morphological analysis of spleen morphology in humanized SLE mice. J Statistical analysis of spleen weight–body weight ratio in humanized SLE mice (n = 4). K H&E staining analysis of spleen pathology in humanized SLE mice. L Immunofluorescence staining analysis of plasma cells (PC) in the spleen of humanized SLE mice. M Statistical analysis of 24-h urinary protein levels in humanized SLE mice (n = 4). N Statistical analysis of plasma urea level in humanized SLE mice (n = 4). O Statistical analysis of plasma creatinine level in humanized SLE mice (n = 4). P H&E, PAS, and MASSON staining analysis of kidney pathology in humanized SLE mice. Q Immunofluorescence staining analysis of IgG and C3 deposition in the kidneys of humanized SLE mice. Results are shown as mean ± SEM. A, B, C, D, E, F, G, H, J, M, N, and O were analyzed by one-way ANOVA. *P < 0.05; **P < 0.01

Fig. 8

MiR-24550 promotes B cell activation by inhibiting KLF5 expression. A Bioinformatic prediction of miR-24550 targeting KLF5. B qRT-PCR analysis of miR-24550 levels in B cells in healthy volunteers and patients with SLE (n = 3). C qRT-PCR analysis of KLF5 levels in B cells from healthy volunteers and patients with SLE (n = 3). D qRT-PCR analysis of KLF5 levels following miR-24550 overexpression in B cells (n = 5). E qRT-PCR analysis of KLF5 levels following miR-24550 knockdown in B cells (n = 5). F qRT-PCR analysis of KLF5 levels after modulation of B-cell miR-24550 level (n = 3). G Flow cytometric analysis of CD80 levels post-KLF5 knockdown in B cells. H Statistical analysis of CD80 levels post-KLF5 knockdown (n = 5). I Flow cytometric analysis of CD86 levels post-KLF5 knockdown in B cells. J Statistical analysis of CD86 levels post-KLF5 knockdown (n = 5). K Statistical analysis of IgG secretion following KLF5 modulation in B cells (n = 5). L Correlation between miR-24550 and KLF5 expression. M Correlation among miR-24550, KLF5, CD80, CD86, and IgG expression levels (n = 15). N Dual-luciferase reporter plasmid design for miR-24550 targeting KLF5. O Dual-luciferase reporter gene analysis of miR-24550 for targeting KLF5 (n = 3). Results are shown as mean ± SEM. B, C, D, E, and O were analyzed by independent-samples t-tests. F, H, J, and K were analyzed by one-way ANOVA. L was analyzed by Spearman correlation analyses. *P < 0.05; **P < 0.01; ***P < 0.001