Allergy Asthma Immunol Res.  2018 Sep;10(5):516-532. 10.4168/aair.2018.10.5.516.

Lactobacillus plantarum-derived Extracellular Vesicles Protect Atopic Dermatitis Induced by Staphylococcus aureus-derived Extracellular Vesicles

• Kim MH
• Choi SJ
• Choi HI
• Choi JP
• Park HK
• Kim EK
• Kim MJ
• Moon BS
• Min TK
• Rho M
• Cho YJ
• Yang S
• Kim YK
• Kim YY
• Pyun BY

• KMID: 2418049
• DOI: http://doi.org/10.4168/aair.2018.10.5.516

Abstract

PURPOSE
The microbial environment is an important factor that contributes to the pathogenesis of atopic dermatitis (AD). Recently, it was revealed that not only bacteria itself but also extracellular vesicles (EVs) secreted from bacteria affect the allergic inflammation process. However, almost all research carried out so far was related to local microorganisms, not the systemic microbial distribution. We aimed to compare the bacterial EV composition between AD patients and healthy subjects and to experimentally find out the beneficial effect of some bacterial EV composition
METHODS
Twenty-seven AD patients and 6 healthy control subjects were enrolled. After urine and serum were obtained, EVs were prepared from samples. Metagenomic analysis of 16s ribosomal DNA extracted from the EVs was performed, and bacteria showing the greatest difference between controls and patients were identified. In vitro and in vivo therapeutic effects of significant bacterial EV were evaluated with keratinocytes and with Staphylococcus aureus-induced mouse AD models, respectively.
RESULTS
The proportions of Lactococcus, Leuconostoc and Lactobacillus EVs were significantly higher and those of Alicyclobacillus and Propionibacterium were lower in the control group than in the AD patient group. Therefore, lactic acid bacteria were considered to be important ones that contribute to the difference between the patient and control groups. In vitro, interleukin (IL)-6 from keratinocytes and macrophages decreased and cell viability was restored with Lactobacillus plantarum-derived EV treatment prior to S. aureus EV treatment. In S. aureus-induced mouse AD models, L. plantarum-derived EV administration reduced epidermal thickening and the IL-4 level.
CONCLUSIONS
We suggested the protective role of lactic acid bacteria in AD based on metagenomic analysis. Experimental findings further suggest that L. plantarum-derived EV could help prevent skin inflammation.

Keyword

Atopic dermatitis; Lactobacillus; microbiome; metagenomics; probiotics

MeSH Terms

Alicyclobacillus
Animals
Bacteria
Cell Survival
Dermatitis, Atopic*
DNA, Ribosomal
Extracellular Vesicles*
Healthy Volunteers
Humans
In Vitro Techniques
Inflammation
Interleukin-4
Interleukins
Keratinocytes
Lactic Acid
Lactobacillus*
Lactococcus
Leuconostoc
Macrophages
Metagenomics
Mice
Microbiota
Probiotics
Propionibacterium
Skin
Staphylococcus*
Therapeutic Uses
DNA, Ribosomal
Interleukin-4
Interleukins
Lactic Acid
Therapeutic Uses

Figure

• 

  Fig. 1 (A) Alpha-diversity as determined by the Chao 1 index. (B) Beta-diversity defined by PCA. (C) Bacterial community analysis of the urine of AD patients and normal controls at the phylum level. (D) Bacterial community analysis of the urine of patients and controls at the genus level (case: AD patients, control: healthy controls). PCA, principal component analysis; AD, atopic dermatitis.

• 

  Fig. 2 (A) Differences in bacterial EVs composition in the urine of AD patients and normal controls. (B) Bacterial community analysis of the urine of AD patients and normal controls at the genus level. EV, extracellular vesicle; AD, atopic dermatitis.

• 

  Fig. 3 Characterization of EVs from Lactobacillus plantarum. (A) TEM images of purified EVs from L. plantarum. (B) The size distribution of EVs from L. plantarum. The size distribution of EVs was measured in a diameter range of 20 to 50 nm. (C) SDS-PAGE analysis of purified proteins (WCL, EVs) from L. plantarum. Standard markers are shown on the left (kDa). EV, extracellular vesicle; TEM, transmission electron microscopy; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; WCL, whole cell lysate.

• 

  Fig. 4 (A) In vitro effects of Lactobacillus plantarum on the secretion of IL-6 from keratinocytes stimulated by Sa EV. NC was treated only with PBS, and PC was treated only with Sa EV. Pre- and co-treatment indicates that Lp EV was treated before Sa EV stimulation, or at the same time, respectively. (B) Keratinocytes cell viability assessment. IL, interleukin; Sa EV, Staphylococcus aureus-induced extracellular vesicle; NC, negative control; PBS, phosphate-buffered saline; PC, positive control; Lp EV, Lactobacillus plantarum-derived extracellular vesicle. *P < 0.05 vs. NC; †P < 0.05 vs. PC; ‡P < 0.01 vs. PC (Mann-Whitney U test).

• 

  Fig. 5 In vivo effects of oral Lp EV on the AD-like skin inflammation. (A) AD mouse model using tape-stripping and Sa EV. (B) Images of H&E-stained skin. (C) Histological analysis of epidermal thickness and infiltrating eosinophil. (D) Levels of IL-4 and IL-5. Lp EV, Lactobacillus plantarum-derived extracellular vesicle; AD, atopic dermatitis; Sa EV, Staphylococcus aureus-induced extracellular vesicle; H&E, hematoxylin and eosin; IL, interleukin; NC, negative control; PC, positive control. *P < 0.05 vs. NC; †P < 0.05 vs. PC; ‡P < 0.01 vs. PC (Mann-Whitney U test).

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