Lactobacillus plantarum-derived extracellular vesicles from dietary barley leaf supplementation attenuate Citrobacter rodentium infection and intestinal inflammation | Journal of Nanobiotechnology

BL attenuates intestinal pathology in C. rodentium-infected mice

To evaluate whether BL conferred protective effects against C. rodentium-induced colonic inflammation, mice were fed with a standard chow diet or an isocaloric diet supplemented with BL for 3 weeks and inoculated or not with C. rodentium (Fig. 1A). Food consumption and water intake were monitored daily prior and during the infection. Our results showed that there was no significant difference in food intake or water intake between BL- and chow diet (CD)-fed mice, suggesting that BL supplementation had no adverse effect on the mouse eating and drinking habits (Figure S1). Compared to control uninfected mice, mice infected with C. rodentium exhibited significant body weight loss and increased disease activity index (DAI) score (Fig. 1B and C). The colon length was shorter and the spleen weight was greater in C. rodentium-infected mice compared to control uninfected mice (Fig. 1D and E; Figure S2). Nevertheless, the above deleterious effects caused by C. rodentium were attenuated in BL-fed mice (Fig. 1B-E). Histological analysis of colonic sections showed that C. rodentium infection led to pathologic changes, such as colonic thickening, epithelial injury, and inflammatory cell infiltration; these histologic damages were remarkably attenuated in BL-fed mice (Fig. 1F and G; Figure S2). We next analyzed the effect of BL on colonic hyperplasia, a hallmark of C. rodentium infection [15]. The number of colonic Ki67⁺ cells was dramatically reduced in C. rodentium-infected mice fed with BL (Fig. 1F and H).

In addition to assessing pathological alterations in the colonic epithelium, the effect of BL on inflammatory factors was also examined. Compared to control uninfected mice, C. rodentium-infected mice showed significantly increased expression of Ifnγ, Il1β, and Tnfα in the colon at day 10 after infection; while BL downregulated these inflammatory cytokines but had no effect on the mRNA level of IL-4 (Fig. 1I). Correspondingly, the elevated levels of IFN-γ, IL-1β, and TNF-α caused by C. rodentium infection were mitigated in BL-fed mice (Fig. 1J). Moreover, BL reduced the upregulated expression of CD4⁺ T cell marker CD4 and macrophage marker F4/80 in the colon of C. rodentium-infected mice, suggesting that BL suppresses C. rodentium-induced inflammatory responses (Fig. 1K). Collectively, these findings suggest that BL alleviates colonic injury and improves intestinal inflammation in C. rodentium-infected mice.

Barley leaf (BL) attenuates C. rodentium (CR)-induced colitis. (A) Study design of in vivo mouse experiment. Mice were fed with a standard chow diet (CD) or an isocaloric BL-supplemented diet for 3 weeks prior to the infection with CR for 10 days. (B) Body weight change. (C) Disease activity index (DAI) scores. (D) Colon length. (E) Spleen weight. (F) Representative hematoxylin and eosin (H&E) staining images of colon tissues. Scale bar, 100 μm. Representative Ki67-stained immunofluorescence images of colon tissues. Scale bar, 50 μm. (G) Histological scores. (H) Quantification of Ki67 positive cells. (I) The mRNA expression of Ifnγ, Il1β, and Tnfα in the colon. (J) The levels of IFN-γ, IL-1β, and TNF-α in the colon. (K) The mRNA expression of CD4 and F4/80 in the colon. In (B) – (H), (n = 8 per group). In (I) – (K), (n = 6 per group). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. For body weight change, a repeated measure two-way analysis of variance (ANOVA) was performed and the rest of the statistics were performed with one-way ANOVA followed by Tukey’s multiple comparison’s test

BL attenuates pathogen colonization and improves gut barrier function

After the oral gavage, C. rodentium initially colonizes the cecum and then migrates to the colon [17]. The severity of intestinal pathology during infection is primarily driven by colonization and virulence potential. To investigate whether BL attenuated C. rodentium colonization during infection, we measured the pathogen burden in C. rodentium-infected mice. BL supplementation significantly reduced the fecal C. rodentium loads on days 1, 4, 7, and 10 post-infection (Fig. 2A). Consistently, the bacterial counts in cecum content were significantly reduced in mice fed with BL on day 10 post-infection (Fig. 2B). Notably, the bacterial levels of C. rodentium in spleen and liver were also significantly lower in BL-fed mice than that in mice fed the control diet (Fig. 2C and D), suggesting that the systemic dissemination of enteric pathogen is mitigated by BL.

