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

Abstract

Autoimmune myocarditis is a severe inflammatory heart disease that can progress to dilated cardiomyopathy and heart failure, while disease-modifying therapies remain limited. Dysregulated activation of antigen-presenting cells, particularly dendritic cells (DCs), disrupts immune tolerance and drives pathogenic cardiac inflammation. Here, we report a nanoparticle-enabled CD40 silencing strategy for immune tolerance programming in experimental autoimmune myocarditis. Using a polymer-lipid hybrid siRNA formulation, we achieved sustained CD40 silencing in myeloid antigen-presenting cells and induced functional reprogramming of DCs toward a tolerogenic state. This intervention established a coordinated DC-centered regulatory circuit, characterized by expansion of Foxp3⁺ regulatory T cells and IL-10-producing regulatory B cells, together with suppression of pathogenic Th17 responses. Functionally, siCD40/NP treatment attenuated myocardial inflammation, limited chronic fibrotic remodeling, and preserved ventricular function without evidence of overt systemic toxicity. Notably, regulatory B cells were enriched in cardiac-draining lymphoid tissues during the chronic phase, suggesting a spatially organized tolerance mechanism that contributes to durable disease control. Collectively, these findings identify nanoparticle-enabled CD40 silencing as a materials-based strategy for immune tolerance programming and highlight antigen-presenting cell reprogramming as a promising design principle for immunoregulatory biomaterials in inflammatory heart disease.

Graphical abstract

Highlights

• •Polymer-lipid nanoparticles enable sustained CD40 silencing in myeloid antigen-presenting cells.
• •CD40 silencing reprograms dendritic cells toward a tolerogenic phenotype.
• •A DC-Treg-Breg regulatory axis mediates durable immune tolerance.
• •Nanoparticle-enabled immune programming limits cardiac inflammation and fibrosis.

1. Introduction

Autoimmune diseases arise from a breakdown of immune tolerance, causing sustained activation of self-reactive immune cells and progressive tissue damage [[1], [2], [3]]. In autoimmune myocarditis, dysregulated immune responses against cardiac antigens drive inflammatory infiltration, cardiomyocyte injury, and eventual cardiac dysfunction [[4], [5], [6]]. Current therapeutic strategies largely rely on systemic immunosuppression, which is effective in controlling inflammation, often compromise host defense and fail to re-establish long-term immune homeostasis [2]. Therefore, developing approaches that can selectively restore immune tolerance without broadly suppressing immunity remains a central challenge in this field.

DCs are key orchestrators of immune responses, functioning as gatekeepers that determine the balance between immune activation and tolerance [7,8]. Through antigen presentation and costimulatory signaling, DCs regulate the priming and differentiation of downstream lymphocyte populations [9,10]. In particular, the CD40⁻CD40L axis plays a pivotal role in DC maturation and T cell activation [[11], [12], [13]]. Perturbations in the pathway contribute to multiple autoimmune diseases, including myocarditis [14,15]. Accordingly, silencing CD40 expression in DCs represents a promising strategy to attenuate pathogenic immune responses and promote tolerance [13,16,17]. However, achieving efficient and selective modulation of DC function in vivo remains challenging due to the lack of delivery systems capable of targeting antigen-presenting cells while preserving systemic immune competence.

Recent advances in nanomaterials have provided new opportunities for modulating immune responses with high precision [[18], [19], [20]]. Beyond serving as passive carriers, emerging evidence suggests that the physicochemical properties of biomaterials can actively shape immune cell fate, influencing cellular uptake, intracellular trafficking, and downstream signaling pathways [21]. In this context, rationally engineered nanocarriers offer the potential to function as active regulators of immune programming, rather than merely vehicles for cargo delivery. Nevertheless, how material design can be leveraged to selectively reprogram dendritic cells toward a tolerogenic phenotype, and how such reprogramming propagates through immune networks in vivo, remain insufficiently understood.

In addition to T cell-centered regulation, accumulating evidence highlights the critical role of regulatory B cells (Bregs) in maintenance of immune tolerance, particularly in the context of cardiac disease [22,23]. Bregs suppress inflammation primarily by secreting immunosuppressive cytokines such as IL-10 to restrain autoimmune responses [24,25]. Notably, the crosstalk between dendritic cells and B cells represents an underexplored axis in immune regulation. Whether targeted modulation of DC function can drive Breg activation and establish a coordinated DC-Breg-mediated tolerance circuit has not been fully elucidated, particularly in the context of nanomaterial-based interventions.

