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Interleukin(IL)-36α and IL-36γ Induce Proinflammatory Mediators from Human Colonic Subepithelial Myofibroblasts.

Kanda T, Nishida A, Takahashi K, Hidaka K, Imaeda H, Inatomi O, Bamba S, Sugimoto M, Andoh A - Front Med (Lausanne) (2015)

Bottom Line: The mRNA expression of proinflammatory mediators induced by IL-36α and/or IL-36γ was significantly suppressed by transfection of siRNA for MyD88 or IRAK1.Both inhibitors of mitogen activated protein kinases and siRNAs specific for NF-κBp65 significantly reduced the expression of IL-6 and CXC chemokines induced by IL-36α and/or IL-36γ.These results suggest that IL-36α and IL-36γ contribute to gut inflammation through the induction of proinflammatory mediators.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Shiga University of Medical Science , Otsu , Japan.

ABSTRACT

Background: Interleukin (IL)-36 cytokines are recently reported member of the IL-1 cytokine family. However, there is little information regarding the association between IL-36 cytokines and gut inflammation. In the present study, we investigated the biological activity of IL-36α and IL-36γ using human colonic subepithelial myofibroblasts (SEMFs).

Methods: The mRNA expression and the protein expression of target molecules in SEMFs were evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The intracellular signaling of IL-36 cytokines was analyzed using Western blot analysis and small interfering RNAs (siRNAs) specific for MyD88 adaptor proteins (MyD88 and IRAK1) and NF-κB p65.

Results: IL-36α and IL-36γ significantly enhanced the secretion of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by SEMFs. The combination of IL-36α/γ and IL-17A or of IL-36α/γ and tumor necrosis factor-α showed a synergistic effect on the induction of IL-6 and CXC chemokines. The mRNA expression of proinflammatory mediators induced by IL-36α and/or IL-36γ was significantly suppressed by transfection of siRNA for MyD88 or IRAK1. Both inhibitors of mitogen activated protein kinases and siRNAs specific for NF-κBp65 significantly reduced the expression of IL-6 and CXC chemokines induced by IL-36α and/or IL-36γ.

Conclusion: These results suggest that IL-36α and IL-36γ contribute to gut inflammation through the induction of proinflammatory mediators.

No MeSH data available.


Related in: MedlinePlus

IL-36α and IL-36γ dose- and time-dependently induce IL-6 and CXC chemokines in human colonic SEMFs. (A) Dose-dependent effects of IL-36α or IL-36γ on IL-6 and CXC chemokines. SEMFs were stimulated for 24 h with increasing concentrations of IL-36α or IL-36γ, and the concentration of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) secreted into the culture medium was determined using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium (0 ng/ml) alone. (B) The kinetics of IL-6 and CXC chemokine induction by IL-36α and IL-36γ. SEMFs were stimulated with 100 ng/ml of IL-36α or 100 ng/ml of IL-36γ for pre-determined times, and the protein levels of IL-6 and CXC chemokines in the culture medium were measured using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values at 0 h.
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Figure 2: IL-36α and IL-36γ dose- and time-dependently induce IL-6 and CXC chemokines in human colonic SEMFs. (A) Dose-dependent effects of IL-36α or IL-36γ on IL-6 and CXC chemokines. SEMFs were stimulated for 24 h with increasing concentrations of IL-36α or IL-36γ, and the concentration of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) secreted into the culture medium was determined using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium (0 ng/ml) alone. (B) The kinetics of IL-6 and CXC chemokine induction by IL-36α and IL-36γ. SEMFs were stimulated with 100 ng/ml of IL-36α or 100 ng/ml of IL-36γ for pre-determined times, and the protein levels of IL-6 and CXC chemokines in the culture medium were measured using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values at 0 h.

Mentions: We investigated the induction of proinflammatory mediators by IL-36α and IL-36γ in further detail. Human colonic SEMFs were incubated for 24 h with increasing concentrations of IL-36α or IL-36γ, and the secretion of CXC chemokines was measured using ELISA. IL-36α or IL-36γ dose-dependently induced the secretion of CXC chemokines (Figure 2A). We next evaluated the protein expression of IL-6 and CXC chemokines. Human colonic SEMFs were incubated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for pre-determined times and the secretion of IL-6 and CXC chemokines was measured using ELISA. As shown in Figure 2B, IL-36α and IL-36γ induced the secretion of proinflammatory mediators in a time-dependent manner (effects of IL-36α and IL-36γ at the mRNA levels were shown in Figure S1 in Supplementary Material).