We speculated that BL might improve intestinal barrier function to inhibit the systemic translocation of C. rodentium. To test this hypothesis, we used real-time PCR to detect the expression of genes encoding tight junction proteins. We found that the mRNA expression of Claudin1, Claudin2 and ZO-1 was lower in C. rodentium-infected mice compared to control uninfected mice; while the expression of these genes was significantly elevated in mice fed with BL (Fig. 2E). Alcian blue staining revealed a reduction of mucus-producing goblet cells in the colon of C. rodentium-infected mice compared to that in control uninfected mice; whereas BL markedly ameliorated the loss of goblet cells caused by C. rodentium (P < 0.05) (Fig. 2F and G).

The transcriptional activation of Lee pathogenicity island encoding T3SS is required for the colonization of C. rodentium [16]. The LEE-encoded T3SS enables pathogens to inject their effectors into colonocytes [16]. We further examined the impact of BL on Lee-encoded virulence factors. As expected, the expression of EspA, Map, and Tir was increased in colon and cecum of C. rodentium-infected mice (Fig. 2H and I). However, these effects were significantly attenuated in BL-fed mice (Fig. 2H and I). In addition, BL supplementation significantly abrogated the reduced colonic expression of chloride anion exchanger Sla26a3 and electrolyte absorption protein Car4 in C. rodentium-infected mice (Fig. 2J). These results indicate that BL improves gut barrier function to prevent C. rodentium colonization and attenuates infection.

Barley leaf (BL) reduces the bacterial burden and improves the gut barrier function in C. rodentium (CR)-infected mice. (A) Number of CR in feces on days 1, 4, 7, and 10 post-infection. (B) Number of CR in cecum content on day 10 post-infection. (C) Number of CR in the spleen on day 10 post-infection. (D) Number of CR in liver on day 10 post-infection. (E) Real-time polymerase chain reaction (PCR) assay for the expression of Occludin, Claudin1, Claudin2, and ZO-1 in the colon. (F) Representative images of alcian blue-stained colonic sections. Scale bar, 100 μm. (G) Quantification of mucus­producing goblet cells. (H) Real-time PCR assay for the expression of EspA, Map, and Tir in the colon. (I) Real-time PCR assay for the expression of EspA, Map, and Tir in the cecum. (J) Real-time PCR assay for the expression of Car4 and Slc26a3 in the colon. (n = 8 per group). Data are mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. For (E), (G), and (J), statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison’s test, and the rest of the statistics were performed with Student’s t-test

BL modulates T cell immune response pathway

To further dissect the mechanisms underlying the beneficial effect of BL, we performed a genome-wide transcriptome sequencing analysis on mouse colonic tissues. There were significant differences in mRNA gene expression profiles among the groups as revealed by PCA analysis and heat map of differentially expressed genes (DEGs) (Fig. 3A; Figure S3). Venn analysis showed that there were 2063 DEGs overlapped between CD group vs. CD + C. rodentium group and CD + C. rodentium group vs. BL + C. rodentium group (Fig. 3B). Volcano plot revealed that a total of 3841 (1815 upregulated genes and 2026 downregulated genes) and 3001 (1647 upregulated genes and 1354 downregulated genes) genes differed significantly in CD versus CD + C. rodentium group and in CD + C. rodentium versus BL + C. rodentium group (P value < 0.05 and Fold change > 2), respectively (Fig. 3C). Notably, Th1/Th2 cell differentiation and Th17 cell differentiation were identified as the top significantly enriched KEGG pathways (Fig. 3D). Heat map analysis of DEGs on these pathways showed that C. rodentium treatment resulted in an increased expression of Jag2, Rela, Cd247, Cd4, Lck, Cd3e, Jak3, Cd3d, Lat, H2-Ea, H2-Aa, H2-Ab1, H2-Eb1, H2-DMb2, H2-DMa, H2-DMb1 and Ifng, and a decreased expression of Gata3, Mapk12 and Mapk10, and this was effectively reversed by BL (Fig. 3E). The interaction network of key genes in pathways of Th1/Th2 and Th17 cell differentiation was depicted by STRING analysis (Fig. 3F). Furthermore, real-time PCR was used to verify the expression of DEGs and the results were consistent with the transcriptome sequencing data (Fig. 3G). These data suggest that T cell immune response may contribute to the attenuated colitis in C. rodentium-infected mice fed with BL.

