Administration of Lactobacillus fermentum KBL375 Causes Taxonomic and Functional Changes in Gut Microbiota Leading to Improvement of Atopic Dermatitis

L. fermentum KBL375, isolated from healthy Korean feces, was cultured as previously described with minor modifications (Jang et al., 2019; Kim et al., 2019). Briefly, L. fermentum KBL375 was cultivated in Lactobacilli MRS Agar (BD Difco, Sparks, MD, USA) supplemented with 0.05% L-cysteine-hydrochloride at 37°C for 24 h under anaerobic conditions using the Anaeropack (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan). The concentration of L. fermentum KBL375 was measured using the cultivation method (Unit: Colony forming unit; CFU) and the optical density (OD)-based method at 600 nm. After cultivation, bacterial cells were harvested using centrifugation (1,200 × g) and washed twice with 1 × phosphate-buffered saline (PBS) prior to administration to mice.

All experimental procedures for the AD-induced in vivo mouse model were approved by the Institutional Animal Care and Use Committee (IACUC: SNU-160928-1-1) of Seoul National University, Republic of Korea. The AD-induced in vivo mouse model was performed as previously described with minor modifications (Kang et al., 2015). Briefly, 5 week-old male NC/Nga mice (Central Lab Animals Inc., Seoul, Republic of Korea), bred from one production colony, were prepared and grouped with nine mice, per experimental condition. Three mice shared a cage. For induction of AD in skin, we removed the ear and dorsal skin hair using electric clipper and hair removal cream and applied 150 μL of 4% sodium dodecyl sulfate (SDS) in the ear and dorsal skin for 3 h to disrupt skin barrier. Then, we administered 100 mg of Dermatophagoides farinae extract (DFE) cream (Biostir® AD; Biostir, Inc, Hiroshima, Japan) twice a week for 21 days. Next, we administered approximately 1 × 10⁹ CFUs of L. fermentum KBL375 in 200 μL of PBS to mice via oral gavage daily for 28 days with weekly application of DFE cream. After treatment with L. fermentum KBL375, mice were sacrificed and cecum, mesenteric lymph nodes (MLNs), serum, and skin were collected for further analyses.

To assess the severity of AD, we anesthetized mice using 2% isoflurane. We then obtained images of the ear or dorsal skin and measured skin thickness (Figure S1A). Dermatitis scores of mice were measured once a week with blinded scoring of four researchers using the following criteria: (1) erythema/hemorrhage, (2) scaling/dryness, (3) edema, and (4) excoriation/erosion (Table S1) (Kang et al., 2015). Total dermatitis scores for mice were calculated with the maximal score of 12. Prior to sacrifice, transepidermal water loss (TEWL), representing the volume of water emitted from the body to the atmosphere through the epidermal layer (skin) by diffusion and evaporation, was measured using a Tewameter TM300 (Courage and Khazaka, Cologene, Germany) at 21–22°C and 50–55% humidity as described previously (Jimbo et al., 2015). The probe was located on the skin for 30 s and the data were measured. Scratching behaviors of mice were also observed for 10 min as described previously with minor modifications (Andoh et al., 2011). Briefly, for acclimation, mice were placed separately in an acrylic case for 1 h. Their behaviors were then recoded individually for 30 min. Total scratching behaviors to the nose, ear, and dorsal skin were counted for 10 min. Repeated and rapid scratching movements over 1 s were counted as one scratching. Blood samples were collected from blood vessels in the eyes of mice. Then, the serum was separated from blood samples by centrifugation at 1,200 × g for 15 min at 4°C. Serum IgE levels were measured using the IgE enzyme-linked immunosorbent assay (ELISA) kit (Komabiotech, Seoul, Korea) according to the manufacturer`s instructions.

Dorsal skin tissues were fixed in 10% formaldehyde and stained with hematoxylin and eosin to confirm neutrophil infiltration (Kang et al., 2015). Congo red or toluidine blue were also used for staining eosinophils or mast cells in tissue, respectively (Kang et al., 2015). The stained tissues were examined using a Panoramic Viewer as described previously (3DHISTECH, Ltd., Budapest, Hungary) (Jang et al., 2019; Kim et al., 2019).

To measure the mRNA expression of various cytokines or chemokines in the skin, total RNA from homogenates of skin samples was extracted using an Easy-spin Total RNA Extraction Kit (Intron, Seoul, Korea) and reverse-transcribed to complementary DNA (cDNA) using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer`s instructions. Cytokine levels were measured using real-time polymerase chain reaction (real-time PCR) using a Rotor-Gene Q (Qiagen, Hilden, Germany) and a QuantiTect SYBR Green PCR kit (Qiagen) with 0.01 mM primers (Table S2) under the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. The relative expression levels of each cytokine or chemokine were calculated using the 2^−ΔΔCT method and normalized to the expression levels of hypoxanthine-guanine phosphoribosyltransferase (HPRT), as described previously (Livak and Schmittgen, 2001).