Here, we report a polymer-lipid hybrid nanoparticle system for the targeted delivery of siRNA against CD40 (siCD40) to dendritic cells, enabling material-driven immune tolerance programming in vivo. This nanoparticle exhibits preferential uptake by myeloid antigen-presenting cells and efficiently suppresses CD40 expression, leading to inhibition of DCs maturation and reconfiguration of costimulatory signaling. Importantly, we demonstrate that this DCs reprogramming extends beyond intrinsic cellular changes and drives the activation of regulatory B cells, establishing a previously underappreciated DC-Treg-Breg axis that amplifies immune tolerance. Using a murine model of experimental autoimmune myocarditis (EAM), we show that this nanomaterial-based strategy effectively suppresses pathogenic T cell responses, alleviates cardiac inflammation, and preserves cardiac function. Collectively, our findings highlight a materials-based approach for reprogramming immune circuits, providing new insights into how engineered nanocarriers can modulate antigen-presenting cell fate and restore immune homeostasis. This work not only advances the design of immunoregulatory biomaterials but also offers a broadly applicable framework for the treatment of autoimmune and immune-mediated diseases.

2. Materials and methods

2.1. Preparation of siRNA-encapsulated nanoparticles

siRNA-loaded polymeric nanoparticles were synthesized by a double-emulsion method. mPEG-PLGA was dissolved in chloroform (62.5 mg/mL) and mixed with DOTAP solution (100 mg/mL) and siRNA prepared in RNase-free water (8 mg/mL). The mixture (mPEG-PLGA, 400 μL; DOTAP, 90 μL; siRNA, 25 μL) was emulsified by probe sonication (60 W, 120 s) to form a primary emulsion, followed by re-emulsification in 5 mL deionized water using the same parameters. Organic solvent was removed by rotary evaporation, and nanoparticles were collected by centrifugation and resuspended in deionized water.

2.2. Cellular uptake and intracellular trafficking

BMDCs were seeded onto coverslips and incubated with Cy5-labeled siRNA nanoparticles or PBS. For uptake studies, cells were fixed after 2 h. For intracellular trafficking analysis, cells were fixed after 2 or 18 h, permeabilized, and stained with antibodies against EEA1 or LAMP1. Actin was labeled with Alexa Fluor 488-phalloidin, with nuclei stained using DAPI.

2.3. CD40 gene silencing and dendritic cell activation

DC1.2 or A20 cells were treated with siCD40/NPs or control formulations at concentrations of 50-150 nM. CD40 expression was assessed at 24 or 48 h by qRT-PCR and flow cytometry. Activation markers on BMDCs or BMDMs exposed to nanoparticles were analyzed after 24 h lipopolysaccharide (LPS, 500 ng/mL) stimulation.

2.4. Experimental autoimmune myocarditis

EAM in male BALB/c mice was generated by subcutaneous injection of α-MyHC peptide emulsified in complete Freund's adjuvant containing Mycobacterium tuberculosis H37Ra. Mice were randomized into four groups: healthy control, PBS-treated EAM, siNC/NPs-treated EAM, and siCD40/NPs-treated EAM. Nanoparticles were administered every other day for nine doses. Myocarditis severity was evaluated in a blinded manner on H&E-stained heart sections using a previously established semi-quantitative scoring system [3]: 0, no leukocyte infiltration; 1, small foci of leukocyte infiltration; 2, involvement of <10% of the myocardial cross-section; 3, 10–30% involvement; 4, 30–50% involvement; and 5, >50% involvement.

2.5. ELISA

Serum levels of α-MyHC-specific IgG, cardiac troponin I, and NT-proBNP were quantified using commercially available ELISA kits, with procedures performed as specified by the manufacturers. A sandwich immunoassay based on plasma-enhanced fluorescence was applied for multiplexed and quantitative detection of cytokines IL-17A, IL-12 [26].

2.6. RNA sequencing

BMDCs treated with siCD40/NPs or control formulations were stimulated with lipopolysaccharide for 24 h. Total RNA was extracted and sequenced on an Illumina platform. Using DESeq2, genes were identified as differentially expressed if the adjusted P value below 0.05 and the absolute log₂ fold change exceeded 1.

2.7. Histopathology and immunofluorescence

Tissue sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, or Picrosirius red. Immunofluorescence staining was performed using antibodies against CD11c, CD4, CD8, CD68, α-SMA, and TUNEL. Images were captured using confocal microscopy, followed by quantitative analysis using Image J by investigators blinded to treatment groups.

2.8. Cell sorting and assays

Naïve CD4⁺ T cells were isolated, labeled with CFSE, and co-cultured with BMDCs pretreated with siCD40/NPs or controls with stimulation. T cell proliferation and differentiation were analyzed by flow cytometry. Regulatory B cells (CD24^hi IgD^lo/-) were sorted by fluorescence-activated cell sorting.