RNA sequencing analysis of the gene expression profiles in colonic tissues. (A) Principle component analysis (PCA) of transcriptional profiling. (B) Venn analysis of the differentially expressed genes (DEGs). (C) Volcano plot of the DEGs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs. (E) Heat map of DEGs in Th1/Th2 cell differentiation signaling pathway. Genes with fold changes of > 1.5 and P adjust (Padj) of < 0.05 were considered to be differentially expressed. (F) Search tool for recurring instances of neighboring genes (STRING) network visualization of the DEGs in the Th1/Th2 cell differentiation signaling pathway. (G) Real-time PCR assay for the DEGs in Th1/Th2 cell differentiation signaling pathway (n = 6 per group). Data are mean ± SEM. *P < 0.05 and **P < 0.01. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison’s test

BL attenuates C. rodentium infection in a CD4⁺ T cell-dependent manner

Previous studies have shown that adaptive T cell immune response is essential for defending against pathogen infection [19, 20]. However, it is unclear whether CD4⁺ T cells were involved in protective effect of BL against C. rodentium infection. To test the involvement of CD4⁺ T cells in beneficial effect of BL, the anti-CD4 antibody was administered prior and during C. rodentium infection (Fig. 4A). Consistent with previous results, mice fed with BL protected against C. rodentium-induced body weight loss, increased DAI scores, colon shortening, and splenomegaly; notably, this was not seen in BL-fed mice treated with anti-CD4 antibody (Fig. 4B-E). Furthermore, C. rodentium-induced colonic epithelial damage and mucosal barrier dysfunction were alleviated in mice fed with BL; while this was not observed when mice were treated with anti-CD4 antibody (Fig. 4F-H). The relieving levels of IFN-γ and IL-1β in the colon were also not observed in BL-fed mice treated with anti-CD4 antibody (Fig. 4I). Consistently, BL failed to reduced C. rodentium loads in feces and liver in mice treated with anti-CD4 antibody (Fig. 4J and K). Together, these results demonstrate that CD4⁺ T cells play a crucial role in modulating BL-mediated protection against C. rodentium infection.

Barley leaf (BL) protects against C. rodentium (CR)-induced colitis in a CD4⁺ T cell-dependent manner. (A) Study design of in vivo mouse experiment. Mice were fed with a standard chow diet (CD) or an isocaloric BL-supplemented diet for 2 weeks prior to the infection with CR for 10 days. For clearance of CD4⁺ T cells, mice were injected intraperitoneally with an anti-CD4 monoclonal antibody or its isotype control antibody once every three days at 500 µg each time for three times before and during CR infection. (B) Body weight change. (C) Disease activity index (DAI) scores. (D) Colon length. (E) Spleen weight. (F) Representative hematoxylin and eosin (H&E) staining images of colon tissues. Scale bar, 100 μm. Representative images of alcian blue-stained colonic sections. Scale bar, 100 μm. (G) Histological scores. (H) Quantification of mucus­producing goblet cells. (I) The levels of IFN-γ, IL-1β, and TNF-α in the colon. (J) Number of CR in feces on days 1, 4, and 7 post-infection. (K) Number of CR in liver on day 7 post-infection. n = 8 per group. Data are mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. For body weight change, a repeated measure two-way analysis of variance (ANOVA) was performed and the rest of the statistics were performed with one-way ANOVA followed by Tukey’s multiple comparison’s test

BL prevents C. rodentium-induced T cell dysregulation

To investigate the effect of BL on T cell immune response, the different CD4⁺ T cell subsets, including Th1 cells (CD4⁺IFN-γ⁺), Th2 cells (CD4⁺IL-4⁺), Th17 cells (CD4⁺IL-17 A⁺) and T regulatory cells (Tregs) (CD4⁺CD25⁺Foxp3⁺), were analyzed by flow cytometry (Figure S4 and Figure S5). Compared to control uninfected mice, C. rodentium infection led to significant expansion of CD4⁺IFN-γ⁺ T cells in mesenteric lymph nodes (P = 0.016), which was attenuated by BL (P = 0.017) (Fig. 5A and B). Consistently, the population of CD4⁺IFN-γ⁺ T cells in the spleen was also significantly increased in C. rodentium-infected mice (P < 0.001), but was reduced in mice fed with BL (P < 0.01) (Fig. 5F and G). However, there was no significant difference in percentage of Th2, Th17, and Tregs between CD + C. rodentium and BL + C. rodentium groups (Fig. 5C-E and H-J). Collectively, these data demonstrate that BL attenuates Th1 immune response, which corresponds with attenuated inflammation and intestinal pathology in C. rodentium-infected mice.

