A Vaccine Using mRNA Nanoparticles Targeting the Spleen to Treat Experimental Asthma

The tolerogenic mRNA‐LNPs vaccine platform was formulated by mixing an ethanol phase containing SM102, DSPC, Chol, DMG‐PEG, stearic acid and celastrol with an aqueous phase containing nucleoside‐modified mRNA. We first evaluated the impact of celastrol inclusion on the physicochemical properties of the stearic acid‐doped LNPs loaded with ovalbumin (OVA) mRNA (sLNP‐OVA). Dynamic light scattering (DLS) analysis revealed that the addition of celastrol (sLNP‐OVA/Cel) did not significantly alter the size (199 ± 21 nm versus 196 ± 24 nm) or zeta potential (−4.5 ± 0.7 mV vs −5.2 ± 0.8 mV) of the mRNA‐LNPs vaccine platform (Figure 2A). Moreover, there were no significant differences in mRNA encapsulation efficiency between sLNPs‐OVA (85.5 ± 4.3%) and sLNPs‐OVA/Cel (84.7 ± 6.3%) (Figure 2A). Agarose gel assays confirmed nearly complete mRNA entrapment within OVA‐mRNA‐loaded sLNPs, regardless of celastrol inclusion (Figure S1A, Supporting Information). Transmission electron microscopy (TEM) images further demonstrated homogeneous spherical nanoparticles in both sLNPs‐OVA and sLNPs‐OVA/Cel formulations (Figure S1B, Supporting Information). Next, we investigated the impact of celastrol on mRNA translation efficiency using the tolerogenic mRNA‐LNPs vaccine platform, substituting OVA‐mRNA with luciferase mRNA (Luc‐mRNA). Imaging analysis confirmed luciferase protein expression exclusively in the spleen (Figure 2B). Quantitatively, there was no significant difference in luciferase protein expression between spleens treated with mRNA‐LNPs vaccines with or without celastrol (Figure 2C).

To examine the splenic DCs selectivity of the tolerogenic mRNA‐LNPs vaccine after intravenous injection, we synthesized mRNA‐LNP delivery systems with or without celastrol, substituting 33.3% of the cholesterol content with Cy5‐labeled cholesterol. These particles were administered intravenously to BALB/c mice at 0.5 mg kg⁻¹ (n = 3), followed by the collection of IVIS images and flow cytometry analysis 6 h later to determine the in vivo biodistribution profiles of the mRNA‐LNPs vaccine platform. The results showed no significant differences in the accumulation of mRNA‐LNPs delivery systems with or without celastrol in the major organs (Figure S1C, Supporting Information). Part of the spleen was submitted to flow cytometry, and the remaining part underwent tissue sectioning. Flow cytometry confirmed the cellular uptake of the tolerogenic mRNA‐LNPs vaccine platform in the spleen 6 h post‐injection (Figure S1D, Supporting Information). Approximately 51.13 ± 1.78% of splenic DCs were Cy5‐positive, which was significantly higher than Cy5‐positive B cells (8.42 ± 0.03%), T cells (7.29 ± 1.71%), and macrophages (28.27 ± 2.61%) (Figure 2D), indicating the excellent splenic DCs selectivity of the stearic acid‐doped LNPs platform. Importantly, the inclusion of celastrol did not affect the uptake of stearic acid‐doped LNP‐based mRNA delivery systems by splenic cells (Figure 2D). After spleen tissue sectioning, digital fluorescence microscopy was performed using FITC‐conjugated anti‐CD11c antibody staining. Confocal laser‐scanning microscopy revealed limited distribution of the mRNA‐LNPs vaccine platform with celastrol in the marginal zone around the white pulp, with co‐localization of mRNA‐LNPs and splenic DCs, similar to that observed with mRNA‐LNPs vaccine platform without celastrol (Figure 2E).

