Harnessing Lactiplantibacillus plantarum EP21 and its membrane vesicles to inhibit myopia development

Abstract

Lactiplantibacillus plantarum, a probiotic that is frequently found in fermented foods, is well known for its numerous health-enhancing properties. This study explored the potential of Lactiplantibacillus plantarum EP21 and its membrane vesicles (MVs) in mitigating myopia progression. In animal models of form-deprivation and TGF-β2-induced myopia, EP21 reduced axial elongation and refractive error shifts. EP21 administration suppressed retinal inflammation by inhibiting nuclear factor-κB activation and decreasing expression levels of tumor necrosis factor-α, NLR family pyrin-domain-containing-3, and interleukin (IL)-1β, while upregulating the expression of anti-inflammatory IL-10 in retinal tissues. To identify the molecular mechanism by which EP21 inhibits myopia, purified MVs were administered by intraperitoneal or intravenous injection or applied directly onto the ocular surface. The MVs crossed the blood - retinal barrier and accumulated in the outer segment of the retina. The MVs exhibited anti-myopic properties, indicated by a reduction in axial length elongation and a corresponding upregulation of refractive error. Mechanistic investigations revealed that EP21-MVs contain bacterial miRNAs that inhibit inflammatory responses and retinal structural remodeling. Furthermore, MV and its miRNAs upregulated genes that are associated with ubiquitination (TNF alpha-induced protein 3, TNFAIP3-interacting protein 1, and Tax1 binding protein 1), which are crucial for maintaining ocular proteostasis and regulating inflammation. EP21 administration, besides MV release, altered systemic metabolites, including tryptophan derivatives and short-chain fatty acids. The findings suggest a role of the gut - eye axis in the development of myopia and elucidate a biological pathway whereby gut-derived probiotics and their vesicles can influence ocular health. This research highlights the potential of EP21- and EP21-derived MVs as noninvasive agents for myopia management, and thereby enhances the comprehension of the gut - eye axis. By targeting inflammation and retinal remodeling, probiotics such as EP21 may contribute to addressing the global myopia epidemic, which offers a promising pathway for both preventive and therapeutic strategies.

Keywords: Lactiplantibacillus plantarum EP21; bacterial membrane vesicle; inflammation; myopia.

Figure 1.

Probiotic treatment suppresses the development of myopia by lowering the inflammatory responses and inflammasome activation. (a) Administration of Lactiplantibacillus plantarum EP21 (EP21) mitigated myopia induced by monocular form deprivation (MFD). EP21 mitigated the elongation of axial length provoked by MFD. (b) EP21 prevented the down-regulation of refractive error caused by MFD. The relative change in refractive error of the right eye was standardized to the refractive error of the left eye, which remained untreated. (c) EP21 prevented the elongation of axial length induced by the sub-junctival injection of transforming growth factor-β2 (TGF-β2). (d) EP21 inhibited the down-regulation of refractive error induced by TGF-β2. The relative change in refractive error of the right eye was standardized to the refractive error of the left eye, which remained untreated. (e) NFκB expression levels determined using western blotting. Relative expression levels of p-NFκB and NF-κB (below the lanes) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the expression levels in lane NC are designated as 1. Immunofluorescence staining of NFκB (f), tumor necrosis factor α (TNFα) (g), nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) (h), and interleukin 1β (IL1β) (i) expression in the eyes of NC, EP21, TGF-β2 and TGF-β2 + EP21-treated rats. Results are shown as mean ± SD. The scale bar indicates 165 μm and 200× magnification for if staining images. Relative expression levels were determined using image J software. Analysis of variance was applied to examine for significant differences (p < 0.05), and Tukey’s multiple comparison tests were used for pairwise comparisons. NC: negative control. INL: inner nuclear layer; ONL: outer nuclear layer; IS: inner segment; OS: outer segment; RPE: retinal pigment epithelium.

Figure 2.

