A20 mRNA therapeutics ameliorate systemic sclerosis by suppressing TRAF-6/NF-KB signaling and DREAM expression and exerting antifibrotic effects

Abstract

Introduction: Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by progressive fibrosis, vascular abnormalities, and immune dysregulation. Decreased expression of A20 (TNFAIP3), a key negative regulator of inflammation, has been shown to aggravate SSc pathogenesis by enhancing fibroblast activation and promoting collagen production. This study explores the therapeutic efficacy of A20 mRNA-lipid nanoparticle (LNP) delivery in restoring A20 expression and mitigating fibrosis through modulation of the TRAF6/NF-κB and Downstream Regulatory Element Antagonist Modulator (DREAM) -SMAD2 signaling pathways.

Methods: Human dermal fibroblasts were transfected with A20 mRNA -LNP and subsequently stimulated with transforming growth factor-beta (TGF-β). Protein expression levels and fibrotic markers were analyzed by Western blotting and quantitative PCR. In vivo, a bleomycin-induced mouse model of SSc received weekly intramuscular injections of A20 mRNA -LNP. The extent of fibrosis was assessed through histological analysis and immunohistochemistry.

Results: Transfection with A20 mRNA-LNP significantly suppressed TRAF6/NF-κB signaling and reduced fibrotic marker expression in vitro. In the SSc mouse model, A20 mRNA-LNP treatment markedly attenuated skin and lung fibrosis and decreased collagen deposition. Importantly, A20 overexpression led to downregulation of DREAM in vivo and inhibition of SMAD2 phosphorylation in vitro, indicating crosstalk between inflammatory and fibrotic pathways.

Discussion: A20 mRNA therapy effectively alleviates fibrosis by restoring A20 expression and inhibiting TRAF6/NF-κB signaling, while also downregulating DREAM, a previously unrecognized target. This dual-pathway regulation underscores the role of A20 and DREAM as central modulators of fibrotic progression. These findings highlight the potential of A20 mRNA-LNP as a novel therapeutic strategy for SSc, offering a multifaceted approach that may surpass current treatment options by simultaneously targeting interconnected pathogenic pathways.

Keywords: A20 (TNFAIP3); dream; fibrosis; immune cells; systemic sclerosis.

Figure 1

A20 mRNA-LNP Production and Expression In Vitro: Suppression of TRAF6-NF-κB Signaling and Fibrosis Markers in Skin Fibroblasts. (A) A20 mRNA was cloned into a linear mRNA expression construct, featuring a loop structure at the poly (A) tail. (B) A20 mRNA-LNP was formulated with four lipids, including the ionizable lipid SM-102. (C) Characterization of A20 mRNA-LNP, including particle size and zeta potential. Human dermal fibroblast (HDF) cells were transfected with either empty mRNA or A20 mRNA and serum-starved for 16–18 h (D, E) A20, TRAF6, pNF-κB, NF-κB, Col1, αSMA protein levels were measured in HDF cells treated with TGF-β (20 ng/mL) for 48 h GAPDH served as the loading control. Bar graphs show the pooled results of three independent experiments, with data presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA (D, E) (*p < 0.05; ns, not significant). (F) mRNA levels of Col1A1 and αSMA in HDFs. Human dermal fibroblasts (HDFs) were transfected with the indicated A20 mRNAs and, stimulated with TGF-β (20 ng/mL) for 24 hours. Total RNA was extracted, and Col1A1 and αSMA mRNA levels were analyzed by real-time PCR. (G, H) Protein expression of A20, p53, and c-Myc in Human dermal fibroblasts (HDFs). HDFs were treated with Actinomycin D (5 μg/mL) and subsequently transfected with the indicated A20 mRNAs. Four hours post-transfection, cells were stimulated for an additional 24 hours. Representative western blots are shown. Bar graphs summarize pooled results from five independent experiments and are presented as mean ± SD (n = 5). Statistical significance was determined by Mann-Whitney test (F–H) (****p < 0.0001; **p < 0.01; *p < 0.05; ns, not significant).

