Nanoparticle-enabled CD40 silencing programs immune tolerance via a DC-Treg-Breg axis in autoimmune myocarditis

Figures

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  Fig. 1. CD40 silencing suppresses dendritic cell maturation and shows enriched uptake in myeloid antigen-presenting cells. (A) Confocal laser scanning microscopy (CLSM) images exhibiting intracellular Cy5 signal in BMDCs after incubation with Cy5-labeled siRNA nanoparticles or PBS for 2 h at 37 °C. Scale bar, 10 μm. (B) CLSM images showing partial co-localization of Cy5-labeled siRNA with early endosomes (EEA1, green) after 2 h, consistent with endosomal trafficking. Scale bar, 10 μm. (C) CLSM images showing reduced co-localization of Cy5-labeled siRNA with late endosomes/lysosomes (LAMP1, green) at 18 h, consistent with endosomal escape and increased cytosolic availability of siRNA cargo. Scale bar, 10 μm. (D) Experimental scheme for BMDC treatment and downstream analyses. (E, F) Representative flow cytometry plots showing surface CD40 expression on BMDCs under different treatments, gating in CD11c+ cells. (G-I) Quantification of CD40, CD80, CD86, and MHC-II (I-A/I-E) expression on BMDCs, indicating reduced maturation/costimulatory phenotype following CD40 silencing (n = 3 independent BMDC preparations). (J) Ex vivo fluorescence imaging (IVIS) of major organs 24 h after intravenous…

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  Fig. 2. CD40 silencing attenuates acute myocardial inflammation and programs a regulatory immune balance in EAM. (A) Schematic of the treatment regimen during the acute phase of EAM. (B) Representative gross morphology and quantification of heart cross-sectional area from healthy controls and EAM mice treated with PBS, siNC/NPs, or siCD40/NPs (n = 9). (C) HW/BW indicated reduced cardiac enlargement following CD40 silencing (n = 8). (D) cTnT levels reflected myocardial injury (n = 4). (E) Representative H&E staining of cardiac sections and inflammation score (healthy, n = 3; EAM groups, n = 8 per group). Scale bar, 50 μm. (F) TUNEL staining showing cardiomyocyte apoptosis (n = 3-5 mice per group; quantification from four randomly selected fields per sample). Scale bar, 50 μm. (G) Flow cytometric gating strategy for CD45+ leukocytes isolated from cardiac tissue during acute EAM (n = 4). (H) Absolute numbers of infiltrating CD4+ T cells in whole hearts (n = 4). (I-K) Flow cytometric analysis of Foxp3+ Tregs and Th17, indicating restoration of the Treg/Th17 balance in the heart (n = 4). (L, M) Frequency of IL-10-producing Bregs in cardiac tissue (n = 4). (N) Cardiac mRNA expression of…

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  Fig. 3. CD40 silencing limits chronic cardiac remodeling and preserves ventricular function in EAM. (A) Experimental timeline illustrating progression from acute to chronic EAM and the treatment schedule. (B) Echocardiographic assessment at the indicated time points, including EF and FS. (C) Representative gross cardiac morphology and quantification of heart cross-sectional area (n = 3). (D) HW/BW at day 56 after immunization (n = 9-10). (E, F) Histological analysis of myocardial fibrosis (n = 3-5). Scale bar, 50 μm. (G) α-Smooth muscle actin (α-SMA) immunostaining showing myofibroblast accumulation, analyzed by Image J (n = 3 mice per group; quantification from four randomly selected fields per sample). Scale bar, 50 μm. (H) TUNEL staining showing cardiomyocyte apoptosis (n = 3; quantification from four randomly selected fields per sample). Scale bar, 50 μm. (I) Serum NT-proBNP levels (n = 5-6). (J) Cardiac myosin-specific IgG titers (n = 8-12). (K) Quantification of cardiac CD4+ T cells counts (per mg myocardial tissue) during the chronic phase (n = 5). (L, M) Frequencies of Tregs and IL-10+ Bregs in the spleen (n = 5). (N, O) The percentage of Tregs and IL-10-producing Bregs in…

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  Fig. 4. CD40 silencing reprograms dendritic cells and indirectly promotes regulatory B cell expansion. (A, B) Flow cytometric analysis of IL-10 production by Bregs and non-regulatory B cell subsets isolated from mouse spleen (n = 4 mice per group). (C) Experimental design for evaluating dose-dependent effects of CD40 silencing on Breg expansion in vivo. (D) Frequencies of Bregs in peripheral blood, spleen, and medLNs following single or repeated administration (n = 3). (E) Workflow for transcriptomic profiling of BMDCs after treatment with siCD40/NPs or control formulations. (F) Principal component analysis of transcriptomic data, demonstrating distinct clustering of CD40-silenced and control BMDCs. (G) Heatmap depicting differentially expressed genes associated with DC activation/inflammatory signaling and immunoregulatory programs. (H-J) Volcano plot depicting suppressed pro-inflammatory pathways along with gene ontology enrichment and pathway analyses, indicating suppression of pro-inflammatory signaling and enrichment of immune tolerance-associated processes following CD40 silencing. (K) Schematic of the DC-T cell co-culture assay. (L, M) Flow cytometric analysis of Tregs and Th17…

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  Fig. 5. In vivo tolerability and safety assessment of CD40 siRNA nanoparticle treatment. (A) Hematological parameters were performed 24 h after three times administrations of PBS, siNC/NPs or siCD40/NPs (n = 5). (B) Representative flow cytometry plots showed platelet activation. (C) Percentage of P selectin (CD62P) in platelets (CD41) at 30 min, 1 h and 24 h after PBS, siNC/NPs or siCD40/NPs administration. (D) Body weight changes during EAM induction and treatment (n = 7). (E) Representative histological sections of major organs (brain, liver, spleen, lung, and kidney) showing preserved tissue architecture without overt pathological abnormalities. Scale bar, 100 μm. Data are presented as mean ± SEM. ***P < 0.001.