Interleukin(IL)-36α and IL-36γ Induce Proinflammatory Mediators from Human Colonic Subepithelial Myofibroblasts.

Kanda T, Nishida A, Takahashi K, Hidaka K, Imaeda H, Inatomi O, Bamba S, Sugimoto M, Andoh A - Front Med (Lausanne) (2015)

IL-36α and IL-36γ dose- and time-dependently induce IL-6 and CXC chemokines in human colonic SEMFs. (A) Dose-dependent effects of IL-36α or IL-36γ on IL-6 and CXC chemokines. SEMFs were stimulated for 24 h with increasing concentrations of IL-36α or IL-36γ, and the concentration of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) secreted into the culture medium was determined using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium (0 ng/ml) alone. (B) The kinetics of IL-6 and CXC chemokine induction by IL-36α and IL-36γ. SEMFs were stimulated with 100 ng/ml of IL-36α or 100 ng/ml of IL-36γ for pre-determined times, and the protein levels of IL-6 and CXC chemokines in the culture medium were measured using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values at 0 h.
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Figure 2: IL-36α and IL-36γ dose- and time-dependently induce IL-6 and CXC chemokines in human colonic SEMFs. (A) Dose-dependent effects of IL-36α or IL-36γ on IL-6 and CXC chemokines. SEMFs were stimulated for 24 h with increasing concentrations of IL-36α or IL-36γ, and the concentration of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) secreted into the culture medium was determined using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium (0 ng/ml) alone. (B) The kinetics of IL-6 and CXC chemokine induction by IL-36α and IL-36γ. SEMFs were stimulated with 100 ng/ml of IL-36α or 100 ng/ml of IL-36γ for pre-determined times, and the protein levels of IL-6 and CXC chemokines in the culture medium were measured using ELISA. Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values at 0 h.
Mentions: We investigated the induction of proinflammatory mediators by IL-36α and IL-36γ in further detail. Human colonic SEMFs were incubated for 24 h with increasing concentrations of IL-36α or IL-36γ, and the secretion of CXC chemokines was measured using ELISA. IL-36α or IL-36γ dose-dependently induced the secretion of CXC chemokines (Figure 2A). We next evaluated the protein expression of IL-6 and CXC chemokines. Human colonic SEMFs were incubated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for pre-determined times and the secretion of IL-6 and CXC chemokines was measured using ELISA. As shown in Figure 2B, IL-36α and IL-36γ induced the secretion of proinflammatory mediators in a time-dependent manner (effects of IL-36α and IL-36γ at the mRNA levels were shown in Figure S1 in Supplementary Material).

Bottom Line: The mRNA expression of proinflammatory mediators induced by IL-36α and/or IL-36γ was significantly suppressed by transfection of siRNA for MyD88 or IRAK1.Both inhibitors of mitogen activated protein kinases and siRNAs specific for NF-κBp65 significantly reduced the expression of IL-6 and CXC chemokines induced by IL-36α and/or IL-36γ.These results suggest that IL-36α and IL-36γ contribute to gut inflammation through the induction of proinflammatory mediators.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Shiga University of Medical Science , Otsu , Japan.

ABSTRACT

Background: Interleukin (IL)-36 cytokines are recently reported member of the IL-1 cytokine family. However, there is little information regarding the association between IL-36 cytokines and gut inflammation. In the present study, we investigated the biological activity of IL-36α and IL-36γ using human colonic subepithelial myofibroblasts (SEMFs).

Methods: The mRNA expression and the protein expression of target molecules in SEMFs were evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The intracellular signaling of IL-36 cytokines was analyzed using Western blot analysis and small interfering RNAs (siRNAs) specific for MyD88 adaptor proteins (MyD88 and IRAK1) and NF-κB p65.

Results: IL-36α and IL-36γ significantly enhanced the secretion of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by SEMFs. The combination of IL-36α/γ and IL-17A or of IL-36α/γ and tumor necrosis factor-α showed a synergistic effect on the induction of IL-6 and CXC chemokines. The mRNA expression of proinflammatory mediators induced by IL-36α and/or IL-36γ was significantly suppressed by transfection of siRNA for MyD88 or IRAK1. Both inhibitors of mitogen activated protein kinases and siRNAs specific for NF-κBp65 significantly reduced the expression of IL-6 and CXC chemokines induced by IL-36α and/or IL-36γ.

Conclusion: These results suggest that IL-36α and IL-36γ contribute to gut inflammation through the induction of proinflammatory mediators.

No MeSH data available.


Related in: MedlinePlus