To further explore the reduction in immunogenicity conferred by celastrol inclusion, we developed an OVA‐mRNA‐loaded stearic acid‐doped mRNA‐LNPs vaccine platform with or without celastrol. Splenocyte suspensions were prepared 24 h post‐injection and stained with antibodies against CD45, CD11c, CD40, CD80, CD86, MHC‐II, CD4, CD8, B220, and CD69 for analysis. Splenocytes from mice treated with sLNP‐OVA/Cel or sLNP/Cel showed lower expression levels of CD40, CD80, CD86, and MHC‐II on splenic DCs compared to those treated with sLNP‐OVA (Figure 2F). Additionally, we evaluated the activation state of splenic B cells, CD4⁺ T cells, and CD8⁺ T cells by gating CD45⁺ B220⁺ cells, CD45⁺ CD4⁺ cells or CD45⁺CD8⁺ cells, respectively (Figure S2A, Supporting Information). Compared to mice injected with sLNP‐OVA, the expression of CD69 on splenic B cells, CD4⁺ T cells and CD8⁺ T cells was also reduced in mice treated with sLNP‐OVA/Cel or sLNP/Cel (Figure S2B,C, Supporting Information). These findings suggest that the inclusion of celastrol restricts the activation splenic immune cells following mRNA‐LNPs delivery systems intravenous injection in vivo.

To investigate whether our particle design promotes the differentiation of tolerogenic rather than inflammatory DCs from immature DCs, we examined the anti‐maturation effect of celastrol‐loaded nucleoside‐modified mRNA‐LNPs delivery systems on bone marrow‐derived dendritic cells (BMDCs). First, we assessed whether the inclusion of celastrol affected the cellular uptake of mRNA‐LNPs delivery systems in vitro. Confocal imaging and flow cytometry were performed on BMDCs incubated with sLNP‐OVA or sLNP‐OVA/Cel. Confocal images showed that BMDCs efficiently internalized the vaccine delivery systems regardless of celastrol addition (Figure S3A, Supporting Information). Furthermore, flow cytometry confirmed similar cellular uptake percentages between sLNP‐OVA/Cel (95.25 ± 2.32%) and sLNP‐OVA (96.18 ± 1.86%) (Figure S3B, Supporting Information). Next, BMDCs were cultured with different mRNA‐LNPs vaccine platforms for 4 h and then activated with lipopolysaccharide (LPS) for 24 h to induce maturation. Flow cytometry analysis revealed that LPS‐treated BMDCs (mature DCs, mDCs) significantly upregulated the expression of co‐stimulatory molecules (CD40, CD80, CD86) compared to PBS‐treated BMDCs (immature DCs, iDCs) (Figure 3A,B). In contrast, BMDCs treated with sLNP‐OVA/Cel or sLNP‐Cel (tolerogenic DCs) showed lower levels of these co‐stimulatory molecules similar to iDCs, significantly different from mDCs and sLNP‐OVA‐treated BMDCs (Figure 3A,B). Thus, celastrol and mRNA co‐loaded vaccine induced a tolerogenic rather than immunogenic phenotype in immature BMDCs following LPS stimulation (Figure 3C). Furthermore, RNA‐seq analysis identified over 194 differentially expressed genes (DEGs) associated with sLNPs‐OVA/Cel or sLNPs‐OVA treatment (Figure 3D). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed enrichment of DEGs in immune‐stimulating pathways such as Cytokine, Chemokine, and NF‐κB signaling pathways with different treatments (Figure 3E). In addition, the key genes involved in pro‐inflammatory and anti‐inflammatory cytokine production were examined. According to the results of quantitative reverse transcription polymerase chain reaction (qRT‐PCR), higher mRNA levels of anti‐inflammatory cytokines (TGF‐β and IL‐10) and lower levels of inflammatory cytokines (IL‐6 and IL‐1β) in sLNP‐OVA/Cel‐treated BMDCs were observed compared to sLNP‐OVA‐treated BMDCs (Figure 3F). Furthermore, flow cytometry (Figure S3C,D, Supporting Information) and enzyme‐linked immunosorbent assay (ELISA) (Figure S3E, Supporting Information) confirmed higher levels of anti‐inflammatory cytokines (TGF‐β and IL‐10) in sLNP‐OVA/Cel‐treated BMDCs compared to sLNP‐OVA‐treated BMDCs. Moreover, the activation state of the NF‐κB signaling pathway in BMDCs was significantly enhanced after stimulation with LPS (Figure 3G). However, when the BMDCs were pretreated with the sLNP‐OVA/Cel, the NF‐κB signaling pathway was significantly suppressed compared to that of BMDC pretreated with sLNP‐OVA or treated solely with LPS, as demonstrated by the significantly decreased phosphorylation of P65 and Ikkβ (Figure 3G–I). Collectively, these data demonstrate that the inclusion of celastrol in nucleoside‐modified mRNA‐LNPs delivery systems can induce tolerogenic DCs differentiation by suppressing the NF‐κB signaling pathway.

