Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: The fabrication of Gel@IVNMs, in vivo application and immunomodulatory mechanism for myocardial infarction (MI) therapy. (A) The stepwise fabrication of the PPM@rapamycin (Rapa) inverse vaccine nanomimics (IVNMs), Gel, and Gel@IVNMs. First, PPMal, PPMan, and Rapa are assembled into the IVNMs via the solvent evaporation method. Concurrently, oDex and gelatin are cross-linked to form a hydrogel network, where IVNMs are encapsulated. (B) The hydrogel is injected into the infarcted myocardium. As the Gel swells, cardiac lysate antigens (CLAs) released by necrotic cardiomyocytes diffuse into it and are captured by Mal on the surface of IVNMs to form antigen-loaded IVNMs (AIVNMs). Lactic acid accumulates at the infarcted site due to the activation of inflammation, followed by a decrease in pH, leading to a mild acidic microenvironment. In response to the mildly acidic pH, the Gel gradually degrades and releases AIVNMs. (C) Released AIVNMs can be taken up by CD206-expressing immune cells, including dendritic cells (DCs), and subsequently modulate DC phenotype by suppressing the mechanistic target of rapamycin (mTOR) pathway (via Rapa-mediated inhibition of mTOR–Rictor–FKBP12 signaling), reducing CD80 and CD86 expression, and increasing programmed death ligand 1 (PD-L1) expression and transforming growth factor-β (TGF-β) secretion. These DCs then present antigens and suppress T cell activation through PD-L1 signaling and TGF-β secretion, fostering regulatory T cell (Treg) expansion. Tregs further modulate macrophage polarization, shifting proinflammatory M1 macrophages toward anti-inflammatory M2 macrophages, thereby balancing proinflammatory and anti-inflammatory cytokine secretion. The established immune tolerance memory can rapidly respond and specifically inhibit inflammatory activation upon restimulation with CLAs. This cascade reduces cardiomyocyte apoptosis, promotes neovascularization, ameliorates myocardial fibrosis, and ultimately enhances heart pumping function, contributing to improved cardiac repair after MI.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: In Vivo, Solvent, Evaporation, Injection, Activation Assay, Expressing, Inhibition

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: The effect of mannose functionalization on the uptake of inverse vaccine nanomimics (IVNMs) by dendritic cells (DCs) in vitro, together with DC phenotypes and cytokine secretion after stimulation with cardiac lysate antigens (CLAs). (A) DC isolation and experimental workflow. The process by which IVNMs capture antigens and are internalized by DCs. After antigen-loaded IVNMs (AIVNMs) are internalized by DCs, the antigens activate mechanistic target of rapamycin (mTOR) signaling, whereas Rapa inhibits mTOR activation, which leads to decreased expression of molecules such as CD80 and CD86, and increased expression of programmed death ligand 1 (PD-L1). Additionally, the secretion of anti-inflammatory factors (such as interleukin-10 [IL-10] and transforming growth factor-β [TGF-β]) is enhanced. (B) Flow cytometry results for the uptake of IVNMs by DCs. (C) Quantification of the uptake of IVNMs by DCs. (D) Fluorescence images of the uptake of IVNMs by DCs, with red fluorescence indicating IVNMs and blue fluorescence indicating cell nuclei. (E and F) Quantification of DC phenotypes by flow cytometry. (G) Representative flow cytometry plots of DC phenotypes. (H) Enzyme-linked immunosorbent assay detection results of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β expression levels in DCs after CLA stimulation and drug administration. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: In Vitro, Isolation, Activation Assay, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Phenotypic changes and inflammatory factor secretion of T cells stimulated by cardiac lysate antigens (CLAs). (A) Coculture protocol for dendritic cells (DCs) and T cells. The tolerogenic DCs (tDCs) present antigens to T cells; meanwhile, programmed death ligand 1 binds to programmed cell death protein 1 on T cells and inhibits T cell activation, promoting the phenotypic conversion of T cells to regulatory T cells (Tregs). Furthermore, due to the lack of activation signals such as CD80 and CD86, some T cells exhibit a weak activation state after receiving antigen presentation, and under the regulation of key factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), they undergo phenotypic transition to Tregs. Under restimulation, tDCs phagocytose antigens and present them to Tregs. Tregs are rapidly activated, express high levels of Forkhead box P3 (FOXP3), and directly inhibit T cell activation, while secreting large amounts of anti-inflammatory factors. (B) Flow cytometry (FC) results for T cell phenotype. (C) Quantification of T cell phenotypes by FC. (D) T cell phenotype immunofluorescence staining results, where blue represents cell nuclei, red represents CD127, and green represents CD4. (E) Expression of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β after coculture of DCs and T cells. (F) Western blot analysis of the activation status of the mechanistic target of rapamycin (mTOR)-p70S6K-S6 pathway after treatment with Gel@PPM, Rapa, inverse vaccine nanomimics (IVNMs), and Gel@IVNMs under CLA stimulation. (G) The percentage of phosphorylation of mTOR, p70S6K, and S6 was statistically analyzed based on the gray values of the western blot results (compared to the unphosphorylated proteins). Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: Activation Assay, Immunopeptidomics, Flow Cytometry, Immunofluorescence, Staining, Expressing, Western Blot, Analysis, Phospho-proteomics

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Changes in myocardial macrophage phenotypes and inflammatory cytokine profiles after myocardial infarction (MI). (A) Representative immunofluorescence images of heart sections from the Control, Gel@PPM, Gel@PP@Rapa, Gel@OVA-IVNMs, and Gel@IVNMs groups at 7 and 56 d after MI. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), macrophages with CD68 (green), proinflammatory macrophage-associated inducible nitric oxide synthase (iNOS) with red fluorescence, and reparative/immunomodulatory macrophage-associated CD206 with purple fluorescence. Scale bars, 200 μm. (B) Quantification of iNOS- and CD206-positive signals in myocardial sections at 7 and 56 d after MI. (C) Enzyme-linked immunosorbent assay quantification of interleukin-2 (IL-2), interferon-γ (IFN-γ), transforming growth factor-β (TGF-β), and IL-10 levels in myocardial tissue at 7 and 56 d after MI. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Tissue