3. Result

3.1. siCD40/NPs enable efficient CD40 silencing and suppress DCs maturation and antigen presentation

To evaluate the effects of CD40 silencing on dendritic cell function, we formulated CD40 siRNA-loaded nanoparticles using an amphiphilic mPEG_5k-PLGA_10k copolymer and cationic DOTAP for systemic delivery with enriched uptake in myeloid antigen-presenting cell (APC) populations (Fig. S1A). The nanoparticles were spherical, ∼115 nm in diameter, with a zeta potential of ∼15 mV (Fig. S1B-D) and high encapsulation efficiency (>90%) (Fig. S1E). Their hydrodynamic size remained stable in water, fetal bovine serum (FBS), and buffers at pH 4.5 and pH 7.4 (Fig. S1F), supporting formulation robustness under physiologically relevant conditions. DOTAP enhanced siRNA encapsulation and formulation stability, enabling reliable nanoparticle preparation for subsequent studies. Confocal microscopy showed that Cy5-labeled siRNA nanoparticles (Cy5-siRNA/NPs) were efficiently internalized by BMDCs, with diffuse intracellular signal detectable by 2 h (Fig. 1A). Colocalization with EEA1 and LAMP1 indicated initial endosomal/lysosomal localization, followed by reduced colocalization over time, consistent with endosomal escape and cytosolic siRNA availability (Fig. 1B and C).

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 administration of Cy5-siRNA/NPs or PBS, showing prominent signals in immune-related organs. (K) CLSM images of spleen sections showing Cy5 signal in proximity to CD11c⁺ cells (green). Scale bar, 10 μm. (L) Flow cytometric quantification of Cy5⁺ cells among splenic dendritic cells, macrophages, T cells, and B cells (n = 3), indicating enriched uptake in myeloid APCs and preferential uptake observed in DCs and macrophages relative to lymphocyte populations. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Following siCD40/NP treatment, CD40 expression in BMDCs or DC1.2 was significantly reduced at both mRNA and protein levels by qRT-PCR and flow cytometry (Fig. 1D). Silencing was dose-dependent and specific, as siNC/NPs did not reduce CD40 expression (Fig. 1E–G, and Fig. S2A). Functionally, CD40 silencing impaired BMDCs maturation, as reflected by reduced surface CD80, CD86, and MHC-II (I-A/I-E), indicating diminished antigen-presenting capacity (Fig. 1H and I).

We further observed reduced generation of DCs from bone marrow progenitor cultures following CD40 silencing (Fig. S2B and C), consistent with prior observations that CD40 signaling supports DCs differentiation [27]. Compared with control groups, siCD40/NP-treated DCs secreted higher levels of IL-10 and TGF-β (Fig. S2D). Together, suppressed maturation markers and reduced DCs generation support a shift toward a tolerogenic-like DC state. In BMDMs, siCD40/NPs similarly reduced costimulatory molecule expression, indicating that CD40 silencing can also dampen activation programs in macrophages (Fig. S2E).

We next assessed in vivo biodistribution and cellular uptake. After intravenous administration of Cy5-siRNA/NPs, fluorescence signals were prominent in immune-related organs, including spleen, lymph nodes, and thymus, with detectable signal in the heart (Fig. 1J and Fig. S3A). Immunofluorescence and flow cytometry indicated that nanoparticle uptake was enriched in myeloid APCs, and preferential uptake was observed in DCs and macrophages, whereas minimal internalization was detected in T and B cells (Fig. 1 K and L and Fig. S3B-D). Notably, the formulation does not incorporate active targeting ligands; thus, the observed uptake pattern likely reflects APC endocytic capacity and in vivo exposure. Collectively, these data support siCD40/NPs as an APC-enriched siRNA strategy to modulate costimulatory signaling.

3.2. siCD40/NPs attenuate acute EAM inflammation and program a regulatory immune balance

We next evaluated whether CD40 silencing in APC compartments could ameliorate autoimmune myocardial injury in vivo using a murine EAM model. Male mice were immunized and treated intravenously with siCD40/NPs or control formulations every other day (Fig. 2A). Compared with PBS- or siNC/NPs-treated EAM mice, siCD40/NPs markedly reduced cardiac enlargement, as reflected by gross cardiac appearance, quantitative measurements of cardiac dimensions, and reduced heart weight-to-body weight ratios (HW/BW) (Fig. 2B and C). Serum cardiac troponin T (cTnT) levels exhibited a consistent downward trend, indicating attenuation of myocardial injury (Fig. 2D). Histopathological analysis further confirmed reduced myocardial inflammation and tissue damage, with diminished leukocyte infiltration and cardiomyocyte apoptosis observed in CD40-silenced mice (Fig. 2E and F).