Flow cytometric analysis of CD4⁺ T cell subsets in mouse mesenteric lymph node and spleen. (A) Representative flow cytometry profile of CD4⁺IFN-γ⁺ cells, CD4⁺IL-4⁺ cells, CD4⁺IL-17 A⁺ cells and CD4⁺CD25⁺Foxp3⁺ cells in mouse mesenteric lymph node. (B) Quantification of CD4⁺IFN-γ⁺ cells. (C) Quantification of CD4⁺IL-4⁺ cells. (D) Quantification of CD4⁺IL-17 A⁺ cells. (E) Quantification of CD4⁺CD25⁺Foxp3⁺ cells. (F) Representative flow cytometry profile of CD4⁺IFN-γ⁺ cells, CD4⁺IL-4⁺ cells, CD4⁺IL-17 A⁺ cells and CD4⁺CD25⁺Foxp3⁺ cells in mouse colon. (G) Quantification of CD4⁺IFN-γ⁺ cells. (H) Quantification of CD4⁺IL-4⁺ cells. (I) Quantification of CD4⁺IL-17 A⁺ cells. (J) Quantification of CD4⁺CD25⁺Foxp3⁺ cells. n = 6 per group. Data are mean ± SEM. **P < 0.01 and ***P < 0.001. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison’s test

BL protects against gut dysbiosis in C. rodentium-infected mice

The gut microbiota plays an essential role in resistance and clearance of C. rodentium [22, 23]. To explore the modulatory effect of BL on the gut microbiota, fecal bacterial composition was evaluated utilizing Illumina sequencing of the V3-V4 region of 16 S rRNA genes. Alpha diversity analysis with Chao1 and Shannon index showed that C. rodentium treatment led to a reduction in gut microbial richness and diversity, which was abolished by BL supplementation (Fig. 6A and B). Partial Least Squares Discriminant Analysis (PLS-DA) of the microbial community showed that there were distinct group-based clustering patterns among the different treatment groups (Fig. 6C). The abundances of predominant phyla and genus were further compared. At the phylum level, the relative abundance of Proteobacteria was increased in C. rodentium-infected mice and was reduced with BL supplementation (Fig. 6D; Figure S6). Further, we observed an overgrowth of Citrobacter in C. rodentium-infected mice, whereas the relative abundance of Lactobacillus was increased in C. rodentium-infected mice fed with BL (Fig. 6E; Figure S6). Correspondingly, Wilcoxon rank-sum test confirmed that BL supplementation induced a significant increase in bacteria belonging to the Lactobacillus genus in both C. rodentium-infected (P < 0.05) or -uninfected (P < 0.05) mice (Fig. 6F).

Next, spearman correlation analysis was performed to identify the bacteria that was responsible for BL-mediated protection against C. rodentium enteric infection. We found that Lactobacillus showed strong negative correlations with DAI, IL-1β, TNF-α, and histological score; while Citrobacter displayed a strong positive association with these pathologic parameters (Fig. 6G). We next assessed whether the impact of BL on gut microbiota was associated with changes in the CD4⁺IFN-γ⁺ T cells during C. rodentium infection. We found that the population of CD4⁺IFN-γ⁺ T cells was positively correlated with Citrobacter and negatively correlated with Lactobacillus (Figure S7).

Short-chain fatty acids (SCFAs) serve as critical microbial metabolites of SCFAs-producing bacteria such as Lactobacillus in modulating the gut homeostasis [31,32,33,34]. We found that the fecal levels of acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were reduced in C. rodentium-infected mice compared to control uninfected mice. However, the levels of SCFAs, which were negatively correlated with colitis-related indexes, were significantly elevated in BL-fed mice (Fig. 6H; Figure S8). Together, these findings suggest that the protective effect of BL against C. rodentium-induced immune dysregulation is closely linked to improvement of gut microbiota dysbiosis.

