Ceramide kinase regulates TNF-α-induced immune responses in human monocytic cells

Abstract

Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1β and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.

Figure 1

CERK inhibition blocks the TNF-α mediated pro-inflammatory changes in THP-1 cells. THP-1 cells were pretreated with CERK inhibitor (NVP-231: 12 nM) or vehicle for 1 h and then incubated with TNF-α for 2 h. Cells were harvested and mRNA expression of CD11c, CD11b and HLA-DR was determined by real-time RT-PCR (A). After 6 h treatment with TNF-α, cells were stained with antibodies against CD11c, CD11b ad HLA-DR along with isotype-matched control antibody. Surface expression was assessed by flow cytometry (B); data are presented as a bar graph of mean staining index, and representative histograms. All data are expressed as mean ± SEM (n ≥ 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 versus vehicle.

Figure 2

CERK inhibition blocks the TNF-α-mediated pro-inflammatory changes in primary human monocytes. Human primary monocytes were pretreated with CERK inhibitor (NVP-231: 12 nM) or vehicle for 1 h and then incubated with TNF-α for 2 h. Cells were harvested and mRNA expression of CD11c and CD11b was determined by real-time RT-PCR (A). After 6 h treatment with TNF-α, cells were stained with antibodies against CD11c, CD11b and CD14 along with isotype-matched control antibody. Surface expression of CD14⁺CD11c⁺ and CD14⁺CD11b⁺ was assessed by flow cytometry (B); data are presented as a bar graph of mean staining index, and representative histograms. All data are expressed as mean ± SEM (n ≥ 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 versus vehicle.

Figure 3

CERK inhibition suppresses the TNF-α-induced expression of IL-1β and MCP-1 in monocytes. Monocytic cells (primary monocytes, THP1 cells) were pretreated with CERK inhibitor (NVP-231: 12 nM) or vehicle for 1 h and then incubated with/without TNF-α for 12 h. Secreted IL-1β and MCP-1 proteins in culture media were determined by ELISA. IL-1β and MCP1 secreted by THP-1 cells (A,B), and primary monocytes (C,D). All data are expressed as mean ± SEM (n ≥ 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 versus vehicle.

Figure 4

TNF-α mediated pro-inflammatory monocytic responses require CERK. THP-1 monocytes and primary human monocytes were transfected with scrambled-siRNA (negative control; NC) or CERK siRNA and incubated for 36 h. Real-time RT-PCR was performed to measure (A) CERK mRNA and protein expression in THP-1 monocytic cells and (B) primary monocytes. CERK-downregulated THP-1 cells were treated with TNF-α and vehicle. Cells were stained with antibodies against CD11c and CD11b along with isotype-matched control antibody and surface expression of these proteins was measured by flow cytometry. Flow cytometry data are presented as a bar graph of mean staining index of the selected inflammatory markers (C). CD11c and CD11b were determined by real-time RT-PCR (D). Surface expression of CD14⁺CD11c⁺ and CD14⁺CD11b⁺ was determined in primary monocytes by flow cytometry (E) CD11c and CD11b mRNA expression was determined by real-time RT-PCR (F). All data are expressed as mean ± SEM (n ≥ 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 versus vehicle.

Figure 5

CERK downregulation attenuates the IL-1β and MCP-1 secretion in TNF-α activated monocytes. THP-1 monocytes and primary human monocytes were transfected with scrambled-siRNA (negative control; NC) or CERK siRNA and incubated for 36 h. CERK-downregulated THP-1 cells and primary human monocytes were treated with TNF-α for 12 h. Secreted IL-1β and MCP-1 proteins were measured in culture media using ELISA. IL-1β and MCP-1 secreted by THP-1 cells (A,B), and primary human monocytes (C,D). All data are expressed as mean ± SEM (n ≥ 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 versus vehicle.

Figure 6

TNF-α induces ceramides in monocytic cells. THP-1 monocytes were treated with vehicle or TNF-α for 12 h. Cellular lipid levels were analyzed. (A) C16 ceramide, (B) total ceramides, (C) C16 C1P, and (D) total C1P by ESI/MS/MS at Stony Brook University Lipidomics Shared Resource Core and data were normalized to total lipid phosphate (Pi). Data represent mean ± SEM, n = 4, *p < 0.05 as compared to vehicle treatment.

Figure 7

CERK inhibition downmodulates TNF-α-induced activation of MAPK and NF-κB signaling pathways in THP-1 cells. THP-1 monocytic cells were pretreated with CERK inhibitor (NVP-231: 12 nM) and then incubated with TNF-α. Cell lysates were prepared as described in “Materials and methods” section. Samples were run on denaturing gels. Immuno-reactive bands were developed using an Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, IL, USA) and visualized by Molecular Imager ChemiDoc MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA). (A) Phosphorylated proteins of SPAK/JNK, (B) p38 and (C) NF-κB are shown in the upper panels with the lower panel representing respective total proteins. The phosphorylation intensity was quantified by using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and presented in bar graphs as arbitrary unit (AU) of corrected protein expression. Signaling proteins were also determined by flow cytometry. Cell were immediately fixed and permeabilize for 20 min at 4 °C, then stained to visualize the JNK, p38 and NF-κB phosphorylation. Flow cytometry data are presented as a bar graph of mean staining index (SI) as well as by representative histograms (D,E). Bar graphs depict the mean values ± SEM of staining intensity (SI). P < 0.05 was considered as statistically significant (*p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The data in all figures are representative of three independent experiments. NF-κB/AP-1 reporter monocytic cells were pretreated with CERK inhibitor (NVP-231: 12 nM) or vehicle for 1 h and then incubated with TNF-α for 12 h. Cell culture media were assayed for SEAP reporter activity, representing NF-κB/AP-1 activation (F). Reporter cells were also tested for surface expression of CD11c (G).

Figure 8

Association between TNF-α and CERK expression in PBMCs from obese individuals. PBMCs were isolated from human peripheral blood samples obtained from lean (n = 13), overweight (n = 14) and obese (n = 13) individuals. CERK and TNF-α mRNA expression was detected by real-time RT-PCR and represented as fold change over controls (A,B). Pearson’s correlation coefficient (r) is shown between CERK and TNF-α (C).

Figure 9

Schematic illustrating the involvement of CERK in TNF-α-mediated inflammatory responses in human monocytic cells. The figure was created using BioRender.com.