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 inflammatory mediators (n = 3-4 mice per group). (O) Serum IgM levels (n = 3-4 mice per group). (P) Frequencies of Tregs and IL-10⁺ Bregs in the spleen (n = 5-7). (Q) The proportion of Bregs in mediastinal lymph nodes (medLNs) (n = 5-7). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To characterize immune cell infiltration and composition within the inflamed myocardium during the acute phase of EAM, we performed flow cytometric profiling of leukocytes isolated from the heart (Fig. 2G and Fig. S4A). Flow cytometric analysis revealed that CD40 silencing markedly reduced the frequency of CD11b⁺ myeloid cells in the myocardium to near-physiological levels (Fig. S4B). In contrast, the relative proportions of antigen-presenting cell populations, notably dendritic cells, macrophages, and B cells, remained largely unchanged. The proportion of CD4⁺ T cells slightly increased, showing performance comparable with healthy controls, while CD8⁺ T cells constituted a minor fraction of the cardiac immune compartment (Fig. S4C).

Importantly, despite relatively stable or modestly increased cellular proportions, CD40 silencing substantially reduced the absolute numbers of infiltrating CD11b⁺ myeloid cells, conventional dendritic cells, macrophages, and CD4⁺ T cells (Fig. 2 H and Fig. S4D and E). A similar reduction was observed in CD8⁺ T cells and B cells (Fig. S4E). This dissociation between relative frequencies and absolute cell numbers is consistent with a global reduction in inflammatory cell infiltration rather than selective expansion of specific immune subsets. Consistent with the flow cytometric findings, immunofluorescence analysis corroborated a marked reduction in CD4⁺ and CD8⁺ T cell infiltration within cardiac tissue (Fig. S4F and G). Moreover, dendritic cells isolated from the myocardium exhibited reduced expression of CD40 and the costimulatory molecules CD80 and CD86, consistent with a suppressed activation and maturation state within the inflamed cardiac tissue (Fig. S4H). These findings indicate that siCD40/NPs treatment limits both recruitment and activation of pathogenic immune cells within the inflamed myocardium.

Beyond reducing inflammatory cell infiltration, CD40 silencing profoundly reshaped the regulatory immune landscape within the heart. The percentage of Tregs (Foxp3⁺ CD4⁺) was significantly increased, while Th17 cells (IL-17A⁺ CD4⁺) were reduced to near-physiological levels (Fig. 2I–K and Fig. S4I). In par6llel, IL-10-producing regulatory B cells were markedly expanded (Fig. 2L and M), resulting in a substantial restoration of the Tregs/Th17 and Bregs/Th17 ratios (Fig. S4J). Consistent with these cellular changes, cardiac expression of proinflammatory cytokines, including IL-17A, IFN-γ, IL-1β, and TGF-β, was reduced by approximately 60-70% compared with PBS-treated EAM mice, approaching levels observed in healthy controls (Fig. 2N). Systemically, circulating IL-17A and IL-12 concentrations were similarly reduced (Fig. S4K), accompanied by a marked decrease in serum IgM levels, indicating suppression of humoral autoreactive responses (Fig. 2O).

Notably, treatment with siNC/NPs resulted in modest reduction of myocardial inflammation and immune cell infiltration, consistent with previous reported baseline immunomodulatory effects of nanoparticle uptake by antigen-presenting cells. However, the effects of siNC/NPs were modest and transient, with no significant changes in dendritic cell activation or differentiation. In contrast, siCD40/NPs specifically suppressed CD40 expression on dendritic cells, leading to a robust inhibition of dendritic cell activation and impaired differentiation. This CD40-specific silencing was associated with reduced co-stimulatory molecule expression and coordinated expansion of regulatory T and B cell populations, a pattern consistent with immune regulatory skewing that was not observed with siNC/NPs (Fig. 2H–M). These findings underscore the distinct role of CD40 silencing in regulating immune responses, which contributed to the observed cardioprotective effects.

We next examined whether CD40 silencing exerted broader effects on systemic immune regulation. In the spleen, treatment significantly expanded Foxp3⁺ Tregs and IL-10-producing Bregs to levels comparable to those of healthy mice (Fig. 2P and Fig. S5A and B). Concomitantly, the frequency of plasma cells was reduced by approximately 50%, suggesting restrained B cell differentiation toward antibody-secreting phenotypes (Fig. S5C and D). In draining mediastinal lymph nodes, overall frequencies of Tregs and Bregs remained largely unchanged; however, Bregs exhibited enhanced immunoregulatory function, as evidenced by increased IL-10 production and reduced plasma cell differentiation (Fig. 2Q and Fig. S6A-D).

