LF41-mediated upregulation of PGE₂ is abrogated by COX-2 blockade, but facilitated by IL-10 blockade in a COX-2-dependent manner.

<p><b>(A)(B)</b> ELISA for PGE₂ secretion by the terminal ileum (A) and PGE₂ amount in the liver (B) of mice (PBS-treated groups: n = 5–6 per group; H-LF41-treated groups: n = 8 per group) treated with a combination of either PBS or H-LF41 for 10 days, either alone or in combination with the COX-2-specific inhibitor celecoxib, its vehicle (vehicle), IL-10-specific antibody (Anti-IL-10), its isotype control (Is-IL-10), or Anti-IL-10 together with either celecoxib or its vehicle. * P < 0.05; <b>&</b> P < 0.05 compared to H-LF41; <b> P > 0.05 compared to PBS; n.s., non-statistical difference. (C)(E) Western blot assay for representative protein levels of COX-2 in epithelial cells (ECs) of the terminal ileum and COX-2 and COX-1 in the liver from mice (n = 4) either given 10 days H-LF41 treatment together with IL-10 blockade (C), or treated with 10 days of PBS, killed-LF41, H-LF41, or killed-LF41 together with H-LF41 (killed-LF41+H-LF41) (E). (D) ELISA for hepatic PGE₂ amount in mice (n = 8) fed PBS or H-LF41 for 10 days or given a combination of 10 days gavage of killed-LF41 or killed-LF41+H-LF41,either singly or combined with COX-2 blockade. * P < 0.05; & P < 0.05 compared to H-LF41; </b> P > 0.05 compared to PBS. All values except that of Western blot are shown as mean ± SEM. Results are representative of 2 similar experiments.</p

Ten days of H-LF41 treatment significantly enhances ileal expression of COX-2 and IL-10.

<p><b>(A)</b> q-PCR for mRNA levels of several factors associated with innate and adaptive immune responses in the terminal ileum collected from mice (n = 10) fed either for 10 days with PBS, L-LF41, or H-LF41 (upper panel), or for 3 weeks with either PBS or H-LF41 (lower panel). Results are expressed as fold change relative to “PBS”. * P < 0.05 compared to PBS. <b>(B)</b> MPO expression in the terminal ileum from mice (n = 6) treated with either PBS or H-LF41 for 10 days. P > 0.05 compared to PBS. <b>(C)</b> Epithelial cells (ECs) from the terminal ileum and its underlying lamina propria cells (LPCs) were isolated from mice (n = 8) orally given10 days supplement of PBS or H-LF41. <i>Cox2</i> and <i>Il10</i> mRNA levels in these cells were determined by q-PCR. Results are expressed as fold change relative to PBS. * P < 0.05compared to PBS. <b>(D)</b> Western blot assay for representative COX-2 protein levels in ECs and LPCs of the terminal ileum of mice (n = 4) fed either PBS or H-LF41 for 10 days. “RI” denotes the mean relative luminous intensity of the targeted protein band, which is positively correlated with the real luminance; the RI in the control group is set at 1.00. All values except that of Western blot are shown as mean ± SEM. Results are representative of 2 similar experiments.</p

Orally-pretreated LF41 attenuates LPS-induced TNF-α expression and hepatic injury.

<p><b>(A)</b> C57BL/6 mice (n = 8) either untreated or treated with antibiotic formula (Ab) were given single IP injection with LPS (500 μg/kg body weight). Mice were killed 2 and 16 h after LPS treatment for determination of hepatic TNF-α gene levels (left panel) by q-PCR and serum ALT activity (right panel), respectively. Results in the left panel are expressed as fold change relative to LPS. P > 0.05 compared to LPS. <b>(B)(C)</b> Mice (LPS-treated groups: n = 8–10 per group; the remainder: n = 6 per group) were given daily IG inoculation either for 10 consecutive days ofL-LF41, H-LF41, killed-LF41, LGG, BC41, or PBS, or for 21 consecutive days of either PBS or H-LF41 (right panel), and then single IP injection with LPS or PBS. Hepatic <i>Tnf</i> mRNA levels by q-PCR (B) and serum ALT activity (C) were determined. Results of (B) are expressed as fold change relative to PBS+PBS. H-LF41+LPS denotes 10 days of oral challenge with H-LF41 and then LPS injection, and other similar abbreviations conform to the same rule. * P < 0.05 compared to PBS+LPS. <b>(D)</b> Mice (LPS-treated groups: n = 8 per group; the remainder: n = 6 per group) were treated for 10 days with either PBS or H-LF41 and then challenged with LPS. Mice were killed 2 h after LPS treatment to test hepatic and serum TNF-α protein levels by ELISA.* P < 0.05 compared to PBS+LPS. <b>(E) (F)</b> Mice(LPS-treated groups: n = 12–14 per group; the remainder: n = 6–7 per group) pretreated for 10 days with PBS or H-LF were challenged with PBS or LPS. 20 h after the challenge, the inflammatory foci in the liver were determined (E), and representative histological outcomes of liver tissue were shown (F). * P < 0.05 compared to PBS+LPS. a: PBS+PBS; b: H-LF41+PBS; c: PBS+LPS; d: LF41+LPS. Values are shown as mean ± SEM. Results of <b>(A)</b> are representative of 2 experiments with similar results, and the remainder 3 experiments with similar results.</p

