DataSheet_1_Maturation of the medaka immune system depends on reciprocal interactions between the microbiota and the intestinal tract.pdf

The interactions between the host immune system and intestinal microorganisms have been studied in many animals, including fish. However, a detailed analysis has not been performed in medaka, an established fish model for biological studies. Here, we investigated the effect of immunodeficiency on the microbiota composition and the effect of gut bacteria on intestinal epithelial development and immune responses in medaka. Chronological analysis of the intestinal microbiota of interleukin 2 receptor subunit gamma (il2rg) mutant medaka showed a gradual decrease in the evenness of operational taxonomic units, mainly caused by the increased abundance of the Aeromonadaceae family. Exposure of wild-type medaka to high doses of an intestine-derived opportunistic bacterium of the Aeromonadaceae family induced an inflammatory response, suggesting a harmful effect on adult il2rg mutants. In addition, we established germ-free conditions in larval medaka and observed large absorptive vacuoles in intestinal epithelial cells, indicating a block in epithelial maturation. Transcriptome analysis revealed a decrease in the expression of genes involved in the defense response, including the antimicrobial peptide gene hepcidin, whose expression is induced by lipopolysaccharide stimulation in normal larvae. These results show that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system.</p

PaCDase-produced S1P induces endothelin-1 and IL-8 production by keratinocytes.

<p>(<i>A</i>) Involvement of SphK and S1P receptor in PaCDase-enhanced endothelin-1 and IL-8 gene expression. Nitrocellulose filters without (-) or with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum, and endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 3 independent experiments. **P<0.01; ***P<0.001. (<i>B</i>) Immunohistochemical analysis. Nitrocellulose filters with Tris-buffered saline alone (-/-), or without (+/−) or with 1 mU/ml PaCDase (+/PaCD) or 5 µM S1P (+/S1P) in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum. The cells were incubated for 24 h, embedded in paraffin, sectioned, and incubated with rabbit anti-endothelin-1 IgG (endothelin-s) or mouse anti-human IL-8 IgG. The data shown represent 3 independent experiments. Bar: 25 µm.</p

PaCDase-induced production of TNF-α and SphK1 by keratinocytes.

<p>PaCDase-induced TNF-α production. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase without or with 10 µM SphK inhibitor in TBS containing 0.1% Triton X-100 were placed on the stratum corneum. After incubation for 24 h, the cells were washed and solubilized and lysates cleared by centrifugation. The equivalent amounts of protein of the lysates were loaded onto polyacrylamide gels. Membranes were incubated with anti-TNF-α (A) or anti-SphK1 (B) antibody. To determine the amount of membrane-bound form TNF-α or SphK1 in each band, the membranes were re-probed with anti-β-actin. The blots shown are representative of 3 independent experiments. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. *P<0.05.</p

PaCDase enhances TNF-α gene expression via S1P and S1P receptors in 3D keratinocytes.

<p>(<i>A</i>) PaCDase induces TNF-α gene expression in 3D keratinocytes. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated), with or without 0.1% Triton X-100, were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 10 independent experiments. **P<0.01. (<i>B</i>) Sphingosine and S1P enhance TNF-α gene expression. Nitrocellulose filters with 1 mU/ml PaCDase, 5 µM sphingosine, phytosphingosine, C14 sphingoid base, S1P, α-hydroxy myristic acid, or 0.1% DMSO (solvent control) in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR and normalized relative to a RNA encoding an S18 ribosomal protein gene. Each bar represents the mean±SD of 5 independent experiments. **P<0.01; ***P<0.001. (<i>C</i>) Involvement of SphK and S1P receptor. Nitrocellulose filters with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 5 independent experiments. *P<0.05; **P<0.01; ***P<0.001.</p

PaCDase-induced endothelin-1 and IL-8 production by keratinocytes.

<p>(<i>A</i>) Expression of endothelin-1 and IL-8 mRNA is dependent on PaCDase enzymatic activity. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml mutant H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated) in TBS containing 0.1% Triton X-100 were placed on the stratum corneum. The cells were incubated for 24 h, and endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. An S18 ribosomal protein gene was used for normalization. Each bar represents the mean±SD of 5 independent experiments. **P<0.01; ***P<0.001. (<i>B</i>) Sphingosine and S1P enhance expression of endothelin-1 and IL-8 mRNA. Nitrocellulose filters without (-) or with 5 µM sphingosine, S1P, or α-hydroxy myristic acid in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum. Endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 3 independent experiments. *P<0.05; ***P<0.001.</p

PaCDase and S1P activate NF-κB-dependent signal pathway.