The generation of tolerogenic DCs by the tolerogenic mRNA vaccine platform prompted us to investigate whether sLNP‐OVA/Cel could induce functional Tregs both in vitro and in vivo. To assess the ability of celastrol addition in the mRNA‐LNPs vaccine platform to promote functional Treg generation in vitro, splenocyte suspensions were used to isolate CD4⁺ T cells by magnetic beads before co‐culturing with differently treated BMDCs for 72 h (Figure 4A). Flow cytometry analysis of non‐adherent cells stained with anti‐mouse CD4 and anti‐mouse Forkhead box P3 (Foxp3) antibodies showed that BMDCs treated with sLNP‐OVA/Cel induced a significantly higher proportion of Tregs compared to sLNP‐OVA treated BMDCs (Figure 4B,C; Figure S4A, Supporting Information). Additionally, mRNA expression analysis revealed higher levels of the Treg‐specific transcription factor Foxp3, as well as the immunosuppressive cytokines IL‐10 and TGF‐β, in the sLNP‐OVA/Cel treated group, while inflammatory cytokine IFN‐γ levels were lower compared to sLNP‐OVA treated BMDCs (Figure 4D–G). Moreover, the IL‐10 and TGF‐β‐producing Treg clusters were further evaluated. The sLNP‐OVA/Cel group showed significantly higher levels of IL‐10 and TGF‐β in Tregs than the other groups (Figure S4B,C, Supporting Information). Functional assays demonstrated that Tregs generated from sLNP‐OVA/Cel treated BMDCs effectively inhibited the differentiation of naive CD4⁺ T cells into Th2 cells compared to those generated from sLNP‐OVA treated BMDCs (Figure S4D,E, Supporting Information). These findings highlight the efficacy of the tolerogenic mRNA vaccine platform in inducing tolerogenic DCs and promoting the differentiation of functional Tregs in vitro.

To assess the generation of Tregs and antigen‐specific Tregs in vivo, the animals were intravenously injected with 0.5 mg kg⁻¹ of the tolerogenic mRNA vaccine once a week for two weeks. Seven days after the final immunization, splenocyte and lung cell suspensions were prepared for analysis (Figure 4H). Flow cytometry analysis revealed a significant increase in the percentage of splenic Tregs within B220⁻CD4⁺ T cells and OVA‐specific tetramer‐positive splenic CD4⁺ Foxp3⁺ Tregs in mice treated with sLNP‐OVA/Cel compared to untreated or sLNP‐OVA treated mice (Figure 4I). Moreover, sLNP‐OVA/Cel treatment resulted in a significant decrease in effector memory T cells compared to sLNP‐OVA treated mice, suggesting potential long‐term immune tolerance (Figure S5A,B, Supporting Information).

To further investigate whether splenic Tregs could migrate to the lung and exert immunosuppressive functions, lung cell suspensions were analyzed by flow cytometry. The results showed that the percentages of Tregs within lung CD4⁺ T cells and OVA‐specific tetramer‐positive lung Tregs were significantly higher in the sLNP‐OVA/Cel treated group compared to PBS or sLNP‐OVA treated groups (Figure 4J). These results demonstrate that intravenous administration of sLNP‐OVA/Cel induced significant generation of OVA‐specific splenic Tregs in situ, and subsequently migrated to the lung to exert immunosuppressive functions.