Barley leaf (BL) prevents C. rodentium (CR)-induced gut microbiota dysbiosis. (A) Chao1 index. (B) Shannon index. (C) Partial least squares-discriminant analysis (PLS-DA) score plots of the gut microbiota composition. (D) Taxonomic distributions of gut bacterial composition at the phylum level. (E) Taxonomic distributions of gut bacterial composition at the genus level. (F) Comparison of gut bacterial composition at the genus level. (G) Spearman correlations analysis between the gut bacteria and colitis-related indexes. (H) Concentrations of short-chain fatty acids in feces. In (A) – (G), (n = 6 per group). In (H), (n = 8 per group). Data are mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. For (F), statistical analysis was performed using a two-tailed Wilcoxon rank-sum test by R Project. For (G), a statistically significant correlation was performed using linear regression analyses. The rest of the statistics were performed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison’s test

Gut microbiota confers protection of BL against C. rodentium-induced enteric infection

To further confirm the role of gut microbiota in the protective effects of BL against C. rodentium, fecal microbiota transplantation (FMT) was performed (Fig. 7A). In this study, antibiotic treatment was used to deplete the gut microbes and the antibiotic-treated recipient mice were then gavaged with the mixture of stool pellets from donor mice during the FMT (Fig. 7A). 16 S rRNA sequencing was used to determine whether the gut microbiota of donor mice was successfully transplanted into the recipient mice (Figure S9). Our result revealed clear discrimination between the FMT-treated two groups (Figure S9). Importantly, there is a high similarity in the gut microbiota community between the donor and the corresponding recipient mice (Figure S9). Consistent with our previous results, mice that received microbiota from BL-fed mice displayed an increased abundance of Lactobacillus compared with control recipient mice (Fig. 7B).

We further studied the functional significance of BL-mediated gut microbiome in the modulation of host response to C. rodentium infection. We found that mice received microbiota from BL-fed mice showed attenuated body weight loss and reduced DAI score compared to mice received microbiota from control CD-fed mice (Fig. 7C and D). Moreover, a longer colon length and a lower spleen weight were detected in mice received microbiota from BL-fed mice compared to mice received microbiota from control mice (Fig. 7E and F). Histological analysis of colon tissues revealed that mice received microbiota from BL-fed mice exhibited attenuated tissue damage and alleviated goblet cell depletion compared to control recipient mice (Fig. 7G-I). The severity of inflammation was improved in recipient mice that were colonized by BL-induced microbiota, as evidenced by reduced levels of IFN-γ and IL-1β in colonic tissues (Fig. 7J). Correspondingly, the bacterial level of C. rodentium in feces, spleen, and liver was reduced in mice received microbiota from BL-fed mice compared to control recipient mice (Fig. 7K-M). Collectively, these results confirm the crucial role of gut microbiota in protective effect of BL against C. rodentium infection.

Transplantation of feces from barley leaf (BL)-fed mice protects against C. rodentium (CR)-induced colitis. (A) Study design of in vivo mouse fecal microbiota transplantation (FMT) experiment. The recipient mice received a two-week treatment of combined antibiotics (neomycin, penicillin, metronidazole, 1000 mg/l; vancomycin and streptomycin 500 mg/l) before FMT to remove the native microbiota. The donor mice were fed a standard chow diet (CD) or an isocaloric BL-supplemented diet for 2 weeks. Fresh feces from donor mice were collected and resuspended in sterile PBS at 1:10 (m/v), vortexed, and rested, then the supernatant was taken for transplantation for three weeks. (B) Body weight change. (C) Disease activity index (DAI) scores. (D) Colon length. (E) Spleen weight. (F) Representative hematoxylin and eosin (H&E) staining images of colon tissues. Scale bar, 100 μm. Representative images of alcian blue-stained colonic sections. Scale bar, 100 μm. (G) Histological scores. (H) Quantification of mucus­producing goblet cells. (I) The levels of IFN-γ, IL-1β, and TNF-α in the colon. (J) Number of CR in feces on days 1, 4, and 7 post-infection. (K) Number of CR in the spleen on day 7 post-infection. (L) Number of CR in the liver on day 7 post-infection. n = 8 per group. Data are mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. For body weight change, a repeated measure two-way analysis of variance (ANOVA) was performed and the rest of the statistics were performed with Student’s t-test

L. plantarum attenuates C. rodentium infection-induced colonic inflammation

Lactobacillus was identified as the most enriched genus in BL-fed mice and mice received microbiota from BL-fed mice (Fig. 6F and Fig. 7B). By using real-time polymerase chain reaction (PCR), 9 common Lactobacillus strains L. rhamnosus, L. brevis, L. murinus, L. fermentum, L. reuteri, L. delbrueckii, L. casei, L. salivarius, and L. plantarum in the genome of the intestinal microbiota were quantified. Notably, L. plantarum was the only species that significantly enriched in BL-fed mice compared to the control CD-fed mice (Figure S10A). We hypothesized that BL may act as a prebiotic to enrich the commensal bacterium L. plantarum and assessed the direct effects of BL on L. plantarum growth. We found that the growth of L. plantarum was significantly promoted by BL treatment (Figure S10B). These data indicate that L. plantarum may play a beneficial role in alleviating C. rodentium-associated pathology.