Collectively, these results demonstrate that CD40 silencing in APC compartments confers robust therapeutic benefit during the acute phase of autoimmune myocarditis by attenuating myocardial inflammation and reprogramming both local and systemic immune responses. The coordinated expansion and functional enhancement of regulatory T and B cell populations establish an immune tolerance state that counteracts pathogenic effector responses and underlies the observed cardioprotective effects.

3.3. siCD40/NPs prevent chronic cardiac remodeling and preserve ventricular function in EAM

We next investigated whether APC-enriched uptake and CD40 silencing could provide sustained protection against chronic inflammatory cardiomyopathy, a hallmark of progressive EAM (Fig. 3A). On day 56 after immunization, echocardiographic analysis revealed that CD40 silencing effectively preserved left ventricular systolic function during the chronic phase of disease. Mice treated with siCD40/NPs exhibited markedly increased ejection fraction (EF) and fractional shortening (FS) compared with PBS- or siNC/NPs-treated EAM mice (Fig. 3B), while treatment effect not detectable at day 21 (Fig. S7A). Left ventricular end-diastolic and end-systolic diameters lingered comparable across all groups at both time points, indicating that CD40 silencing prevented pathological ventricular dilation and preserved cardiac performance (Fig. S7B). These findings indicate that CD40 silencing effectively prevents the progressive functional deterioration characteristic of chronic autoimmune myocarditis.

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 cardiac-draining medLNs. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Morphological and histological analyses further demonstrated that CD40 silencing markedly mitigated pathological cardiac remodeling during the chronic phase of EAM. Compared with PBS- or siNC/NPs-treated EAM mice, siCD40/NPs-treated mice exhibited attenuated pathological cardiac enlargement, as evidenced by reduced gross cardiac enlargement and decreased HW/BW (Fig. 3C and D). Quantitative analysis of cardiac cross-sectional area further confirmed the reduction in pathological cardiac expansion in the siCD40/NPs group (Fig. 3C, right panel). At the tissue level, histological assessment revealed marked attenuation of myocardial fibrotic remodeling. Masson's trichrome staining showed a substantial reduction in interstitial and perivascular collagen deposition in siCD40/NPs-treated mice compared with control EAM groups (Fig. 3E). This antifibrotic effect was further corroborated by Picrosirius red staining, which demonstrated decreased collagen accumulation and a less dense fibrotic network in the myocardium following CD40 silencing (Fig. 3F). At the cellular level, immunostaining for α-smooth muscle actin (α-SMA) revealed a pronounced reduction in myofibroblast accumulation in the hearts of siCD40/NPs-treated mice (Fig. 3G), indicating suppressed fibroblast activation. In parallel, cardiomyocyte apoptosis was significantly reduced, with an approximately 80% decrease in TUNEL-positive cells compared with PBS-treated EAM mice (Fig. 3H), indicating preserved cardiomyocyte survival during the chronic phase. These observations indicate that CD40 silencing limits pathological cardiac remodeling through coordinated suppression of fibro-inflammatory remodeling and preservation of cardiomyocyte integrity. Notably, siNC/NPs also conferred partial attenuation of myocardial fibrosis and apoptosis compared with PBS-treated EAM mice, as reflected by intermediate reductions in collagen deposition and TUNEL-positive cardiomyocytes (Fig. 3 E-H). This observation is consistent with previous reports indicating that nanoparticle uptake by antigen-presenting cells can exert modest, nonspecific immunomodulatory effects [28,29]. In contrast, these effects were significantly less pronounced than those achieved by siCD40/NPs and were not associated with sustained suppression of DC-lineage activation programs or expansion of regulatory immune populations.

Serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were lessened in siCD40/NPs-treated mice, indicating reduced cardiac stress during the chronic phase of EAM (Fig. 3I). In parallel, cardiac myosin-specific IgG titers, which were markedly elevated in PBS- and siNC/NP-treated EAM mice, were substantially reduced following CD40 silencing and restored toward near-physiological levels (Fig. 3J). Given the established contribution of autoantibody responses to myocardial injury in EAM, these findings suggest that CD40 silencing attenuates humoral autoreactivity associated with disease progression.

The total numbers of cardiac immune cells were comparable across treatment groups during the chronic phase (Fig. 3K and Fig. S7C and D), indicating resolution of overt myocardial inflammation rather than sustained immunosuppression. Similarly, the frequencies of Tregs and Bregs within the heart and spleen remained stable (Fig. 3L and M, and Fig. S8A). Notably, Tregs are known to play a central role in controlling acute T cell-mediated inflammation in EAM [30,31]. In contrast, the role of Bregs in EAM has been less clearly defined. Stable Treg frequencies together with a selective expansion of IL-10-producing Bregs in cardiac-draining lymph nodes suggests that these regulatory populations contribute in a temporally and spatially distinct manner during chronic disease progression.