<p>(<i>A</i>) PaCDase and S1P significantly increase phosphorylated NF-κB p65 levels. Nitrocellulose filters with Tris-buffered saline (-/-) or Tris-buffered saline plus 0.1% Triton X-100 alone (−/+) or with 1 mU/ml PaCDase (PaCD/+), 5 µM S1P (S1P/+), or 1 mU/ml PaCDase with 10 µM VPC 23019 (PaCD/VCP23019/+) were placed onto the stratum corneum and incubated for 4 h. The cells were washed and solubilized, lysates were cleared by centrifugation, and the equivalent amounts of protein of the supernatants were subjected to SDS-PAGE/immunoblotting with anti-phospho-NF-κB p65 (Ser536). To determine the amount of NF-κB p65 in each band, the membranes were re-probed with anti-NF-κB p65. The blots shown are representative of 3 independent experiments. The band intensity of phosphorylated NF-κB p65 is shown relative to that of NF-κB p65 in each lane. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001. (<i>B</i>) PaCDase and S1P decrease IκBα protein concentration in cytosolic extracts in keratinocytes. The cells were incubated for 4 h as described in (A), washed and solubilized, the lysates were cleared by centrifugation, and the supernatants were subjected to SDS-PAGE/immunoblotting with anti- IκBα. To determine the amount of IκBα in each band, the membranes were re-probed with anti-β-actin. The blots shown are representative of 3 independent experiments. The band intensity of IκBα is shown relative to that of βactin in each lane. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. **P<0.01; ***P<0.001.</p

Infliximab inhibits PaCDase-induced production of IL-8, but not TNF-α, by keratinocytes.

<p>(<i>A</i>) PaCDase-induced TNF-α gene expression is not inhibited by infliximab. Nitrocellulose filters with Tris-buffered saline with 0.1% Triton X-100 (-/-) or with 1 mU/ml PaCDase (-), PaCDase plus 100 µg/ml normal human IgG or 10 or 100 µg/ml infliximab were placed on the stratum corneum. The cells were incubated for 4 h or 24 h, and the level of TNF-α mRNA was determined. An S18 ribosomal protein gene was used for normalization. Each bar represents the mean±SD of 3 independent experiments. **P<0.01. (<i>B</i>) Infliximab inhibition of PaCDase-induced IL-8 gene expression. IL-8 mRNA levels were assayed in the samples described in A. Each bar represents the mean±SD of 3 independent experiments. **P<0.01; ***P<0.001.</p

Infliximab inhibits the PaCDase-activated NF-κB-mediated signaling pathway.

<p>(<i>A, B</i>) Infliximab suppresses the phosphorylation of NF-κB p65. Nitrocellulose filters with 1 mU/ml PaCDase in the absence or presence of 100 µg/ml normal human IgG or 100 µg/ml infliximab in Tris-buffered saline with 0.1% Triton X-100 were placed on the stratum corneum. The cells were incubated for 30 min (A) or 4 h (B), washed, and solubilized. The lysates were cleared by centrifugation, and the supernatants were subjected to SDS-PAGE/ immunoblotting with anti-phospho-NF-κB p65 (Ser536) and then re-probed with anti-NF-κB p65. The blots shown represent 3 independent experiments. The band intensity of phosphorylated NF-κB p65 is shown relative to that of NF-κB p65 in each lane. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. ***P<0.001. (<i>C</i>) Infliximab increases IκBα protein concentration in keratinocyte cytosolic extracts. Cells were incubated for 4 h as described in (A), washed and solubilized. The lysates were cleared by centrifugation, and the supernatants were subjected to SDS-PAGE/immunoblotting with anti-IκBα antibody. To determine the amount of IκBα in each band, the membranes were re-probed with anti-βactin. The blots shown are representative of 3 independent experiments. Shown is the band intensity of IκBα relative to that of βactin. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. *P<0.05.</p

The 20 genes most strongly upregulated by PaCDase in 3D cultured keratinocytes.

<p>The results shown are representative data of 3 experiments.</p

Effect of PaCDase on levels of expression of sphingosine kinase and S1P receptors genes.

<p>Nitrocellulose filters with 1 mU/ml PaCDase or 0.1% DMSO (-) in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and DNA microarray analysis was performed. The results shown are representative of 3 experiments.</p