To evaluate the prophylactic efficacy of the tolerogenic mRNA vaccine, BALB/c mice were given two intravenous injections of 0.5 mg kg⁻¹ sLNP‐OVA, sLNP‐OVA/Cel, or sLNP‐Luc/Cel once a week. The animals were sensitized intraperitoneally with OVA twice a week for two weeks, followed by an intratracheal challenge to induce experimental allergic asthma models (Figure 5A). At the end of the prophylactic study, half of each lung was fixed in 4% formaldehyde and sectioned for subsequent experiments. Hematoxylin and eosin (H&E) staining revealed that asthma induction led to lung inflammation, with sLNP‐OVA/Cel injection showing more effective inhibition of lung inflammation compared to other treatments (Figure 5B). The inflammation score of the sLNP‐OVA group was significantly lower than that of the PBS group (Figure 5C). Importantly, the inflammation score of the sLNP‐OVA/Cel group was significantly lower than that of the sLNP‐OVA group (Figure 5C). Mucus production in lung tissues, assessed by periodic acid‐Schiff (PAS) staining and scored accordingly, was reduced in the sLNP‐OVA/Cel treated group compared to PBS, sLNP‐OVA, or sLNP‐Luc/Cel treated groups (Figure 5D,E). Pulmonary cell suspensions were also analyzed. Notably, sLNP‐OVA treatment significantly reduced lung neutrophil infiltration compared to PBS‐treated asthma mice (Figure 5F,G), while sLNP‐OVA/Cel treatment resulted in even less neutrophil infiltration compared to sLNP‐OVA‐treated mice (Figure 5F,G). Additionally, analysis of bronchoalveolar lavage fluid (BALF) cell counts showed that total immune cells, lymphocytes, eosinophils, and neutrophils were lowest in the tolerogenic vaccine group compared to other treatment groups (Figure S5C,H, Supporting Information). Furthermore, levels of OVA‐specific IgE and Th2‐associated cytokines (IL‐4, IL‐5, IL‐13) in BALF were significantly reduced in the sLNP‐OVA/Cel treated group compared to other asthma treatment groups (Figure S5D,E, Supporting Information). These findings confirm the potent prophylactic immunotherapy efficacy of the tolerogenic sLNP‐OVA/Cel vaccine against experimental allergic asthma.

The infiltration of immune cells into the lungs plays an essential role in the progression of inflammatory asthma. Here, we investigated whether sLNP‐OVA/Cel injection could mitigate asthma by modulating the infiltration of inflammatory and suppressive immune cells in the lung through immunostaining analyses of lung sections (Figure 6A,D) and flow cytometry of lung cell suspensions (Figure 6B,C,E,F). The results showed that sLNP‐OVA/Cel immunization significantly suppressed the infiltration of inflammatory monocytes in the lung compared to Control groups (Figure 6A–C). Additionally, the accumulation of CD45⁺ immune cells and CD4⁺ T cells was markedly reduced in mice treated with sLNP‐OVA/Cel compared to those treated with sLNP‐OVA (Figure S6A–C, Supporting Information). Importantly, sLNP‐OVA/Cel immunization led to a significant increase in the accumulation of Tregs in the lung by the end of the study (Figure 6D–F). These results indicate that our splenic DCs‐targeted tolerogenic mRNA‐LNPs vaccine could effectively mitigate the severity of allergic asthma by modulating the inflammatory microenvironment in the lungs.

Considering the immune cells' reservoir role of the spleen, the activation of DCs and other immune cells such as T cells and B cells, and the number of splenic Tregs were accompanied by flow cytometry of splenic cell suspension at the end of the study. We observed that sLNP‐OVA immunization significantly suppressed the over‐activation of DCs (Figure 7A,B), which correlated with the reduced lung inflammation score observed (Figure 5C). Moreover, sLNP‐OVA/Cel immunization further suppressed the over‐activation of DCs (Figure 7A,B), as well as B cells (Figure 7C,D), CD4⁺ T cells (Figure 7E), and CD8⁺ T cells (Figure 7F), compared to PBS or sLNP‐OVA treatment. Importantly, sLNP‐OVA/Cel immunization increased the number of splenic Tregs (Figure 7G,H), demonstrating its role in inducing a tolerogenic immune response in the spleen. Overall, the immunotherapeutic efficacy of the tolerogenic mRNA‐LNPs vaccine was accompanied by the induction of tolerogenic DCs and Tregs in the spleen, as well as an increase in the percentage of Tregs in the lung.