However, a selective and sustained expansion of IL-10-producing Bregs was observed in the cardiac-draining mediastinal lymph nodes, with their frequencies nearly doubling compared with control EAM mice (Fig. 3N and O, and Fig. S8B). In contrast, Tregs in the mediastinal lymph nodes remained unchanged, indicating that the immune regulatory effects were spatially restricted to Bregs in lymphoid tissues. This preferential enrichment of Bregs in cardiac-draining lymphoid tissues likely contributes to long-term suppression of autoreactive immune responses by regulating lymph node-based immune activation and humoral responses, thereby limiting chronic inflammatory remodeling.

Collectively, these results indicate that siCD40/NPs treatment provides durable cardioprotection in autoimmune myocarditis by preventing fibrotic remodeling, preserving ventricular function, and maintaining immune homeostasis. Rather than inducing sustained immunosuppression, this strategy establishes a targeted and spatially restricted immune regulatory program that constrains chronic disease progression.

3.4. Dendritic cell reprogramming indirectly drives regulatory B cell expansion following CD40 silencing

Given the sustained expansion of regulatory B cells observed during CD40 silencing in vivo, we next sought to define the cellular and mechanistic basis underlying Breg induction. We first assessed whether Bregs exhibit enhanced immunoregulatory capacity relative to non-regulatory B cell subsets. Analysis of splenic B cells by flow cytometry in wild-type mice supported that IL-10-producing cells were significantly enriched within the Breg population compared with non-Bregs (Fig. 4A and B), validating their functional relevance in immune regulation.

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 after co-culture with CD40-silenced BMDCs (n = 3-4). (N) Schematic illustration of Transwell indirect co-culture system. (O) Percentage of Tregs in CD4⁺ T cells. (P) Percentage of Bregs and IL-10 production by Bregs. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To determine whether siCD40/NPs directly act on B cells, we examined nanoparticle uptake and CD40 expression in B cell subsets following systemic administration. Cy5-siNC/NPs exhibited minimal internalization by both Bregs and non-Bregs (Fig. S9A and B). Consistently, CD40 expression of B cells and proliferative capacity of purified IgD ^lo/- CD24 ^hi Bregs remained unchanged following siCD40/NPs treatment (Fig. S9C-E). These findings indicate that Breg expansion is not mediated by direct engagement of B cells, suggesting an indirect immunoregulatory mechanism.

We next assessed the tissue distribution and dynamics of Breg expansion following siCD40/NPs treatment (Fig. 4C). Flow cytometric analysis across multiple compartments revealed a dose-dependent increase in IL-10-producing Bregs in peripheral blood, spleen, and cardiac-draining lymph nodes, with repeated dosing resulting in approximately twofold expansion in these tissues compared with control groups (Fig. 4D). Although siNC/NPs induced modest increases in IL-10⁺ Bregs, this effect was transient and occurred in the absence of dendritic cell reprogramming or coordinated T cell modulation. In contrast, Breg expansion induced by siCD40/NPs was more pronounced and sustained, suggesting involvement of upstream immune modulation mediated by antigen-presenting cells rather than direct B cell activation.

Because DCs are major upstream modulators of adaptive immunity [32], we next investigated whether siCD40/NPs reprogram dendritic cell function to indirectly promote Breg expansion (Fig. 4E). Transcriptomic profiling of BMDCs treated with siCD40/NPs or control formulations revealed clear separation of treatment groups by principal component analysis, indicating pronounced transcriptional reprogramming in dendritic cells following CD40 silencing (Fig. 4F). Following CD40 silencing, genes associated with dendritic cell activation, migration, and Th17-polarizing signaling were markedly downregulated, including Ccl2, Ccl7, Myd88, and Tlr3 (Fig. 4G and Fig. S9F). In parallel, genes linked to immunoregulatory programs, such as Tgfbr2, Tgfbi, Jag2, and Traf4, were upregulated (Fig. 4G and Fig. S9F). Gene ontology and pathway enrichment analyses further supported broad suppression of pro-inflammatory signals, including Toll-like receptor, IL-17, and cytokine-cytokine receptor interactions, accompanied by enrichment of immune tolerance-associated processes (Fig. 4H–J and Fig. S9G). These data indicate that siCD40/NPs induce a transcriptional program in dendritic cells consistent with a tolerogenic phenotype [[33], [34], [35]].