To investigate the role of L. plantarum in C. rodentium-induced colitis, we administered phosphate-buffered saline (PBS), live L. plantarum or heat-inactivated L. plantarum to C. rodentium-infected mice (Fig. 8A). The qPCR analysis confirmed that administration of live L. plantarum resulted in increased abundance of L. plantarum in mouse feces (Fig. 8B). We found that mice administered with L. plantarum has significantly attenuated manifestations of colitis compared to mice gavaged with PBS, as demonstrated by body weight loss and DAI score (Fig. 8C and D). Although L. plantarum had no impact on the spleen weight, the colon length was significantly longer in mice administered with L. plantarum compared to mice gavaged with PBS (Fig. 8E and F). Histological analysis revealed that C. rodentium-induced pathological damage and mucosal barrier dysfunction were also abrogated by L. plantarum (Fig. 8G-I). The colonic expression of Sla26a3 and Car4 was increased and T-bet expression was reduced in mice received L. plantarum administration (Fig. 8J and K). Furthermore, C. rodentium infection led to elevated levels of C. rodentium in feces, liver, and cecum, which were significantly reduced by L. plantarum (Fig. 8L-N). Notably, heat-inactivated L. plantarum failed to confer the above beneficial effects on C. rodentium infection (Fig. 8C-N). Collectively, these results indicate that L. plantarum enriched with BL supplementation plays a crucial role in protection against C. rodentium-induced intestinal inflammation.

Lactobacillus plantarum (L. plantarum) attenuates colonic inflammation in C. rodentium (CR)-infected mice. (A) Study design of in vivo mouse experiment. Mice were gavaged daily with phosphate-buffered saline (PBS), 10⁹ CFU of live L. plantarum or 10⁹ CFU of heat-inactivated L. plantarum (H-L. plantarum) for 2 weeks prior to the infection with CR for 12 days. (B) Real-time polymerase chain reaction (PCR) assay for the quantification of L. plantarum in mouse feces. (C) Body weight change. (D) Disease activity index (DAI) scores. (E) Colon length. (F) Spleen weight. (G) Representative hematoxylin and eosin (H&E) staining images of colon tissues. Scale bar, 100 μm. Representative images of alcian blue-stained colonic sections. Scale bar, 100 μm. (H) Histological scores. (I) Quantification of mucus­producing goblet cells. (J) Real-time PCR assay for the expression of T-bet in the colon. (K) Real-time PCR assay for the expression of Slc26a3 and Car4 in the colon. (L) Number of CR in feces on days 1 post-infection. (M) Number of CR in liver on day 1 post-infection. (N) Number of CR in cecum content on day 1 post-infection. n = 6 per group. Data are Mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. For (B), Student’s t-test was performed. For (C), a repeated measure two-way analysis of variance (ANOVA) was performed and the rest of the statistics were performed with one-way ANOVA followed by Tukey’s multiple comparison’s test

L. plantarum ameliorates C. rodentium-induced colitis through its secreted EVs

Bacterial EVs are membrane-enclosed lipid bilayer nanoparticles that play essential roles in communication between gut microbiome and human health [35]. Recently, the EVs derived from probiotics particularly Lactobacillus have emerged as potential mediators of host immune response and anti-inflammatory effect [35].