An important question is whether the generation of Tregs by sLNP‐OVA/Cel can prevent allergic asthma once allergic sensitization has occurred, even if prophylactic intervention before sensitization is observed. We investigated this by administering two intravenous doses of sLNP‐OVA, sLNP‐OVA/Cel, or sLNP‐Luc/Cel after two rounds of intraperitoneal sensitization (Figure 8A). Subsequent intratracheal challenge induced asthma in untreated animals. Notably, both Cel‐loaded delivery platforms mitigated asthma development, with sLNP‐OVA/Cel demonstrating significantly greater efficacy than sLNP‐Luc/Cel. Suppression of asthma manifestations coincided with reduced lung inflammation (Figure 8B,C), mucus production (Figure 8D,E), and eosinophil infiltration (Figure 8F,G). Additionally, cell counts from BALF revealed significantly lower levels of total immune cells, lymphocytes, eosinophils, and neutrophils in the sLNP‐OVA/Cel group compared to PBS, sLNP‐OVA, or sLNP‐Luc/Cel treated groups (Figure 8H).

Furthermore, we evaluated this approach in a therapeutic model by administering mRNA vaccines after establishing experimental asthma (Figure , Supporting Information). Lungs from PBS‐treated asthmatic mice exhibited pronounced inflammation (Figure , Supporting Information). The inflammation score decreased in the sLNP‐OVA group compared to PBS‐treated groups (Figure , Supporting Information). Importantly, the inflammation score of the sLNP‐OVA/Cel group was significantly lower than that of the sLNP‐OVA group (Figure , Supporting Information), indicating the potent therapeutic effects of sLNP‐OVA/Cel against experimental allergic asthma. These findings highlight the potential of our splenic DCs‐targeted tolerogenic mRNA‐LNPs vaccine as a novel strategy for asthma treatment, warranting further research. S6D S6E S6F S6F

Lastly, we comprehensively evaluated the in vitro cytotoxicity and preliminary organ toxicity of sLNP‐OVA/Cel in vivo. Analysis of the cytotoxic effect of celastrol on BMDCs showed that even at high concentrations of up to 1 mg mL⁻¹, celastrol did not induce significant cytotoxicity (Figure S7A, Supporting Information). Subsequently, BMDCs treated with PBS, sLNP‐OVA, sLNP‐OVA/Cel, or sLNP‐Cel for 4 h followed by LPS stimulation for 24 h showed no significant differences in cytotoxicity among the treatment groups (Figure S7B, Supporting Information). Histological examination of major organs at the end of the study on the generation of antigen‐specific Tregs in vivo revealed no signs of organ damage (Figure S8A, Supporting Information), indicating that intravenous administration of sLNP‐OVA/Cel did not result in overt organ toxicity. Additionally, there were no significant changes in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), albumin (ALB), and uric acid (UA) among mice treated with PBS, sLNP‐OVA, sLNP‐OVA/Cel, or sLNP‐Luc/Cel (Figure S8B, Supporting Information). These findings demonstrate the promising biocompatibility of our tolerogenic mRNA‐LNPs vaccine platform, supporting its further exploration in preclinical studies.

SM102, DSPC, DMG‐PEG and cholesterol were purchased from Shanghai A.V.T. Pharmaceutical Co., Ltd (Shanghai, China). Citrate buffer and phosphate buffer (PBS) were purchased from Solorbio (Beijing, China). Stearic acid, bovine serum albumin (BSA), and ovalbumin (OVA) were supplied by Sigma–Aldrich (USA). The T7 High Yield RNA Transcription Kit, N1‐Me‐Pseudo UTP, Cap 1 Capping System, and E.coli Poly(A) Polymerase were acquired from Novoprotein (China). D‐Luciferin (sodium salt) was purchased from Biovision Biotechnology (USA).

