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A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Kueanjinda P, Roytrakul S, Palaga T - Sci Rep (2015)

Bottom Line: Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK.In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb.A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

View Article: PubMed Central - PubMed

Affiliation: 1] Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand [2] Center of Excellence in Immunology and Immune-mediated Diseases, Chulalongkorn University, Bangkok 10330, Thailand.

ABSTRACT
Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

No MeSH data available.


Related in: MedlinePlus

Expression of pro-inflammatory cytokines in macrophages lacking both Numb and Notch signaling after LPS stimulation.(A–F) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were pretreated with DAPT for 1 hr prior to LPS stimulation. The levels of intracellular TNFα, IL-6, and IL-12 were determined by flow cytometry (A–C) or ELISA (D–F). N.D. = not detectable. (G) Expression level of Rbpjκ from macrophages transduced with siRbpjκ, or scrambled siRNA as control, was measured by qPCR. (H–J) Expression levels of TNFα, IL-6, and IL-12p70 from GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors which were transfected with scramble siRNA or siRbpjk were measured by ELISA. N.D. = not detectable.
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f4: Expression of pro-inflammatory cytokines in macrophages lacking both Numb and Notch signaling after LPS stimulation.(A–F) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were pretreated with DAPT for 1 hr prior to LPS stimulation. The levels of intracellular TNFα, IL-6, and IL-12 were determined by flow cytometry (A–C) or ELISA (D–F). N.D. = not detectable. (G) Expression level of Rbpjκ from macrophages transduced with siRbpjκ, or scrambled siRNA as control, was measured by qPCR. (H–J) Expression levels of TNFα, IL-6, and IL-12p70 from GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors which were transfected with scramble siRNA or siRbpjk were measured by ELISA. N.D. = not detectable.

Mentions: Our earlier observation revealed that Notch signaling was up-regulated in Numb-deficient macrophages following LPS stimulation. Previous reports have also demonstrated that Notch regulates pro-inflammatory cytokines in macrophages, especially IL-6 and IL-12p4027283240. Therefore, to elucidate the regulatory mechanism downstream of Numb, we used a γ-secretase inhibitor, DAPT, to pharmacologically inhibit Notch signaling in macrophages prior to and during LPS stimulation. As expected, intracellular and secreted TNFα, IL-6, and IL-12p40 protein level in DAPT-treated, Numb-intact macrophages decreased after LPS stimulation (Fig. 4A–F). However, there was not a significant difference in TNFα levels in Numb-silenced macrophages treated with DMSO or DAPT (Fig. 4A,D). In contrast, the levels of intracellular and secreted IL-6 (Fig. 4B,E) and IL-12p40 (Fig. 4C,F) in DAPT-treated, Numb-deficient macrophages were lower than those of the DMSO-treated, Numb-deficient cells. These results suggest that Notch and Numb counter-regulate expression of IL-6 and IL-12p40, but not TNFα. To confirm this, we silenced CSL/RBP-Jκ, which plays a central role in canonical Notch signaling41. The mRNA level of CSL/Rbp-jκ was reduced by ~70% (Fig. 4G). Consistent with the results obtained by the treatment with DAPT, the levels of IL-6 and IL-12p40, but not of TNFα, in Numb-deficient macrophages were further decreased, compared to the those in macrophages transfected with scramble siRNA (Fig. 4H–J). Despite increased activation of Notch signaling in Numb-deficient macrophages, a reduction in TNFα expression that results from silencing Numb, demonstrates TNFα is not under the direct influence of Notch signaling. By contrast, it appears IL-6 and IL-12p40 expression are, at least partially, directly regulated by canonical Notch signaling.


A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Kueanjinda P, Roytrakul S, Palaga T - Sci Rep (2015)

Expression of pro-inflammatory cytokines in macrophages lacking both Numb and Notch signaling after LPS stimulation.(A–F) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were pretreated with DAPT for 1 hr prior to LPS stimulation. The levels of intracellular TNFα, IL-6, and IL-12 were determined by flow cytometry (A–C) or ELISA (D–F). N.D. = not detectable. (G) Expression level of Rbpjκ from macrophages transduced with siRbpjκ, or scrambled siRNA as control, was measured by qPCR. (H–J) Expression levels of TNFα, IL-6, and IL-12p70 from GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors which were transfected with scramble siRNA or siRbpjk were measured by ELISA. N.D. = not detectable.
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Related In: Results  -  Collection

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Show All Figures
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f4: Expression of pro-inflammatory cytokines in macrophages lacking both Numb and Notch signaling after LPS stimulation.(A–F) GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors were pretreated with DAPT for 1 hr prior to LPS stimulation. The levels of intracellular TNFα, IL-6, and IL-12 were determined by flow cytometry (A–C) or ELISA (D–F). N.D. = not detectable. (G) Expression level of Rbpjκ from macrophages transduced with siRbpjκ, or scrambled siRNA as control, was measured by qPCR. (H–J) Expression levels of TNFα, IL-6, and IL-12p70 from GFP+ macrophages containing control (open bars) or shNumb (closed bars) vectors which were transfected with scramble siRNA or siRbpjk were measured by ELISA. N.D. = not detectable.
Mentions: Our earlier observation revealed that Notch signaling was up-regulated in Numb-deficient macrophages following LPS stimulation. Previous reports have also demonstrated that Notch regulates pro-inflammatory cytokines in macrophages, especially IL-6 and IL-12p4027283240. Therefore, to elucidate the regulatory mechanism downstream of Numb, we used a γ-secretase inhibitor, DAPT, to pharmacologically inhibit Notch signaling in macrophages prior to and during LPS stimulation. As expected, intracellular and secreted TNFα, IL-6, and IL-12p40 protein level in DAPT-treated, Numb-intact macrophages decreased after LPS stimulation (Fig. 4A–F). However, there was not a significant difference in TNFα levels in Numb-silenced macrophages treated with DMSO or DAPT (Fig. 4A,D). In contrast, the levels of intracellular and secreted IL-6 (Fig. 4B,E) and IL-12p40 (Fig. 4C,F) in DAPT-treated, Numb-deficient macrophages were lower than those of the DMSO-treated, Numb-deficient cells. These results suggest that Notch and Numb counter-regulate expression of IL-6 and IL-12p40, but not TNFα. To confirm this, we silenced CSL/RBP-Jκ, which plays a central role in canonical Notch signaling41. The mRNA level of CSL/Rbp-jκ was reduced by ~70% (Fig. 4G). Consistent with the results obtained by the treatment with DAPT, the levels of IL-6 and IL-12p40, but not of TNFα, in Numb-deficient macrophages were further decreased, compared to the those in macrophages transfected with scramble siRNA (Fig. 4H–J). Despite increased activation of Notch signaling in Numb-deficient macrophages, a reduction in TNFα expression that results from silencing Numb, demonstrates TNFα is not under the direct influence of Notch signaling. By contrast, it appears IL-6 and IL-12p40 expression are, at least partially, directly regulated by canonical Notch signaling.

Bottom Line: Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK.In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb.A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

View Article: PubMed Central - PubMed

Affiliation: 1] Interdisciplinary Program in Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand [2] Center of Excellence in Immunology and Immune-mediated Diseases, Chulalongkorn University, Bangkok 10330, Thailand.

ABSTRACT
Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

No MeSH data available.


Related in: MedlinePlus