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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

Activation of MAPKs by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with or without IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for the indicated pre-determined times. Expression of phosphorylated (p-) and total MAPKs were sequentially evaluated by Western blotting. The data are representative of two independent experiments. (B) SEMFs were pretreated with 20 μM of a p38 MAPK inhibitor (SB203580) or an MEK1/2 inhibitor (U0126 or PD98059) for 1 h, and were then incubated with or without 100 ng/ml of IL-36α (top panels) or IL-36γ (bottom panels) for 24 h. The mRNA expression of IL-6 and chemokines was then analyzed. The mRNA expression for IL-6 and chemokines 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 IL-36α or IL-36γ stimulation.
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Figure 5: Activation of MAPKs by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with or without IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for the indicated pre-determined times. Expression of phosphorylated (p-) and total MAPKs were sequentially evaluated by Western blotting. The data are representative of two independent experiments. (B) SEMFs were pretreated with 20 μM of a p38 MAPK inhibitor (SB203580) or an MEK1/2 inhibitor (U0126 or PD98059) for 1 h, and were then incubated with or without 100 ng/ml of IL-36α (top panels) or IL-36γ (bottom panels) for 24 h. The mRNA expression of IL-6 and chemokines was then analyzed. The mRNA expression for IL-6 and chemokines 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 IL-36α or IL-36γ stimulation.

Mentions: It has previously been reported that IL-36 cytokines activate MAPKs and NF-κB in Jurkat cells (14). To assess whether the activations of MAPKs are involved in the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ, we evaluated the phosphorylation of MAPKs in IL-36-stimulated human colonic SEMFs by Western blot analysis. As shown in Figure 5A, both IL-36α and IL-36γ rapidly induced the phosphorylation of p42/44 MAPK, p38 MAPK, and JNK within 5 min after stimulation. Next, we examined the involvement of MAPKs in the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ using specific inhibitors of p38 MAPK (SB203580) and of MEK1 (PD98059 and U0126) that is directly upstream of p42/44MAPK. As shown in Figure 5B, inhibition of p38 MAPK or of p42/44MAPK significantly reduced the mRNA expression of IL-6 or CXC chemokines induced by IL-36α or IL-36γ. These results indicated that the activation of MAPKs is involved in the induction of proinflammatory mediators by both IL-36α and IL-36γ 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)

Activation of MAPKs by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with or without IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for the indicated pre-determined times. Expression of phosphorylated (p-) and total MAPKs were sequentially evaluated by Western blotting. The data are representative of two independent experiments. (B) SEMFs were pretreated with 20 μM of a p38 MAPK inhibitor (SB203580) or an MEK1/2 inhibitor (U0126 or PD98059) for 1 h, and were then incubated with or without 100 ng/ml of IL-36α (top panels) or IL-36γ (bottom panels) for 24 h. The mRNA expression of IL-6 and chemokines was then analyzed. The mRNA expression for IL-6 and chemokines 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 IL-36α or IL-36γ stimulation.
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Figure 5: Activation of MAPKs by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with or without IL-36α (100 ng/ml) or IL-36γ (100 ng/ml) for the indicated pre-determined times. Expression of phosphorylated (p-) and total MAPKs were sequentially evaluated by Western blotting. The data are representative of two independent experiments. (B) SEMFs were pretreated with 20 μM of a p38 MAPK inhibitor (SB203580) or an MEK1/2 inhibitor (U0126 or PD98059) for 1 h, and were then incubated with or without 100 ng/ml of IL-36α (top panels) or IL-36γ (bottom panels) for 24 h. The mRNA expression of IL-6 and chemokines was then analyzed. The mRNA expression for IL-6 and chemokines 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 IL-36α or IL-36γ stimulation.
Mentions: It has previously been reported that IL-36 cytokines activate MAPKs and NF-κB in Jurkat cells (14). To assess whether the activations of MAPKs are involved in the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ, we evaluated the phosphorylation of MAPKs in IL-36-stimulated human colonic SEMFs by Western blot analysis. As shown in Figure 5A, both IL-36α and IL-36γ rapidly induced the phosphorylation of p42/44 MAPK, p38 MAPK, and JNK within 5 min after stimulation. Next, we examined the involvement of MAPKs in the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ using specific inhibitors of p38 MAPK (SB203580) and of MEK1 (PD98059 and U0126) that is directly upstream of p42/44MAPK. As shown in Figure 5B, inhibition of p38 MAPK or of p42/44MAPK significantly reduced the mRNA expression of IL-6 or CXC chemokines induced by IL-36α or IL-36γ. These results indicated that the activation of MAPKs is involved in the induction of proinflammatory mediators by both IL-36α and IL-36γ 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