The antibodies used for flow cytometry were listed in Extended Data Table (Supporting Information), and the sequences of all primers utilized for qRT‐PCR were shown in Extended Data Table (Supporting Information). ELISA kits for Mouse IL‐10 (EK0417), Mouse TGF‐β (EK0515), Mouse IL‐4 (EK0405), Mouse IL‐5 (EK0408), and Mouse IL‐13 (EK0425) were procured from BOSTER Biological Technology Co., Ltd. (China). Mouse anti‐IgE antibodies were sourced from Invitrogen (USA). Antibodies specific for Ikkβ (8943S), p‐Ikkβ (2697S), p65 (3033S), and p‐p65 (8242S) were purchased from Cell Signaling Technology. HRP‐conjugated anti‐rabbit antibody (AS014) was obtained from ABclonal, and antibodies against ACTB (GB11001‐100) were acquired from Servicebio (China). S1 S2

Nucleoside‐modified OVA‐mRNA and Luc‐mRNA were synthesized using T7 RNA polymerase (Novoprotein) with N1‐Me‐Pseudo UTP substituted for UTP, following the manufacturer's instructions. All mRNAs were stored frozen at −80 °C.

The splenic DCs‐targeted mRNA‐loaded LNPs were prepared as previously described.^[34^] Briefly, an ethanolic lipid solution containing SM‐102, DSPC, cholesterol, DMG‐PEG, and stearic acid was combined with an aqueous solution of mRNA in citrate buffer (pH 4.5) by rapid pipetting. LNPs loaded with celastrol were prepared similarly, with celastrol (1% of the lipid mass) added to the ethanolic lipid solution. The final sLNPs‐OVA and sLNPs‐OVA/Cel formulations were obtained by dialysis against 1× PBS.

The mRNA loading efficiency of sLNPs‐OVA and sLNPs‐OVA/Cel was initially assessed using denaturing formaldehyde agarose gel electrophoresis. Quantitative mRNA encapsulation efficiency was determined using the Quant‐iT RiboGreen RNA assay Kit. Particle size and zeta potential of sLNPs‐OVA and sLNPs‐OVA/Cel were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, United Kingdom). The morphology of the nanoparticles was examined by transmission electron microscopy (Hitachi, Tokyo, Japan).

The distribution of Cy5‐labeled sLNPs‐Luc/Cel in the spleen was analyzed ex vivo 6 h after intravenous injection. The expression of luciferase and the fluorescent signal density of LNPs‐based mRNA vaccine delivery systems in the spleen were quantified using PerkinElmer equipment. Single‐cell suspensions were prepared from a portion of the spleen and analyzed by flow cytometry. Splenic cell subsets were labeled with antibodies against CD45, CD3, B220, CD11b, and CD11c, with DAPI used for viability staining to exclude dead cells. The proportions of Cy5⁺/CD11c⁺ DCs, Cy5⁺/CD11b⁺ macrophages, Cy5⁺/CD3⁺ T cells, and Cy5⁺/B220⁺ B cells were determined. Additionally, frozen spleen sections from another portion of the spleen were acquired and stained with DAPI and CD11c to investigate the intrasplenic spatial distribution of the tolerogenic mRNA‐LNPs vaccine.

To investigate the induction of tolerogenic splenic DCs in vivo, BALB/c mice were intravenously injected with PBS, sLNPs‐OVA, sLNPs‐OVA/Cel (at mRNA dosages of 0.5 mg kg⁻¹), or sLNPs‐Cel. Spleens were excised 24 h post‐injection, and splenic cell subsets were labeled with antibodies against CD11c, CD40, CD80, CD86, MHC‐II, CD4, CD8, B220, and CD69. Flow cytometry was used to determine the proportions of CD40⁺, CD80⁺, CD86⁺, or MHC‐II⁺ cells within the CD11c⁺ DC population, as well as CD69 expression in CD4⁺ T cells, CD8⁺ T cells, and B220⁺ B cells. Data analysis was performed using FlowJo software (V10).

BMDCs were prepared as previously described.^[32^] Briefly, bone marrow cells were obtained from 6 to 8‐week‐old female mice on Day 0 and cultured in RPMI 1640 supplemented with 10% heat‐inactivated FBS, 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin, 55 µM β‐mercaptoethanol, and 20 ng mL⁻¹ GM‐CSF. After three days, an equal volume of complete medium with supplements was added, and on Day 7, suspended cells were used for experiments.