Finally, we assessed whether siCD40/NPs-treated dendritic cells exert functional immunomodulatory effects on downstream lymphocyte differentiation. In co-culture assays, BMDCs pretreated with siCD40/NPs promoted a significant expansion of Foxp3⁺ Tregs while concurrently decreasing Th17 cells when incubated with naïve CD4⁺ T cells derived from EAM mice (Fig. 4K–M). Similar skewing toward regulatory T cell differentiation was detected using wild-type naïve CD4⁺ T cells (Fig. S9H). To investigate whether regulatory lymphocytes induction depends on soluble mediators or direct cell-cell contact, we established Transwell co-culture systems. siCD40/NP-treated DCs in the upper chamber significantly promoted the differentiation of CD4⁺ T cells into Foxp3⁺ Tregs in the lower chamber (Fig. 4N and O), indicating that soluble factors released by tolerogenic DCs are sufficient for Treg induction. Correspondingly, the frequency of IL-10-producing Bregs was significantly increased in the lower chamber (Fig. 4P), suggesting that DC-induced Tregs contribute to downstream Breg differentiation. When DCs, CD4⁺ T cells, and B cells were co-cultured in direct contact (Fig. S9I), siCD40/NP-treated DCs significantly enhanced both Treg expansion and IL-10 production by Bregs (Fig. S9J and Fig. S9K). In contrast, when DCs and CD4⁺ T cells were confined to the upper chamber and B cells were cultured separately in the lower chamber, no significant differences in B cellsIL-10 production were observed among groups (Fig. S9L), indicating that while soluble factors released by tolerogenic DCs are sufficient to induce Tregs differentiation, the subsequent enhancement of Breg function partially requires direct Treg-B cell interactions.

Collectively, these results indicate that siCD40/NPs treatment induces Breg expansion through an indirect, dendritic cell-initiated immune regulatory mechanism. Rather than acting directly on B cells, CD40 silencing reprograms DCs toward a tolerogenic state that reshapes downstream T cell differentiation and establishes an immune environment permissive for sustained Breg expansion and peripheral immune tolerance.

To evaluate the in vivo tolerability of siCD40/NP treatment, hematological and splenocytes analyses were performed 24 h following systemic administration three times (Fig. S10A). Hematological parameters, including red and white blood cell counts, hemoglobin levels, platelet and neutrophil counts, stayed within normal physiological limits in all groups (Fig. 5A). Flow cytometry analyses of splenocytes showed that the siCD40/NPs primarily affected DCs differentiation and maturation (Fig. S10B and C). whereas macrophages exhibited minimal alterations in frequency and expression of activation-associated surface markers (Fig. S10D and E). B cells did not exhibit evidence of functional suppression and instead showed a modest increase in frequency (Fig. S10F and G). CD40 silencing did not overtly impair macrophage responsiveness to inflammatory stimulation (Fig. S10H). These findings suggest that siCD40/NPs preferentially modulate dendritic cell function rather than induce broad immunosuppression across antigen-presenting cell populations. Furthermore, Platelet activation was evaluated by flow cytometry (Fig. 5B), The DOTAP concentration used in the nanoparticle did not significantly induce platelet activation compared with control groups at 30 min, 1 h and 24 h after administration (Fig. 5C). Body weight changes during EAM induction were comparable among treatment groups and fully recovered by the end of the study (Fig. 5D). Histological examination of major organs revealed preserved tissue architecture without evidence of off-target toxicity (Fig. 5E). These findings indicate that siCD40/NPs are well tolerated in vivo and do not induce overt systemic toxicity or broad immunosuppression.

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.

4. Discussion

Autoimmune myocarditis can progress from acute immune-mediated myocardial injury to chronic cardiomyopathy and heart failure [36,37], yet currently available therapies remain dominated by nonspecific immunosuppression and are often insufficient to provide durable disease control [2,38]. In this study, we show that nanoparticle-enabled CD40 silencing establishes a sustained immunoregulatory trajectory in EAM. Importantly, this effect is not limited to suppression of acute myocardial inflammation, but extends into the chronic phase to restrain fibrotic remodeling and preserve cardiac function. This feature is particularly important in autoimmune disease, where transient inhibition of inflammation is often inadequate to prevent long-term tissue damage and organ dysfunction [39]. Our findings therefore support the concept that effective treatment of autoimmune myocarditis requires not only control of acute immune injury, but also durable reprogramming of the immune response that can reshape later disease evolution.