However, the role of L. plantarum-derived EVs (L-EVs) in C. rodentium infection and intestinal inflammation remains unclear. We thus isolated EVs from the supernatants of L. plantarum using an ultracentrifugation-based system (Fig. 9A). The scanning electron microscopy and transmission electron microscopy showed that the isolated L-EVs were double-layer membrane-enclosed nanoparticles with spherical morphology (Fig. 9B and C). The nanoparticle tracking analysis (NTA) analysis showed that the mean particle size of L-EVs ranged from 100 nm to 200 nm in diameter (Fig. 9D), which was consistent with the size of previous reports [35]. We further performed proteomic analysis to characterize the protein composition of L-EVs. A total of 380 proteins were identified to be present in L-EVs. The biological functions of these proteins were analyzed by using the Gene Ontology (GO) database. The top ranked proteins are localized to the plasma membrane (26.61%), suggesting that L-EVs might originate directly from the budding of plasma membrane. KEGG pathway enrichment analysis further indicated that L-EVs-enriched proteins were primarily associated with amino acid metabolism, energy metabolism, antimicrobial and infectious disease. Specifically, most of them were found to be significantly involved in the modulation of signal peptide, or via JAK/STAT pathway, NF-κB pathway and TLR pathway [36,37,38,39].

Preparation and characterization of L. plantarum-derived EVs (L-EVs). (A) Isolation and purification procedures of L-EVs. (B) Representative image of the scanning electron microscope for L. plantarum showing membrane vesicles on the bacterial cell surface. (C) Representative image of the transmission electron microscopy for L-EVs. (D) Nanoparticle tracking analysis was performed to detect the size distribution of L-EVs. (E) Go secondary classification statistical charts of L-EVs. (F) KEGG annotated statistical charts of L-EVs

We evaluated the biosafety of L-EVs. There is no notable difference in daily food intake, body weight changes, and weight of organs (colon, liver, and spleen) between the L-EVs group and the control group (Figure S11A-E). Histological analysis showed that oral administration of L-EVs at the different dose did not cause any colon, liver, and spleen damage (Figure S11F). Furthermore, cytokine profiling analysis indicated no significant difference in serum levels of IFN-γ and TNF-α (Figure S11G and H). In addition, we supplemented in vivo studies with DiR-labeled L-EVs and demonstrated that these vesicles remain stable in circulation and effectively reach and persist in the colon without significant off-target distribution (Figure S12). These results suggest that L-EVs exhibit favourable stability and safety profiles.

To investigate whether the beneficial effects of L. plantarum were associated with their secreted EVs, L-EVs were administered to mice by oral gavage followed by the infection with C. rodentium (Fig. 10A). Our results showed that the dramatic loss of body weight and increased DAI score in C. rodentium-infected mice were significantly reduced by L-EVs intervention (Fig. 10B and C). Moreover, L-EVs mitigated C. rodentium-induced colon length shortening and increased spleen weight (Fig. 10D and E). Histopathological analysis showed that C. rodentium-induced increase in pathology score and mucosal barrier injury could be alleviated by L-EVs treatment (Fig. 10F-H). Similar trends were obtained with the expression of Ifnγ, Tnfα, Sla26a3 and Car4 in colon tissues (Fig. 10I and J). The C. rodentium loads in feces, liver, and cecum were significantly reduced in L-EVs-treated mice (Fig. 10K-M). We then further analyzed the population of CD4⁺ T cells. The increased population of CD4⁺IFN-γ⁺ T cells in the spleen was significantly reduced by L-EVs intervention (Fig. 10N and O). Collectively, these results suggest that L. plantarum attenuates C. rodentium infection and intestinal inflammation via its secreted EVs.

L. plantarum-derived EVs (L-EVs) ameliorates C. rodentium (CR)-induced colitis. (A) Study design of in vivo mouse experiment. Mice were gavaged daily with phosphate-buffered saline (PBS) or 50 µg of L-EVs for 2 weeks prior to the infection with CR for 12 days. (B) Body weight change. (C) Disease activity index (DAI) scores. (D) Colon length. (E) Spleen weight. (F) Representative hematoxylin and eosin (H&E) staining images of colon tissues. Scale bar, 100 μm. Representative images of alcian blue-stained colonic sections. Scale bar, 100 μm. (G) Histological scores. (H) Quantification of mucus­producing goblet cells. (I) Real-time PCR assay for the expression of Ifnγand Tnfα in the colon. (J) Real-time PCR assay for the expression of Slc26a3 and Car4 in the colon. (K) Number of CR in feces on days 1 post-infection. (L) Number of CR in liver on day 1 post-infection. (M) Number of CR in cecum content on day 1 post-infection. (N) Representative flow cytometry profile of CD4⁺IFN-γ⁺ cells. (O) Quantification of CD4⁺IFN-γ⁺ cells. n = 5–6 per group. Data are Mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. For (B), a repeated measure two-way analysis of variance (ANOVA) was performed and the rest of the statistics were performed Student’s t-test