Therapeutic Potential of Arginine-Loaded Red Blood Cell Nanovesicles Targeting Obese Asthma

Abstract

Purpose: The role of the gut microbiomes has been emphasized in the pathogenesis of obese asthma (OA). However, the molecular mechanism of airway dysfunction underlying OA has not yet been fully elucidated. The effects of microbiomes on arginine metabolism in relation to lung functions and a novel method for delivering arginine to lung tissue based on arginine-loaded red blood cell (RBC)-derived nanovesicles (NVs) (NV^Arg) will be investigated. Materials and Methods: Inflammatory status, amino acid profiles, and microbial diversity were evaluated in 20 adult patients with OA compared to 30 adult patients with non-OA (NOA) and 10 healthy control (HC) groups. Changes in gut or lung microbial composition that altered arginine metabolism in relation to airway inflammation were investigated in an OA mouse model in vivo. Additionally, this study evaluated the delivery of arginine to lung tissue utilizing NV^Arg in vivo and in vitro. Results: Significantly increased Bacteroides abundance but decreased serum arginine concentration with lower forced exhaled volume at 1 s (FEV₁) (%) was noted in the OA group compared to the NOA and HC groups. In mouse experiments, when OA mice were given living bacteria from normal control (NC) mice, lung arginine concentration and airway resistance were restored. However, the administration of arginine or its metabolite (citrulline) did not increase the arginine levels in the lung tissues. We therefore created NV^Arg, which successfully delivered arginine into the cytoplasm of the airway epithelial cell line in vitro. Oral administration of NV^Arg for OA mice significantly induced the AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) pathways in airway epithelial cells, which reduced airway resistance and inflammation. Conclusion: These findings suggest that microbiomes contribute to airway dysfunction by regulating arginine metabolism, whereas NV^Arg treatment may be a potential option for managing OA.

Keywords: arginine; asthma; microbiome; nanovesicles; obesity.

Figure 1

Microbial diversity and composition in blood samples of the study subjects. (A) Chao1 diversity index. (B) Microbial composition at the phylum level. (C) Heatmap plot of the microbial communities at the genus level. (D and E) Correlations between the relative abundance of Bacteroides and arginine/citrulline concentration. (F) A positive correlation between arginine concentration and FEV₁%. The data are presented as the Pearson's correlation coefficient r (p value). Horizontal lines indicate median values, and whiskers indicate minimum to maximum values. p values were adjusted for age as a covariate using a generalized linear model. HC, healthy control; NOA, non-obese asthma; OA, obese asthma.

Figure 2

Functional gene profiles of the microbiome based on metagenomic analysis. (A) Heatmap plot of multiple genes associated with cellular processes, metabolism, and genetic information processing. (B) Evaluation of the relative abundance of microbial genes related to L-arginine biosynthesis and L-arginine degradation involved in arginine metabolism. (C–E) Genes contributing to L-arginine biosynthesis and L-arginine degradation as well as arginine, ornithine, and proline interconversion. Horizontal lines indicate median values, and whiskers indicate minimum to maximum values. p values were adjusted for age as a covariate using a generalized linear model. AST, arginine succinyltransferase; ATP, adenosine triphosphate; HC, healthy control; NOA, non-obese asthma; OA, obese asthma.

Figure 3

Role of the gut microbiome in arginine metabolism in obese asthma (OA). Live or heated bacteria were isolated from normal control (NC) mice and transplanted into mice with OA. (A) Changes in the body weight of mice. (B) Changes in airway resistance (R_L). (C) Arginine concentration in the lung tissues. (D) Correlations between airway resistance and arginine concentration in the lungs. The data are presented as the Pearson's correlation coefficient r (p value). (E) Phosphorylation of AMPK and eNOS. Horizontal lines indicate median values, and whiskers indicate minimum to maximum values. p values were determined by the one-way ANOVA with Bonferroni's post hoc test. AMPK, AMP-activated protein kinase; eNOS, endothelial nitric oxide synthase.

Figure 4

Effect of NV^Arg on airway epithelial cells to produce NO via the AMPK–eNOS pathway. (A) Schematic protocol of NV^Arg construction. (B) Transmission electron microscopic images of NV^Arg. Scale bar, 100 nm. (C) Size of NV^Arg measured using dynamic light scattering. (D) Protein band patterns of NV and NV^Arg. (E) Ratio of arginine to the total protein concentration of NV^Arg. (F) Confocal images of airway epithelial cells treated with or without NV^Arg-DiO. (G) Changes in cell viability after NV^Arg treatment. (H and I) The levels of IL-8 and nitric oxide produced by airway epithelial cells. (J) The expression of phosphorylated AMPK and eNOS in airway epithelial cells. Horizontal lines indicate median values, and whiskers indicate minimum to maximum values. p values were determined by the Mann–Whitney U test or by the one-way ANOVA with Bonferroni's post hoc test or by the Kruskal–Wallis with Dunn's post hoc test. Arg, arginine; DAPI, 4′,6-diamidino-2-phenylindole; eNOS, endothelial nitric oxide synthase; IL, interleukin; MPK, AMP-activated protein kinase; NV, RBC-derived nanovesicles; NV^Arg, arginine-loaded RBC-derived nanovesicles; NV^Arg-DiO, DiO-labeled NVArg; RBC, red blood cell; Sup, supernatant.

Figure 5

Efficacy of NV^Arg in mice with obese asthmatic mice compared to normal control mice. (A) Fluorescence assay for the detection of NV^Arg-Cy7 in dissected organs after oral administration. (B) The presence of NV^Arg-DiI in the lung tissues of mice. (C) Changes in airway resistance (R_L). (D) The levels of arginine in the lungs. (E) Nitric oxide production in the lungs. (F) Phosphorylation of AMPK and eNOS in the lungs. (G) The proposed mechanisms by which NV^Arg reduces airway resistance in OA via the AMPK–eNOS pathway with nitric oxide production. Horizontal lines indicate median values, and whiskers indicate minimum to maximum values. p values were determined by one-way ANOVA with Bonferroni's post hoc test or Kruskal–Wallis with Dunn's post hoc test. AMPK, AMP-activated protein kinase; Arg, arginine; DAPI, 4′,6-diamidino-2-phenylindole; eNOS, endothelial nitric oxide synthase; NV^Arg, arginine-loaded RBC-derived nanovesicles; NV^Arg-DiI, DiI-labeled NV^Arg; NVs, RBC-derived nanovesicles; RBC, red blood cell.