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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 cytokines in macrophages lacking Numb after LPS stimulation.(A–D) Secretion levels of TNFα (A), IL-6 (B), IL-12p70 (C), and IL-10 (D) from control- (open bars) or shNumb-infected macrophages (closed bars) following LPS stimulation were measured by ELISA. (E–H) mRNA levels of Tnfα (E), Il6 (F), Il12p40 (G), and Il10 (H) from control- or shNumb-infected macrophages following LPS stimulation were measured by qPCR. Data are the mean ± SEM from the representative experiment of two independent experiments performed in triplicates.
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f3: Expression of cytokines in macrophages lacking Numb after LPS stimulation.(A–D) Secretion levels of TNFα (A), IL-6 (B), IL-12p70 (C), and IL-10 (D) from control- (open bars) or shNumb-infected macrophages (closed bars) following LPS stimulation were measured by ELISA. (E–H) mRNA levels of Tnfα (E), Il6 (F), Il12p40 (G), and Il10 (H) from control- or shNumb-infected macrophages following LPS stimulation were measured by qPCR. Data are the mean ± SEM from the representative experiment of two independent experiments performed in triplicates.

Mentions: Our group and others reported that abrogating Notch signaling diminishes the ability of macrophages to secrete pro-inflammatory cytokines and perform other effector functions27282940. Therefore, we hypothesized that pro-inflammatory cytokines under the regulation of Notch signaling would increase in macrophages wherein Numb was silenced. Surprisingly, we found that Numb-silenced macrophages secreted significantly less TNFα, IL-6, and IL-12 after LPS stimulation (Fig. 3A–C). In contrast, the anti-inflammatory cytokine, IL-10, was higher in Numb-silenced macrophages (Fig. 3D). Furthermore, the expression of the costimulatory molecule CD86 decreased significantly (Supplementary Figure 1B) after LPS stimulation, whereas the expression of MHCII was not different between LPS-activated Numb-sufficient and Numb-deficient macrophages (Supplementary Figure 1C). These results suggest that Numb positively regulates pro-inflammatory programs in macrophages. We next investigated cytokine mRNA expression. We observed no difference in the levels of Tnfα between Numb-deficient and control macrophages (Fig. 3E). In contrast, the levels of Il6 and Il12p40 were readily down-regulated in Numb-deficient macrophages at all time points tested (Fig. 3F,G). In addition, the mRNA level of Il10 and Ifnβ was increased at 4 hrs after stimulation (Fig. 3H and data not shown). From these results, we conclude that the expression of Il6, Il12p40 and Il10, but not Tnfα, are regulated by Numb at the transcriptional level.


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 cytokines in macrophages lacking Numb after LPS stimulation.(A–D) Secretion levels of TNFα (A), IL-6 (B), IL-12p70 (C), and IL-10 (D) from control- (open bars) or shNumb-infected macrophages (closed bars) following LPS stimulation were measured by ELISA. (E–H) mRNA levels of Tnfα (E), Il6 (F), Il12p40 (G), and Il10 (H) from control- or shNumb-infected macrophages following LPS stimulation were measured by qPCR. Data are the mean ± SEM from the representative experiment of two independent experiments performed in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Expression of cytokines in macrophages lacking Numb after LPS stimulation.(A–D) Secretion levels of TNFα (A), IL-6 (B), IL-12p70 (C), and IL-10 (D) from control- (open bars) or shNumb-infected macrophages (closed bars) following LPS stimulation were measured by ELISA. (E–H) mRNA levels of Tnfα (E), Il6 (F), Il12p40 (G), and Il10 (H) from control- or shNumb-infected macrophages following LPS stimulation were measured by qPCR. Data are the mean ± SEM from the representative experiment of two independent experiments performed in triplicates.
Mentions: Our group and others reported that abrogating Notch signaling diminishes the ability of macrophages to secrete pro-inflammatory cytokines and perform other effector functions27282940. Therefore, we hypothesized that pro-inflammatory cytokines under the regulation of Notch signaling would increase in macrophages wherein Numb was silenced. Surprisingly, we found that Numb-silenced macrophages secreted significantly less TNFα, IL-6, and IL-12 after LPS stimulation (Fig. 3A–C). In contrast, the anti-inflammatory cytokine, IL-10, was higher in Numb-silenced macrophages (Fig. 3D). Furthermore, the expression of the costimulatory molecule CD86 decreased significantly (Supplementary Figure 1B) after LPS stimulation, whereas the expression of MHCII was not different between LPS-activated Numb-sufficient and Numb-deficient macrophages (Supplementary Figure 1C). These results suggest that Numb positively regulates pro-inflammatory programs in macrophages. We next investigated cytokine mRNA expression. We observed no difference in the levels of Tnfα between Numb-deficient and control macrophages (Fig. 3E). In contrast, the levels of Il6 and Il12p40 were readily down-regulated in Numb-deficient macrophages at all time points tested (Fig. 3F,G). In addition, the mRNA level of Il10 and Ifnβ was increased at 4 hrs after stimulation (Fig. 3H and data not shown). From these results, we conclude that the expression of Il6, Il12p40 and Il10, but not Tnfα, are regulated by Numb at the transcriptional level.

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