To assess the tolerogenic characteristics of BMDCs treated with sLNP‐OVA/Cel followed by LPS stimulation, the following procedures were performed: After the incubation period, the cells were harvested by scraping and then treated with FACS buffer (PBS containing 2% FBS). They were subsequently stained with antibodies against CD11c, CD40, CD80, CD86, IL‐10, and TGF‐β at 4 °C for 30 min. After staining, the cells were washed twice with FACS buffer and resuspended in FACS buffer using a BD flow cytometer, and the data were assessed using FlowJo V10.

Gene expression analysis in BMDCs treated with sLNPs‐OVA/Cel (or sLNPs‐OVA) was conducted using RNA sequencing. RNA samples were extracted and processed for sequencing at OE Biotech Co., Ltd. (Shanghai, China) using a BGI sequencer. The DESeq2 software was used for data analysis to identify differential gene expression patterns. Additionally, a portion of the extracted RNA samples was used to investigate the transcription levels of pro‐inflammatory and anti‐inflammatory cytokines.

Whole‐cell lysates from BMDCs treated with sLNP‐OVA/Cel followed by stimulation with LPS were analyzed for the activation of the NF‐κB signaling pathway. Protein samples were subjected to Western blotting by incubating with a 1:10 000 dilution of monoclonal antibodies against p65, phosphorylated p65 (pp65), IKKβ, or phosphorylated IKKβ (pIKKβ) overnight at 4 °C. Subsequently, donkey anti‐mouse or anti‐rabbit horseradish peroxidase‐conjugated secondary antibodies (1:5000 dilution) were used to detect the primary antibody signals. The blots were visualized using the Amersham ECL Western Blotting Detection Reagent and an Amersham Imager 600 system (both from GE Healthcare).

To assess the ability of whether celastrol addition in the mRNA‐LNPs vaccine platform could facilitate the differentiation of Tregs from naïve CD4⁺ T cells in vitro, splenocyte suspensions from 6 to 8‐week‐old female BALB/c mice were used to isolate CD4⁺ T cells via magnetic beads. BMDCs from 6 to 8‐week‐old female BALB/c mice were treated with different formulations for 4 h followed by stimulation with LPS for 24 h. These cells were then co‐cultured with splenic CD4⁺ T cells for 72 h. Non‐adherent cells were harvested and stained with anti‐mouse CD4, anti‐mouse Foxp3, IL‐10, and TGF‐β antibodies. Flow cytometry analysis was performed to quantify the differentiation of CD4⁺ T cells into Tregs induced by BMDCs treated with tolerogenic nano vaccines. Additionally, qRT‐PCR was employed to assess the mRNA expression levels of Foxp3, IL‐10, TGF‐β, and IFN‐γ in the non‐adherent Tregs.

The functional suppression assays of Tregs generated from sLNP‐OVA/Cel‐treated BMDCs were further explored. Specifically, splenic cells from normal mice were stained with CFSE and co‐incubated with Tregs generated from sLNP‐OVA/Cel‐treated BMDCs for 3 days. CFSE staining was performed to rule out Treg generated from sLNP‐OVA/Cel‐treated BMDCs. To assess the differentiation of naive CD4⁺ T cells into Th2 cell clusters, cells were restimulated with Cell Activation Cocktail (PMA/Ionomycin with Brefeldin A) for 4 h. After washing twice with PBS, FVD staining was performed at a 1:1000 dilution to exclude dead cells. Surface antibody staining was conducted at a 1:100 dilution against CD4, followed by intracellular IL‐4 staining after fixation and permeabilization.

To investigate the induction of Tregs, antigen‐specific Tregs, and memory T cells in vivo, BALB/c mice received intravenous injections of PBS, sLNPs‐OVA, sLNPs‐OVA/Cel, or sLNPs‐Luc/Cel (mRNA dosages of 0.5 mg kg⁻¹) on days 0 and 7. On day 14, spleen and lung tissues were collected. Erythrocytes were lysed using red blood cell lysis buffer, and splenic or lung cell subsets were labeled with antibodies against B220, CD4, and Foxp3, as well as the H‐2IAd/AAHAEINEA tetramer (OVA329‐337 tetramer). Additionally, splenic cell subsets were stained with CD4, CD8, CD44, and CD62L antibodies to analyze effector memory T cells and central memory T cells. Flow cytometry was performed to assess the Treg population (B220‐CD4⁺Foxp3⁺) and antigen‐specific Treg population (H‐2IAd/AAHAEINEA⁺/B220‐CD4⁺Foxp3⁺). Data analysis was performed using FlowJo software (V10).

