Ceramide kinase regulates TNF-\u03b1-induced immune responses in human monocytic cells

Fatema Al-Rashed,Rasheed Ahmad,Sardar Sindhu,Lina M Obeid,Ashley J Snider,Motasem Melhem,Fahd Al-Mulla,Reeby Thomas,Yusuf A Hannun,Shihab Kochumon,Zunair Ahmad

doi:10.1038/s41598-021-87795-7

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.

Highlights

Ceramide kinase (CERK) is an enzyme that regulates phosphorylation of ceramide and produces ceramide1-phosphate (C1P), a sphingolipid which is commonly implicated in inflammation[1]
To investigate whether CERK is involved in TNF-α-mediated immune responses, THP-1 monocytic cells were pre-treated with specific CERK inhibitor NVP-231(12 nM; Supplementary Fig. S1) before exposure to TNF-α
To further validate the physiological relevance of these data obtained from THP-1 cells, primary human monocytes were preincubated with NVP-231, followed by exposure to TNF-α

Summary

Introduction

Ceramide kinase (CERK) is an enzyme that regulates phosphorylation of ceramide and produces ceramide1-phosphate (C1P), a sphingolipid which is commonly implicated in inflammation[1]. The CERK- knockout (KO) mice expressed lower levels of MCP-1 which is a well-known inflammatory marker in the adipose tissue. MCP-1-induced infiltration of macrophages into the adipose tissue was significantly reduced in CERK KO mice, which associated with reduced diet-induced obesity and metabolic inflammation, as well as with increased insulin sensitivity in these m ice[7]. Monocytes exhibit an inflammatory phenotype associated with the increased expression of CD11b, CD11c and HLA-DR surface markers[8,9], along with higher secretion of proinflammatory cytokines/chemokines such as TNF-α, IL-6, IL-1β, and MCP-18,10. TNF-α is a proinflammatory cytokine overexpressed in obese humans and rodents and it has been identified as a key regulator of inflammation and insulin r esistance[11]. TNF-α activates the Scientific Reports | (2021) 11:8259

Methods

Results

Conclusion