PGE₂-EP4 pathway is in charge of LF41-mediated attenuation of hepatic TNF-α expression.

<p><b>(A)</b> ELISA for PGE₂ secretion by the terminal ileum and total PGE₂ levels in the liver of mice (n = 8) orally treated either for 10 days with PBS, L-LF41, or H-LF41, or for 3 weeks with PBS or H-LF41. * P < 0.05 PBS. <b>(B)</b> ELISA for hepatic IL-10 protein concentration of mice (n = 8) fed either PBS or H-LF41 for 10 days. P > 0.05 compared to PBS. <b>(C)</b> q-PCR for hepatic <i>Cox1</i> or <i>Cox2</i> mRNA levels of mice (n = 8) fed either PBS or H-LF41 for 10 days. Results are expressed as fold change relative to PBS. P > 0.05 compared to PBS. <b>(D)</b> Western blot assay for representative hepatic COX-1 and COX-2 protein levels of mice (n = 4) orally treated with either PBS or H-LF41 for 10 days. Hepatic COX-2 protein levels from a mouse receiving 2 h of stimulation with LPS (0.5 mg/kg BW; single IP injection) were determined as a positive control (left lane). <b>(E)</b> Mice (H-LF41-treated groups: n = 10 per group; PBS-treated groups: n = 8 per group) were pretreated with 10 days of PBS or H-LF41, either alone or combined with administration of either a specific inhibitor for PGE₂ receptor EP-4, ONA-AE3-208 (I-EP4), or its vehicle (Vehicle). Hepatic <i>Tnf</i> mRNA levels were assayed by q-PCR 2 h after LPS treatment. Results are expressed as fold change relative to PBS+LPS.* P < 0.05; <b>&</b> P < 0.05 compared to H-LF41+LPS; <b>n.s.</b>, non-statistical difference. All values except that of Western blot are shown as mean ± SEM. Results are representative of 2 similar experiments.</p

Validation of q-PCR for quantitation of LF and effect of LF41 administration on LF-specific 16S rRNA levels in intestinal tissues.

<p><b>(A)</b> LF41, BC41, or LGG was cultured in MRS broth at 37°C overnight. An aliquot of culture from each culture was dilution-plated on MRS agar (to enumerate each strain). Total bacterial genomic DNA was isolated from an aliquot of each culture and analyzed by q-PCR using the primers specific to the 16S rRNA of either LF or LGG. Black and white triangles denote log numbers of 16S rRNA gene copies determined by LF- and LGG-specific q-PCR, respectively; black squares denote log numbers of bacteria determined by serial dilution. <b>(B)</b> MRS broth was co-inoculated with LGG and low, middle, or high dose of LF41, grown at 37°C overnight. Total bacterial genomic DNA was isolated from an aliquot of each sample and analyzed by q-PCR using the primers specific to 16S rRNA of <i>Lactobacillus</i>, LF, or LGG. The samples of a, b, and c denote the co-cultures of LGG with low, middle, and high dose of LF41, respectively; “R(LF)” and “R(LGG)” denote the ratios of the respective 16S rRNA gene copies determined by LF- and LGG-specific q-PCR to the gene copies by <i>Lactobacillus</i>-specific q-PCR. <b>(B)(C)(D)</b> Mice (n = 8) were orally inoculated either for 10 days with PBS, L-LF41, or H-LF41, or for 3 weeks with PBS or H-LF41, and LF-specific 16S rRNA gene levels in terminal ileum <b>(B)</b>, proximal colon <b>(C)</b>, and distal jejuna <b>(D)</b> determined by q-PCR. Results are expressed as log₁₀ of the 16S rRNA gene copies per mg of tissue samples. Values of are shown as mean ± SEM. * P < 0.05 compared to L-LF41 or H-LF41 (21 days); <b>+</b> P < 0.05 compared to H-LF41 (10 days); nd, not detected. Results are representative of 2 experiments with similar results.</p

Effect of COX-2 or IL-10 blockade on TNF-α expression and intestinal permeability in LF41-fed mice.