Figure 2

A20 mRNA Suppressed Immune Cell Activation and Skin and Lung Fibrosis in a Mouse Model of Systemic Sclerosis (SSc). (A) A murine model of SSc was generated as described in the schematic. Mice were injected subcutaneously (S.C.) with bleomycin daily for 3 weeks to induce fibrosis. Mice received a total of seven intramuscular (I.M.) injections of A20 mRNA-LNP (n = 5) or vehicle control (n = 5), administered once or twice weekly over 6 weeks. All mice were sacrificed at week 6 for subsequent analyses. (B) At the 6-week sacrifice time point, the frequency of Th2 (IL-4+ CD4+) and Th17 (IL-17+ CD4+) cells in the spleen was analyzed by flow cytometry. (C) Immunofluorescence analysis of CD4⁺ T cell subsets in skin and lung tissues. Tissue sections from WT mice, bleomycin-induced fibrotic mice treated with vehicle, and bleomycin-induced fibrotic mice treated with A20 mRNA-LNP were stained for CD4 (green), IL-4 or IL-17 (red), and nuclei (DAPI, blue). Representative confocal images are shown (scale bar, 50 μm). Quantification of CD4⁺IL-4⁺ and CD4⁺IL-17⁺ cells per high-power field (HPF) is shown in the adjacent bar graphs. Original magnification: 200×; scale bar: 50 μm. (D, E) Representative skin sections stained with hematoxylin and eosin (H&E) showing dermal thickening, Masson’s Trichrome (MT) demonstrating collagen deposition, and Sirius Red highlighting fibrotic area. Dermal thickness and percentage of fibrotic area were quantified. (F, G) Representative lung sections stained with H&E showing fibrotic morphology and alveolar wall thickening, MT demonstrating collagen deposition, and Sirius Red highlighting fibrotic area. Fibrotic area was quantified accordingly. Original magnification: 200×; scale bar: 100 μm. (H) Hydroxyproline levels were measured in skin (top) and lung (bottom) tissues from WT mice, bleomycin-induced fibrotic mice treated with vehicle, and bleomycin-induced fibrotic mice treated with A20 mRNA-LNP. For quantification, three fields per mouse were analyzed (n = 5 mice per group). Data are presented as mean ± SD from one representative experiment of three independent experiments. The statistical significance of all graphs was determined by one-way ANOVA (B–H). (*p < 0.01; p < 0.05; ns, not significant).

Figure 3

A20 mRNA Therapy Inhibited TRAF6/NF-κB Signaling and TGF-β Cytokine Expression in Skin and Lung Tissues from Systemic Sclerosis. Skin and lung tissues collected at the 6-week sacrifice time point were analyzed. (A) Skin sections were immunohistochemically stained for A20, TRAF6, NF-κB, and TGF-β. (B) Lung sections were stained in parallel with the same antibodies to evaluate A20 expression and fibrotic signaling molecules. For quantification, three fields per mouse were analyzed (n = 5 mice per group), and the number of antibody-positive cells was presented as mean ± SD. Original magnification: 400×; scale bar: 100 μm. Bar graphs represent data from one of three independent experiments, with statistical significance determined by one-way ANOVA (*P < 0.05, **P < 0.01; (A, B).

Figure 4

A20 mRNA Therapy Suppressed the Expression of DREAM, a Known A20 Inhibitory Molecule, and Reduced SMAD2, αSMA, and Procollagen in Systemic Sclerosis Skin Tissue and Human Skin Fibroblasts. (A) Skin and lung sections were stained with antibodies against DREAM, SMAD2, procollagen, and αSMA. The number of antibody-positive cells (mean ± SEM) is plotted. Original magnification: 400×; scale bar: 100 μm. (B) The protein levels of DREAM and SMAD2 were measured in cells treated with TGF-β (20 ng/mL) for 48 h GAPDH served as the validation control. For quantification, three fields per mouse were analyzed (n = 5 mice per group). Data are presented as mean ± SD from a representative experiment of three independent experiments. Statistical significance was determined by one-way ANOVA (A, B). (*P < 0.05; **P < 0.01).

Figure 5

A20 mRNA Therapy Reduces Fibrosis in Systemic Sclerosis Lung Tissue by Inhibiting DREAM Expression. Lung sections were stained with antibodies against DREAM, SMAD2, procollagen, and αSMA. For quantification, three fields per mouse were analyzed (n = 5 mice per group), and the number of antibody-positive cells is plotted as mean ± SEM. Original magnification: 400×; scale bar: 100 μm. Bar graph shows data from one representative experiment (**P < 0.01). The statistical significance of all graphs was determined by one-way ANOVA (A). The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/bAoEK8.

Figure 6

Proposed mechanism by which A20 mRNA therapy attenuates fibrosis in systemic sclerosis. In the systemic sclerosis condition (left), activation of TLR4 initiates TRAF6-mediated NF-κB signaling and upregulates TGF-β, IL-8, collagen type I (Col1), and α-smooth muscle actin (α-SMA), thereby promoting fibroblast activation and extracellular matrix (ECM) accumulation. In contrast, A20 mRNA encapsulated in lipid nanoparticles (LNPs) (right) restores A20 protein expression, leading to inhibition of TRAF6-dependent NF-κB phosphorylation, suppression of DREAM and SMAD pathways, and subsequent reduction of fibrotic markers in skin and lung fibroblasts. Downward arrows indicate decreased expression following A20 mRNA treatment. The schematic was created with BioRender.com.