Exosomes from Limosilactobacillus fermentum Ameliorate Benzalkonium Chloride-Induced Inflammation in Conjunctival Cells

Abstract

Dry eye is characterized by persistent instability and decreased tear production, which are accompanied by epithelial lesions and inflammation on the surface of the eye. In our previous paper, we reported that supplementation with Limosilactobacillus fermentum HY7302 (HY7302) could inhibit corneal damage in a benzalkonium chloride (BAC)-induced mouse model of dry eye, through its effects in gut microbiome regulation. The aim of this study was to determine what functional extracellular substances can alter the inflammatory response of conjunctival cells. We isolated exosomes from HY7302 probiotic culture supernatant, analyzed their morphological characteristics, and found that their average size was 143.8 ± 1.1 nm, which was smaller than the exosomes from the L. fermentum KCTC 3112 strain. In addition, HY7302-derived exosomes significantly reduced the levels of genes encoding pro-inflammatory cytokines, including interleukin (IL)-20, IL-8, IL-6, and IL-1B, in BAC-treated human conjunctival cells. Moreover, HY7302-derived exosomes significantly increased the levels of genes encoding tight junction proteins, including TJP1, TJP2, and occludin-1, in Caco-2 cells. Lastly, the HY7302 exosomes reduced mRNA expression levels of IL1B, IL20, IL6, IL8, and NFAT5 in a transwell coculture system. Our findings indicate that HY7302 exosomes have potential for use in the treatment of ocular inflammation-related dry eye disease, through gut-eye axis communication via exosomes.

Keywords: Limosilactobacillus fermentum HY7302; conjunctiva cell; exosomes; ocular inflammation.

Figure 1

Images showing the morphology of exosomes from Limosilactobacillus fermentum HY7302 (HY7302). (A) HY7302 exosomes isolated by high-speed centrifugation. (B) Negative staining transmission electron microscopy images of exosomes isolated from L. fermentum HY7302. Scale bar, 50 nm. Arrowheads, exosomes.

Figure 2

Characterization of exosomes from Limosilactobacillus fermentum. Nanoparticle tracking analysis of exosomes isolated from L. fermentum HY7302 (A) and KCTC3112 (B). Table box below show size distribution data obtained using a Malvern NanoSight NS300 and NanoSight NTA 3.4 Analytical software exosomes isolated from L. fermentum HY7302 (C) and KCTC3112 (D). Nanoparticle image of the isolated exosome from (E) HY7302 and (F) KCTC3112 were obtained using NTA analysis.

Figure 3

Effect of exosomes from Limosilactobacillus fermentum HY7302 (7302E) on cytotoxicity. (A,C) Lactate dehydrogenase (LDH) release cytotoxicity assay and (B,D) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Control cells or cells exposed to 0.0005% benzalkonium chloride (BAC) for 3 h were treated with L. fermentum HY7302 exosomes (7302E; 0, 0.01, 0.1, 0.5, 1.0, or 5.0 µg/10⁴ cells). Data are expressed as mean ± standard deviation (SD) (n = 3). Different letters indicate significantly different values (p < 0.05) (a > ab > b > c).

Figure 4

Effect of Limosilactobacillus fermentum HY7302 exosomes on tight junction molecules in Caco-2 cells. (A) Tight junction protein 1 (TJP1) and (B) occludin-1 were normalized to those of GAPDH and relative fold changes in their levels calculated. Levels of mRNA encoding (C) TJP1, (D) TJP2, and (E) occludin-1 were normalized to those of GAPDH and relative fold changes in their levels calculated. CON, control; HY7302, 10⁶ CFU/mL HY7302; 7302EL, 0.5 μg/mL of HY7302 exosomes; 7302EH, 1 μg/mL of HY7302 exosomes. Data are expressed as mean ± standard deviation (SD) (n = 3). Different letters indicate significantly different values (p < 0.05) (a > ab > b > c > d).

Figure 5

Effects of Limosilactobacillus fermentum HY7302 extracellular vesicles (7302E) on pro-inflammatory factors in conjunctival cell lines treated using 0.0005% BAC. Levels of (A) interleukin-20 (IL20), (B) IL8, (C) IL1B, (D) IL6, (E) nuclear factor of activated T cells 5 (NFAT5), and (F) nuclear factor kappa B subunit 1 (NFKB1) mRNA were normalized to those of GAPDH and calculated as relative fold-change values. CON, control; BAC, 0.0005% (v/v) BAC; 7302, 10⁶ CFU/mL HY7302; 7302E, 1 μg/mL of HY7302 exosomes; 3112, 10⁶ CFU/mL KCTC3112; 3112E, 1 μg/mL of KCTC3112 exosomes. Data are expressed as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05) (a > b > c > d).

Figure 6

(A) Schematic of the experimental protocol to test the effect of exosomes of Limosilactobacillus fermentum HY7302 (7302E) on pro-inflammatory cytokines production. Clone 1-5c-4 cells were seeded in transwell plates and, once they reached confluence, exposed to Caco-2 cells. Caco-2 cells were co-cultured either with or without exosomes, using transwells, and culture medium samples of conjunctiva cell collected for analysis. Levels of (B) IL1B, (C) IL20, (D) IL6, (E) IL8, and (F) NFAT5 mRNA in cells were normalized to those of GAPDH and relative fold-change values calculated. CON, control; BAC, 0.0005% (v/v) BAC; 7302, 10⁶ CFU/mL HY7302; 7302E, 1 μg/mL of HY7302 exosomes; 3112, 10⁶ CFU/mL KCTC3112; 3112E, 1 μg/mL of KCTC3112 exosomes. Data are expressed as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05) (a > ab > b > bc > c).