Limits...
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

Differentiation of bone marrow cells to macrophages upon silencing of Numb.(A,B) Expression of macrophage markers, CD11b and F4/80, from GFP+ population of macrophages that were retrovirally infected with control (A) or shNumb (B) vectors was determined by flow cytometry. (C,D) Expression level of F4/80 (C) and CD11b (D) from (A) and (B) were shown as fold-change of mean fluorescence intensity (MFI). Data are the mean ± SEM from three independent experiments. N.S. = not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4542673&req=5

f2: Differentiation of bone marrow cells to macrophages upon silencing of Numb.(A,B) Expression of macrophage markers, CD11b and F4/80, from GFP+ population of macrophages that were retrovirally infected with control (A) or shNumb (B) vectors was determined by flow cytometry. (C,D) Expression level of F4/80 (C) and CD11b (D) from (A) and (B) were shown as fold-change of mean fluorescence intensity (MFI). Data are the mean ± SEM from three independent experiments. N.S. = not statistically significant.

Mentions: A report by Wilson et al.16 demonstrated that hematopoietic stem cell compartments, including macrophages, developed normally in Numb and Numb-like double-knockout mice. Our system employed an insertion of the retroviral vector in hematopoietic cells from the bone marrow, causing constitutive silencing of Numb. We, therefore, tested whether silencing of Numb affects the development of macrophages from bone marrow hematopoietic stem cells in vitro. After retroviral transduction of pMKO.1-shNumb-GFP or control vectors, bone marrow cells were induced to differentiate to macrophages. Cells containing the vectors were gated for GFP+ and assayed for the expression of two macrophage-specific surface markers, F4/80 and CD11b. Our results showed that the expression levels both of F4/80 and CD11b were comparable between control and Numb-silenced macrophages (Fig. 2A–D), indicating that Numb was dispensable for macrophage differentiation in vitro.


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)

Differentiation of bone marrow cells to macrophages upon silencing of Numb.(A,B) Expression of macrophage markers, CD11b and F4/80, from GFP+ population of macrophages that were retrovirally infected with control (A) or shNumb (B) vectors was determined by flow cytometry. (C,D) Expression level of F4/80 (C) and CD11b (D) from (A) and (B) were shown as fold-change of mean fluorescence intensity (MFI). Data are the mean ± SEM from three independent experiments. N.S. = not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542673&req=5

f2: Differentiation of bone marrow cells to macrophages upon silencing of Numb.(A,B) Expression of macrophage markers, CD11b and F4/80, from GFP+ population of macrophages that were retrovirally infected with control (A) or shNumb (B) vectors was determined by flow cytometry. (C,D) Expression level of F4/80 (C) and CD11b (D) from (A) and (B) were shown as fold-change of mean fluorescence intensity (MFI). Data are the mean ± SEM from three independent experiments. N.S. = not statistically significant.
Mentions: A report by Wilson et al.16 demonstrated that hematopoietic stem cell compartments, including macrophages, developed normally in Numb and Numb-like double-knockout mice. Our system employed an insertion of the retroviral vector in hematopoietic cells from the bone marrow, causing constitutive silencing of Numb. We, therefore, tested whether silencing of Numb affects the development of macrophages from bone marrow hematopoietic stem cells in vitro. After retroviral transduction of pMKO.1-shNumb-GFP or control vectors, bone marrow cells were induced to differentiate to macrophages. Cells containing the vectors were gated for GFP+ and assayed for the expression of two macrophage-specific surface markers, F4/80 and CD11b. Our results showed that the expression levels both of F4/80 and CD11b were comparable between control and Numb-silenced macrophages (Fig. 2A–D), indicating that Numb was dispensable for macrophage differentiation in vitro.

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