Membrane vesicles secreted by Lactiplantibacillus plantarum EP21 (MV) inhibit inflammatory responses and pass across blood-retinal-barrier. (a) Lactiplantibacillus plantarum EP21 and its derived MVs (red arrows) detected by cryo-scanning electron microscopy. (b) DIL-labelled MVs were internalized by retinal pigment epithelial (RPE-1) cells. (c) EP21 MVs inhibited interleukin (IL) 1β induced nuclear factor (NF)κB activation in RPE-1 cells. NFκB expression levels determined using western blotting. Relative expression levels of p-NF-κB and NF-κB (below the lanes) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the expression levels in lane NC are designated as 1. (d) EP21 MVs inhibited IL1β induced IL6 expression in RPE-1 cells. Cells were treated with phosphate buffered saline (NC), IL1β or IL1β + MV for 24 hours. The IL6 concentration was then determined by enzyme-linked immunosorbent assay. (e) EP21 MVs inhibited tumor necrosis factor α (TNFα) induced IL6 expression in RPE-1 cells. Cells were treated with NC, TNFα or TNFα + MV for 24 hours. The IL6 concentration was then determined by enzyme-linked immunosorbent assay. For (d) and (e), analysis of variance was applied to examine for significant differences (p < 0.05), and Tukey’s multiple comparison tests were used for pairwise comparisons. (f) Dil-labeled or non-labeled EP21 MVs were inject intravenously into C57BL/6 mice. After 24 hours, eyes were collected to determine the fluorescence intensities in the retina. (g) EP21 was stained with or without carboxifluorescein diacetate succinimidyl ester (CFSE) and orally fed C57BL/6 mice for 7 days. Eyes were collected to determine the fluorescence intensities in the retina. For (f) and (g), relative expression levels were determined using image J software. T-test was used to evaluate the significant difference between non-labeled (NL) and EP21.

Figure 3.

Lactiplantibacillus plantarum EP21 membrane vesicles (EP21 MVs) treatment suppresses the development of myopia by lowering the inflammatory responses and inflammasome activation. Myopia was induced by sub-junctival injection of transforming growth factor-β2 (TGF-β2). (a) Intraperitoneal injection of EP21 MVs (3.3 × 10⁸ or 3.3 × 10⁹ particles) prevented the elongation of axial length caused by the sub-junctival injection of TGF-β2. (b) Intraperitoneal injection of EP21 MVs inhibited the down-regulation of refractive error induced by TGF-β2. The relative change in refractive error of the right eye was standardized to the refractive error of the left eye, which remained untreated. (c) Intravenously injection of EP21 MVs (3.3 × 10⁸ or 3.3 × 10⁹ particles) prevented the elongation of axial length caused by the sub-junctival injection of TGF-β2. (d) Intravenously injection of EP21 MVs inhibited the down-regulation of refractive error induced by TGF-β2. The relative change in refractive error of the right eye was standardized to the refractive error of the left eye, which remained untreated. Immunofluorescence staining of NFκB (e), tumor necrosis factor α (TNFα) (f), nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) (g), and interleukin 1β (IL1β) (h) expression in the eyes of negative control (NC), TGF-β2 and TGF-β2 + EP21 MV-treated (intraperitoneally administered) rats. Results are shown as mean ± SD. The scale bar indicates 165 μm and 200× magnification for if staining images. Relative expression levels were determined using image J software. Analysis of variance was applied to examine for significant differences (p < 0.05), and Tukey’s multiple comparison tests were used for pairwise comparisons. INL: inner nuclear layer; ONL: outer nuclear layer; IS: inner segment; OS: outer segment; RPE: retinal pigment epithelium. (h) The expression levels of phospho-NFκB (p-NFκB), NFκB, NLRP3, pro-IL1β, and TNFα in the retina were determined using western blotting. Relative expression levels (below the lanes) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Figure 4.

RNA in the Lactiplantibacillus plantarum EP21 membrane vesicles (EP21 MVs) inhibited inflammatory reactions in the retinal pigment epithelial cell. (a) Mediators inside the EP21 MVs but not DNA or RNA associated with MVs inhibited the activation of nuclear factor κB (NFκB). Intact EP21 MVs were treated with DNase I or RNase a to remove DNA or RNA binds to the surface of EP21 MVs. RPE-1 cells were treated with phosphate buffered saline (NC), interleukin 1β (IL1β), MV, DNAase I or RNase a treated MVs for 30 min and the activation of NFκB was determined by western blot. Relative expression levels (below the lanes) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (b) The activation of NFκB by transforming growth factor β2 (TGF-β2) or IL1β in RPE-1 cells was inhibited by EP21 MV (MV). RPE-1 cells were treated with indicated treatments for 30 min and the activation of NFκB was determined by western blot. Relative expression levels (below the lanes) were normalized to GAPDH. (c) The increased in expression levels of NFκB and IL1β induced by IL1β in RPE-1 cells were inhibited by transfecting EP21 MV total RNA (MVR). RPE-1 cells were treated with indicated treatments for 24 hours and the expression of NFκB was determined by western blot. Relative expression levels (below the lanes) were normalized to GAPDH. (d) The increased in expression levels of NFκB and IL1β induced by tumor necrosis factor α (TNFα) in RPE-1 cells were inhibited by transfecting EP21 MV total RNA (MVR). RPE-1 cells were treated with indicated treatments for 24 hours and the expression of NFκB was determined by western blot. Relative expression levels (below the lanes) were normalized to GAPDH. (e) MVR inhibited IL1β induced IL6 expression in RPE-1 cells. Cells were treated with phosphate buffered saline (NC), IL1β, MVR, or IL1β + MVR for 24 hours. The IL6 concentration was then determined by enzyme-linked immunosorbent assay. (f) MVR inhibited TNFα induced IL6 expression in RPE-1 cells. Cells were treated with phosphate buffered saline (NC), TNFα, MVR, or TNFα + MVR for 24 hours. The IL6 concentration was then determined by enzyme-linked immunosorbent assay. (g) EP21 MVs inhibited the expression of inflammatory related markers in RPE-1 cells determined by realtime-quantitative polymerase chain reaction. TGF-Beta activated kinase 1 (MAP3K7) binding protein 3:TAB3; tumor necrosis factor; TNFA; RELA proto-oncogene, NF-KB subunit: RELA; caspase 1: CASP1; interleukin 1β: IL1B; interleukin 6:IL6.

