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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 induce IL-6 and CXC chemokine mRNA expression in human colonic SEMFs. (A) SEMFs were stimulated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The mRNA expression for IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) was evaluated using real-time PCR. The mRNA expression for IL-6 and chemokine was converted to a value relative to β-actin mRNA expression and presented as fold-increase relative to the results for medium alone (no stimulation). Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium alone. (B) SEMFs were incubated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The protein levels of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) in the culture medium were 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 alone.
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Figure 1: IL-36α and IL-36γ induce IL-6 and CXC chemokine mRNA expression in human colonic SEMFs. (A) SEMFs were stimulated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The mRNA expression for IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) was evaluated using real-time PCR. The mRNA expression for IL-6 and chemokine was converted to a value relative to β-actin mRNA expression and presented as fold-increase relative to the results for medium alone (no stimulation). Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium alone. (B) SEMFs were incubated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The protein levels of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) in the culture medium were 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 alone.

Mentions: To investigate the function of IL-36 cytokines in human colonic SEMFs, the cells were stimulated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h, and the mRNA expression of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) was evaluated using real-time PCR. The secretion of IL-6 and CXC chemokine proteins was also determined using ELISA. As it has been reported that IL-36α, IL-36β, and IL-36γ can be activated by the removal of N-terminal residues, we used spliced recombinant IL-36 cytokines (30). As shown in Figures 1A,B, both IL-36α and IL-36γ induced a significant increase in the mRNA and protein expression of these proinflammatory mediators compared to the medium control. These results indicate that IL-36α and IL-36γ are strong inducers of proinflammatory mediators in human colonic SEMFs.


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 induce IL-6 and CXC chemokine mRNA expression in human colonic SEMFs. (A) SEMFs were stimulated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The mRNA expression for IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) was evaluated using real-time PCR. The mRNA expression for IL-6 and chemokine was converted to a value relative to β-actin mRNA expression and presented as fold-increase relative to the results for medium alone (no stimulation). Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium alone. (B) SEMFs were incubated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The protein levels of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) in the culture medium were 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 alone.
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Figure 1: IL-36α and IL-36γ induce IL-6 and CXC chemokine mRNA expression in human colonic SEMFs. (A) SEMFs were stimulated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The mRNA expression for IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) was evaluated using real-time PCR. The mRNA expression for IL-6 and chemokine was converted to a value relative to β-actin mRNA expression and presented as fold-increase relative to the results for medium alone (no stimulation). Data are expressed as means ± SD of four independent experiments. *P < 0.05, **P < 0.01; significant differences from the values for medium alone. (B) SEMFs were incubated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h. The protein levels of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) in the culture medium were 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 alone.
Mentions: To investigate the function of IL-36 cytokines in human colonic SEMFs, the cells were stimulated with IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for 24 h, and the mRNA expression of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) was evaluated using real-time PCR. The secretion of IL-6 and CXC chemokine proteins was also determined using ELISA. As it has been reported that IL-36α, IL-36β, and IL-36γ can be activated by the removal of N-terminal residues, we used spliced recombinant IL-36 cytokines (30). As shown in Figures 1A,B, both IL-36α and IL-36γ induced a significant increase in the mRNA and protein expression of these proinflammatory mediators compared to the medium control. These results indicate that IL-36α and IL-36γ are strong inducers of proinflammatory mediators in human colonic SEMFs.

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