<p><b>(A)(B)</b> Mice (PBS-treated groups: n = 5 per group; H-LF41-treated groups: n = 7 per group) were given 10 days treatment of PBS or H-LF41, either alone or in combination with blockade of EP4, COX-2, or IL-10, or co-blockade of TNF-α with COX-2 or IL-10, and then given IG inoculation with FITC-Dextran. Three hours later, the FITC-Dextran amount in the blood was determined. I-EP4, EP4-specific inhibitor; vehicle, the vehicle for celecoxib; celecoxib, COX-2-specific inhibitor; Anti-TNF, TNF-α-specific antibody; Is-TNF, the isotype control for Anti-TNF; Anti-IL-10, IL-10-specific antibody; Is-IL-10, the isotype control for Anti-IL-10.* P < 0.05; & P < 0.05compared to H-LF41; <b>n.s.</b>, non-statistical difference. <b>(C)</b> ELISA for TNF-α secretion by the terminal ileum collected from mice (PBS-treated groups: n = 5 per group; H-LF41-treated groups: n = 7 per group) fed for 10 days PBS or H-LF41, either singly or in combination with blockade of COX-2, IL-10, or EP4. * P < 0.05; & P < 0.05 compared toH-LF41. <b>(D)</b> q-PCR for <i>Tnf</i> mRNA levels in the epithelial cells (ECs) and lamina propria cells (LPCs) of the terminal ileum from mice (n = 7) treated for 10 days with PBS or H-LF41 in the presence of celecoxib administration. Results are expressed as fold change relative to PBS. * P < 0.05. <b>(E)</b> q-PCR for <i>Tnf</i> mRNA levels in the HMNCs isolated from mice (PBS-treated groups: n = 6 per group; H-LF41-treated groups: n = 8–10 per group) given 10 days treatment of PBS or H-LF41, either alone or together with blockade of EP4, COX-2, or IL-10, or with co-blockade of IL-10 and COX-2. Results are expressed as fold change relative to PBS. * P < 0.05; <b>&</b> P < 0.05 compared to H-LF41. Values are shown as mean ± SEM. Results are representative of 2 similar experiments.</p

The gene location and predicted topology of <i>S. coelicolor</i> SecDF homologs.

<p>(A) Schematic diagrams of the location of <i>secDF</i> homologous genes in <i>S. coelicolor</i> genome. The core region of <i>S. coelicolor</i> is indicated in gray box. (B) The predicted topology of <i>S. coelicolor</i> SecDF protein. The conserved domains D1–D6 and F1–F4, which are presented in all known SecD and SecF proteins, are highlighted in black color.</p

Strains and plasmids used in this study.

<p><i>ermEp*</i>: the enhanced promoter region of the erythromycin resistance gene (<i>ermE</i>) of <i>Streptomyces erythraeus</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105237#pone.0105237-Bibb1" target="_blank">[48]</a>.</p

Proposed schematic model of <i>secDF</i> homologs evolution in <i>Streptomyces</i>.

<p>Proposed schematic model of <i>secDF</i> homologs evolution in <i>Streptomyces</i>.</p

Assay of extracellular XlnA and AmlC activity and protein amounts.

<p>(A) Detecting XlnA protein secretion by SDS-PAGE and Western blot. WB: Western blot; CB: Commassie Blue Staining. ZJUZ23-26 strains were cultivated for 96 h. (B) Assay of extracellular XlnA activity. ZJUZ23-26 strains were cultivated for 72 h and 96 h. *: p<0.05, **: p>0.05, Chi-squared test. (C) Relative amylase activity from ZJUZ27, ZJUZ28, ZJUZ29 and ZJUZ30. The diameters of the transparent zones around colonies were measured to determine the relative activity.</p