Edible exosome-like nanoparticles from portulaca oleracea L mitigate DSS-induced colitis via facilitating double-positive CD4+CD8+T cells expansion

Abstract

Plant-derived exosome-like nanoparticles (PDENs) have been paid great attention in the treatment of ulcerative colitis (UC). As a proof of concept, we isolated and identified Portulaca oleracea L-derived exosome-like nanoparticles (PELNs) from edible Portulaca oleracea L, which exhibited desirable nano-size (~ 160 nm) and a negative zeta potential value (-31.4 mV). Oral administration of PELNs effectively suppressed the expressions of pro-inflammatory cytokines (TNF-α, IL-6, IL-12, and IL-1β) and myeloperoxidase (MPO), increased levels of the anti-inflammatory cytokine (IL-10), and alleviated acute colitis in dextran sulfate sodium (DSS)-induced C57 mice and IL-10^-/- mice. Notably, PELNs exhibited excellent stability and safety within the gastrointestinal tract and displayed specific targeting to inflamed sites in the colons of mice. Mechanistically, oral administration of PELNs played a crucial role in maintaining the diversity and balance of gut microbiota. Furthermore, PELNs treatment enhanced Lactobacillus reuteri growth and elevated indole derivative levels, which might activate the aryl-hydrocarbon receptor (AhR) in conventional CD4⁺ T cells. This activation downregulated Zbtb7b expression, leading to the reprogramming of conventional CD4⁺ T cells into double-positive CD4⁺CD8⁺T cells (DP CD4⁺CD8⁺ T cells). In conclusion, our findings highlighted the potential of orally administered PELNs as a novel, natural, and colon-targeted agent, offering a promising therapeutic approach for managing UC. Schematic illustration of therapeutic effects of oral Portulaca oleracea L -derived natural exosome-like nanoparticles (PELNs) on UC. PELNs treatment enhanced Lactobacillus reuteri growth and elevated indole derivative levels, which activate the aryl-hydrocarbon receptor (AhR) in conventional CD4⁺ T cells leading to downregulate the expression of Zbtb7b, reprogram of conventional CD4⁺ T cells into double-positive CD4⁺CD8⁺T cells (DP CD4⁺CD8⁺ T cells), and decrease the levels of pro-inflammatory cytokines.

Keywords: Double-positive CD4+CD8+T; Lactobacillus reuteri; Portulaca oleracea L-derived exosome-like nanoparticles; Ulcerative colitis.

Fig. 1

Oral administration of PELNs protects mice against DSS-induced colitis. a, Protocol for DSS-induced colitis and PELNs administration; b, Changes of body weight over time, normalized to the percentage of the day-zero body weight; c, Disease activity index (DAI); d, e, Colon length; f, Histological scores; g, H&E-stained colon sections. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001

Fig. 2

Pro-inflammatory cytokines and anti-inflammatory cytokine expression profiles. a, b, c, d, e, qRT-PCR detecting the levels of IL-6, IL-12, IL-1β, TNF-α and IL-10 in colonic samples; f, g, h, i, j, k, ELISA testing the expression profiles of IL-6, IL-12, IL-1β, TNF-α, IL-10 and MPO in blood serum. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001

Fig. 3

Oral administration of PELNs protects IL-10^−/− mice spontaneous colitis. a, Changes of body weight; b, Disease activity index (DAI); c, Colon length; d, Histological scores; e, H&E-stained colon sections

Fig. 4

In vivo distribution of PELNs. Fluorescence images of the gastrointestinal tract revealing in vivo bio-distribution of orally administered IRDye 800CW-labeled PELNs at different time points (3, 6, 12, and 24 h) in acute colitis mice (a) and health mice (b)

Fig. 5

PELNs alters diversity of gut microbiota in mice models of colitis. a, Upset Polt of OTUs; b, ACE index; c, Chao 1 index; d, PD-tree index; e, Simpson index; f, Shannon index; g, PCA (Principal Component Analysis); h, PCoA (principal co-ordinate analysis). Sum of PCoA1 and PCoA2 more than 50%, indicating significant difference; i, NMDS (Non-metric Multi-Dimensional Scaling). Stress value less than 0.1, indicating significant difference. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

PELNs alters gut microbiota structures across different levels in mice with colitis. a, Stacked bar plot depicts the structure of gut microbiota in each group of mice. Left to right: phylum, family, genus, and species; b-e, Welch’s t test analysis of PELNs mediated differential microbial changes at the phylum, family, genus, and species level

Fig. 7

PELNs inducing the differentiation of DP CD4⁺CD8⁺T cells. a, Schematic representation of mechanisms for Lactobacillus reuteri metabolizing tryptophan (L-Trp) into indole derivatives; b, PELNs promote the growth of Lactobacillus reuteri; c, PELNs treatment significantly increased the levels of indole derivatives; d, e, PELNs treatment significantly decreased the levels of Zbtb7b protein using WB and IHC assay; f, Schematic representation for the derivatives of Lactobacillus reuteri activating the aryl hydrocarbon receptor (AhR) in conventional CD4⁺ T cells, and leading to the downregulation of Zbtb7b expression and the reprogramming of conventional CD4⁺ T cells into DP CD4⁺CD8⁺ T cells; g, Flow cytometry detecting the population of DP CD4⁺CD8⁺ T cells in colonic samples; h, Represent immunofluorescence showing the population of DP CD4⁺CD8⁺ T cells in colonic samples. Data are representative FCM images or expressed as the mean ± SEM of each group. *P < 0.05, **P < 0.01

Fig. 8

Biosafety of orally administered PELNs. a, H&E-stained of vital organs (heart, liver, spleen, lung, and kidney) in control, DSS, PELNs-L, and PELNs-H group; b, c, Liver function; d, e, Kidney function. ns p > 0.5