Flow cytometry analyses were performed as described previously (Jang et al., 2019; Kim et al., 2019). Briefly, MLN tissues were smashed and filtered using a cell strainer (100 μm pore size (SPL Life Sciences Co., Ltd., Pocheon-si, Gyeonggi-do, Republic of Korea) and isolated T cells were subjected to a FC gamma receptor blocking and surface staining for 30 min at 4°C. To identify the live cells, T cells were stained with Fixable Viability Stain 510 (FVS510; BD Biosciences, San Jose, CA, USA) following the manufacturer`s instructions. Moreover, T cell surface were stained with CD3+ fluorescein isothiocyanate (145-2C11; BD Biosciences), CD4+ Percep-Cyanine 5.5 (RM4-5; BD Biosciences) and CD25+ phycoerythrin (PC61; BD Biosciences) following the manufacturer`s instructions. T cells were permeabilized with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for intracellular staining and were stained with Foxp3+ Alexa Flour 647 (MF23; BD Biosciences) following the manufacturer`s instructions. IgG isotypes (BD Biosciences) were used as a control. The CD4+CD25+Foxp3+ T cell population was counted using a BD FACSVerse™ Flow Cytometer (BD Biosciences).

Cecum microbiota were analyzed as previously described with minor modifications (Lim et al., 2016; Jang et al., 2019; Kim et al., 2019). Briefly, total DNA was collected using a QIAmp Fast DNA stool mini kit (Qiagen) following the manufacturer`s protocol. The V3-4 hypervariable region of the 16S rRNA gene was amplified using the barcoded primers 341F and 805R as described previously with minor modifications. The amplicons were purified subsequently using a QIAquick PCR Purification Kit (Qiagen) and sequenced using a MiSeq platform (Illumina. Inc., San Diego, CA, USA). Data were analyzed by using Quantitative Insights into Microbial Ecology 1.8.0 (QIIME) software (QIIME development team; http://qiime.org/)(Caporaso et al., 2010) and Greengenes version 13_5 database (http://greengenes.secondgemone.com). First, the sequences were clustered using an open-reference operational taxonomic unit (OTU) picking protocol at least 97% nucleotide identity with exclusions of singletons and rare OTUs, which were founded in 3.0) with Galaxy ver. 2.1.1 (Hutlab; http://huttenhower.org/galaxy) (Segata et al., 2011) and comparisons of abundances for significantly different microbial taxa among experimental groups were also measured.

AAs in cecum samples were analyzed as previously described (Jang et al., 2019). Briefly, approximately 1 mL of cecum extracts (concentration: 20 mg/mL) in liquid chromatography-grade methanol were derivatized by 70 μL of AccQ•Tag™ Ultra Borate Buffer (Waters Corporation., Milford, MA, USA) and 20 μL of AccQ•Tag™ Ultra reagent (Waters Corporation) for 10 min at 55°C. Samples were subsequently analyzed using an Acquity ultra-performance liquid chromatography (UPLC) (Waters Corporation) and a SYNAPT G2-Si mass spectrometer (Waters Corporation) with an ESI probe under the following conditions: 1.5 kV of capillary voltage, 600 and 50 L/h of desolvation or cone gas flow, respectively, and 250°C of desolvation temperature. An AA-S-18 analytical standard mixture (Sigma-Aldrich, St. Louis, MO, USA) was used to identify AAs, and data analyses were performed using MassLynx software 4.1 (Waters Corporation) (Roucher et al., 2013).

To measure SCFAs in samples, ceca were homogenized in distilled water and centrifuged at 13,000 × g for 5 min. Supernatant was collected and internal standards (1% 2-methylpentanoic acid for volatile acids or benzoic acid for non-volatile acids, respectively) were added. Then, extraction solvents (ethyl ether for volatile acids or chloroform for non-volatile acids, respectively) were added to the solution and centrifuged at 13,000 × g for 5 min. The organic layer was collected and analyzed using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) under the following conditions; 1.5 kV of capillary voltage. Six hundred and fifty liter per hour of desolvation or cone gas flow, respectively, 170°C of oven temperature and 225°C for a flame ionization detector (FID) and an injection port temperature. The standard mixture was used as a reference for retention times and peak areas for extracts (David et al., 2014).

Experimental data were expressed as means ± standard error of the mean (SEM) of experimental groups (nine mice per group). When appropriate, data were analyzed using the Mann-Whitney U test. P-values < 0.05 were considered as statistically significant. GraphPad PRISM 5 (GraphPad software, San Diego, CA) were used for statistical analyses and visualizations.