Figure 5.

Lactiplantibacillus plantarum EP21 membrane vesicles (EP21 MV) modify ubiquitination pathways. (a) Primary retinal pigment epithelial cells were treated with EP21 MVs for 2 hours. The total RNA of treated cells was subjected to whole transcriptome sequencing analysis. Pathway analysis on the six EP21 miRNAs regulated genes. EP21 miRNAs modulated inflammation and ubiquitination pathways (red arrows). (b) RPE-1 cells were transfected with the mixture of six EP21 miRNAs for 16 hours and the expression level of phospho-nuclear factor κB (p-NFκB), NFκB, nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) and interleukin 1β (IL1β) was determined by western blot. Relative expression levels were determined using image J software. Relative expression levels (below the lanes) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Figure 6.

Lactiplantibacillus plantarum EP21 membrane vesicles containing miRNAs modify inflammation and ubiquitination pathways. (a) Ingenuity pathway analysis identified the gene networks associated with inflammation and ubiquitination pathways. (b) The levels of genes associated with ubiquitination, specifically TNF alpha induced protein 3 (TNFAIP3), TNFAIP3-interacting protein 1 (TINP1), and Tax1 binding protein 1 (TAX1BP1), were significantly elevated in RPE-1 cells following treatment with EP21 MV. NC refers to the negative control condition. (c) Total protein was extracted from the retina of eyes subjected to negative control, transforming growth factor β2 (TGF-β2), and EP21 MVs treatment, and the extent of protein ubiquitination was assessed using western blot analysis. (d) RPE-1 cells underwent treatment with tumor necrosis factor (TNF) α, transfection with miRNAs or co-treatment with TNFα and miRNAs for a duration of 24 hours. The level of protein ubiquitination was evaluated through western blotting. Poly-ub: polyubiquitinated protein; free-ub: free ubiquitin. Relative free ubiquitin expression levels were determined using image J software. Relative expression levels (below the lanes) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Figure 7.

Inhibition of the ubiquitination pathway genes upregulated by Lactiplantibacillus plantarum EP21 membrane vesicles exacerbates inflammation. (a) Validation of the gene silencing efficacy of siRNAs targeting TNFAIP3, TNIP1, and TAX1BP1. Mixture of siRNA against TNFAIP3, TNIP1, and TAX1BP1 were transfect into RPE-1 cells and determined the gene silencing efficacy by real-time PCR. T-test was used to evaluate the significant difference between control siRNA (siNC) and siRNA. (b) Pooled siRNAs targeting TNFAIP3, TNIP1, and TAX1BP1 or control siRNA (siNC) were transfected into retinal pigment epithelial cell, RPE-1 and then treated with tumor necrosis factor α (TNFα) for 24 hours. The expression of interleukin 6 (IL6) and IL8 in the culture supernatant was determined by enzyme-linked immunosorbent assay. (c) Pooled siRNAs targeting TNFAIP3, TNIP1, and TAX1BP1 or control siRNA (siNC) were transfected into RPE-1 cell which was treated with EP21 MVs for 24 hours and then treated with TNFα for 24 hours. The expression of IL6 and IL8 in the culture supernatant was determined by enzyme-linked immunosorbent assay. Analysis of variance was applied to examine for significant differences (p < 0.05), and Tukey’s multiple comparison tests were used for pairwise comparisons.