BALB/c mice were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and housed in a specific pathogen‐free environment at the Experimental Animal Facility, Academy of Medical Science, Zhengzhou University. All animal procedures were approved by the First Affiliated Hospital of Zhengzhou University Animal Care and Use Committee (Protocol No. 2021‐KY‐0634‐001). To induce experimental allergic asthma, BALB/c mice were sensitized intraperitoneally with 50 µg of OVA with aluminum adjuvant once a week for two weeks. Two weeks after the final sensitization, mice were challenged intranasally with 20 µg of OVA daily for three consecutive days.

For the prophylactic studies in experimental asthma, BALB/c mice received intravenous injections of PBS, sLNPs‐OVA, sLNPs‐Luc/Cel, or sLNPs‐OVA/Cel (mRNA dosages of 0.5 mg kg⁻¹) on days 0 and 7. On day 14, mice were intraperitoneally sensitized with ovalbumin once a week for two weeks. Subsequently, mice were intratracheally challenged with OVA to induce asthma models for three consecutive days starting on day 35. Animals were euthanized 2 days after the final intratracheal challenge, and lung tissues were harvested post‐mortem. BALF was collected by lavaging the lungs with 1 mL of PBS supplemented with 2% newborn calf serum twice. BALF IgE levels and cytokine profiles were quantified using ELISA following the manufacturer's protocols.

In the therapeutic vaccination model following sensitization, 6‐8‐week‐old BALB/c mice received two doses of sLNP‐OVA, sLNP‐OVA/Cel, or sLNP‐Luc/Cel via intravenous injection once a week, commencing one day after two rounds of intraperitoneal sensitization with ovalbumin. Six days after the final immunization, mice were intratracheally challenged with OVA to induce asthma models for three consecutive days. Animals were euthanized 2 days after the last intratracheal challenge for subsequent analysis.

The lung tissues were harvested from each mouse at the end of the study. Then, half of each lung was embedded in paraffin and serially sectioned frontally at 5 µm thickness. Inflammatory cell infiltration was assessed using hematoxylin and eosin (H&E) staining, while mucus secretion was visualized with periodic acid‐Schiff (PAS) staining and photographed using a Slide Scan System SQS (TEKSQRAY). Additionally, infiltration of inflammatory monocytes was evaluated using anti‐CD11b primary antibody followed by fluorescence‐labeled secondary antibody staining. Similarly, the presence of Tregs was examined with anti‐Foxp3 primary antibody and fluorescence‐labeled secondary antibody staining. Imaging was performed using a Perkin Elmer Vectra machine. Furthermore, single‐cell suspensions of lung tissues were stained with CD45, CD11b, CD4, and Foxp3 antibodies and analyzed by flow cytometry.

BMDCs were seeded at a density of 1 × 10⁴ cells well⁻¹ and treated with varying concentrations of celastrol for 24 h at 37 °C and 5% CO2. Cell viability was assessed using a CCK8 kit. The cytotoxicity of sLNP‐OVA/Cel on BMDCs was evaluated similarly to the differentiation of tolerogenic DCs, also employing the CCK8 kit.

Organs, including the liver, spleen, lungs, kidneys and heart, were excised from BALB/c mice at the conclusion of OVA‐specific Treg immune response experiments. These organs were immediately fixed in PBS containing 4% paraformaldehyde for three days, embedded in paraffin, and sectioned transversely at 5 µm thickness. Inflammatory tissue reactions were examined using H&E staining. Blood samples were collected for measurement of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), albumin (ALB), and uric acid (UA) levels.

Quantitative data were presented as mean ± SEM unless otherwise specified. Statistical analyses of data from two or multiple groups were performed using two‐sided Student's t‐test or nonparametric one‐way ANOVA with Tukey's test, respectively, using GraphPad Prism 8.0. Statistical significance was indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and NS indicating no significant differences.