Limits...
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

The activation of IκBα by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with 100 ng/ml of IL-36α or IL-36γ for the indicated pre-determined times. Expression of phosphorylated (p-) and total IκBα was sequentially evaluated by Western blotting. The data are representative of three independent experiments. (B) SEMFs were transfected with NF-κBp65 siRNA and cultured for 2 days. The cells were then stimulated 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 the indicated chemokines was then 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 IL-36α or IL-36γ stimulation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585048&req=5

Figure 6: The activation of IκBα by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with 100 ng/ml of IL-36α or IL-36γ for the indicated pre-determined times. Expression of phosphorylated (p-) and total IκBα was sequentially evaluated by Western blotting. The data are representative of three independent experiments. (B) SEMFs were transfected with NF-κBp65 siRNA and cultured for 2 days. The cells were then stimulated 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 the indicated chemokines was then 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 IL-36α or IL-36γ stimulation.

Mentions: The transcription factor NF-κB plays critical roles in the mRNA expression of inflammatory mediators. To investigate whether IL-36α or IL-36γ induce the activation of NF-κB, we evaluated the effects of both IL-36α and IL-36γ on IκBα phosphorylation and total IκBα degradation in human colonic SEMFs using western blot analysis. As shown in Figure 6A, both IL-36α and IL-36γ induced the phosphorylation and degradation of IκB. We also examined the involvement of NF-κB activation in the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ using a siRNA specific for NF-κBp65. Blockade of the expression of NF-κBp65 significantly suppressed the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ (Figure 6B) [we confirmed that the gene expression of NF-κBp65 was suppressed by the transfection of siRNA specific for NF-κBp65 using real-time PCR (Figure S2 in Supplementary Material)]. These results indicated that the activation of NF-κB is involved in the induction of proinflammatory mediators by both IL-36α and IL-36γ.


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)

The activation of IκBα by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with 100 ng/ml of IL-36α or IL-36γ for the indicated pre-determined times. Expression of phosphorylated (p-) and total IκBα was sequentially evaluated by Western blotting. The data are representative of three independent experiments. (B) SEMFs were transfected with NF-κBp65 siRNA and cultured for 2 days. The cells were then stimulated 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 the indicated chemokines was then 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 IL-36α or IL-36γ stimulation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585048&req=5

Figure 6: The activation of IκBα by IL-36α or IL-36γ in human colonic SEMFs. (A) SEMFs were stimulated with 100 ng/ml of IL-36α or IL-36γ for the indicated pre-determined times. Expression of phosphorylated (p-) and total IκBα was sequentially evaluated by Western blotting. The data are representative of three independent experiments. (B) SEMFs were transfected with NF-κBp65 siRNA and cultured for 2 days. The cells were then stimulated 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 the indicated chemokines was then 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 IL-36α or IL-36γ stimulation.
Mentions: The transcription factor NF-κB plays critical roles in the mRNA expression of inflammatory mediators. To investigate whether IL-36α or IL-36γ induce the activation of NF-κB, we evaluated the effects of both IL-36α and IL-36γ on IκBα phosphorylation and total IκBα degradation in human colonic SEMFs using western blot analysis. As shown in Figure 6A, both IL-36α and IL-36γ induced the phosphorylation and degradation of IκB. We also examined the involvement of NF-κB activation in the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ using a siRNA specific for NF-κBp65. Blockade of the expression of NF-κBp65 significantly suppressed the induction of IL-6 and CXC chemokines (CXCL1, CXCL2, and CXCL8) by IL-36α or IL-36γ (Figure 6B) [we confirmed that the gene expression of NF-κBp65 was suppressed by the transfection of siRNA specific for NF-κBp65 using real-time PCR (Figure S2 in Supplementary Material)]. These results indicated that the activation of NF-κB is involved in the induction of proinflammatory mediators by both IL-36α and IL-36γ.

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