A central finding of this work is that nanoparticle-mediated CD40 silencing reprograms dendritic cells toward a tolerogenic state and thereby reshapes the wider immune network. The induced DC phenotype is characterized not only by reduced MHC-II and costimulatory molecule expression, but also by altered immunoregulatory programs that change how downstream lymphocyte responses are instructed. Functionally, these DCs dampen pathogenic immune activation, reduce CD8⁺ T cell accumulation and effector polarization, suppress Th17 responses, and promote Treg expansion. Thus, the effect of CD40 silencing is not simply the weakening of one costimulatory pathway, but the rewiring of a broader antigen-presenting cell program that redefines how adaptive immune responses are initiated and maintained. In this context, dendritic cells function as a key regulatory node through which nanoparticle-enabled intervention can reset immune balance.

Notably, scrambled siRNA-loaded nanoparticles also exhibited modest immunomodulatory activity in both acute and chronic EAM. This observation is consistent with previous reports showing that nanoparticle uptake by antigen-presenting cells can itself influence immune cell function through intracellular trafficking and endosomal processing. Although these effects were substantially weaker than those induced by CD40 silencing and were insufficient to establish durable immune tolerance or long-term cardioprotection, they suggest that the carrier system contributes a baseline level of immune modulation. This intrinsic biological activity should therefore be considered when interpreting the therapeutic contribution of nanoparticle-mediated gene delivery.

An additional notable aspect of this study is the activation of IL-10-producing regulatory B cells. Compared with Tregs, the biology of Bregs in autoimmune myocarditis remains much less well defined, yet their immunosuppressive potential is increasingly recognized [40,41]. In our study, Breg activation was not achieved through direct nanoparticle engagement of B cells, but instead emerged as an indirect consequence of dendritic cell tolerization. This finding is particularly meaningful for two reasons. First, Bregs represent a relatively rare and heterogeneous B cell subset, making them intrinsically difficult to target directly with delivery systems. Second, induction of Bregs through tolerogenic DC programming has rarely been demonstrated in related nanomedicine-based immune tolerance studies. The ability of our strategy to activate Bregs indirectly therefore suggests that the tolerogenic DC phenotype induced by siCD40/NPs is functionally distinctive, with a broader capacity to organize immunosuppressive circuitry. Rather than functioning solely as weakened antigen-presenting cells, these reprogrammed DCs appear to actively create an immune environment permissive for sustained regulation [42]. DC-Treg-Breg regulatory axis may indirectly contribute to the attenuation of chronic inflammatory remodeling.

From a biomaterials perspective, these findings highlight that nanoparticle systems can do more than deliver siRNA cargo [43]. By enabling effective CD40 silencing in peripheral myeloid antigen-presenting cell compartments, the polymer-lipid nanoparticle used here serves as a platform for immune tolerance programming in vivo. The biological consequences extend beyond gene knockdown itself and include reorganization of downstream immune circuits involving dendritic cells, T cells, and B cells. This supports a broader design principle for immunoregulatory biomaterials: modulation of upstream antigen-presenting cell fate can be leveraged to generate durable therapeutic effects in autoimmune disease [44].

Several limitations should be acknowledged. Although our data support durable immune regulation, the degree of cardiac autoantigen specificity versus broader immune modulation remains to be further clarified. In addition, while overt systemic toxicity and broad immunosuppression were not observed, extended evaluation under infection-prone conditions will be required to assess long-term immune competence.

In summary, nanoparticle-enabled CD40 silencing provides more than transient suppression of myocarditis-associated inflammation; it establishes a sustained immune-regulatory program that protects against both acute injury and chronic remodeling. By reprogramming dendritic cells and engaging a DC-Treg-Breg regulatory circuit, this strategy restores immune homeostasis and provides durable cardioprotection, offering a promising biomaterials-based approach for autoimmune myocarditis and related inflammatory diseases.

Funding

This work was supported by the National Key Research and Development Program of China (2024YFA0918600); NSFC (82325029, U22A20156, W2441022); Youth Development Fund of the First Hospital of Jilin University (JDYY16202522); Special Project on Health Talents of Jilin Province (JLSRCZX2025-140); Fundamental Research Funds for the Central Universities, Jilin University.

CRediT authorship contribution statement

Meiling Yu: Conceptualization, Formal analysis, Methodology, Visualization, Writing – original draft. Huizhu Tan: Formal analysis, Methodology. Kuirong Mao: Data curation, Formal analysis. Yanbao Xin: Methodology, Software. Xiandi Meng: Investigation, Methodology. Yue Lv: Investigation, Validation. Yiting Bai: Methodology. Jialiang Wang: Investigation, Methodology. Mengfei Zhao: Methodology. Xiuxiu Cong: Formal analysis. Haorui Wang: Validation. Yong-Guang Yang: Conceptualization, Funding acquisition, Writing – review & editing. Tianmeng Sun: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.