Complement component 5a receptor 1 and leukotriene B4 receptor 1 regulate neutrophil extracellular trap (NET) formation through Rap1a/B-Raf/ERK signaling pathway and their deficiency in term low birth weight newborns leads to deficient NETosis.

BACKGROUND

Neutrophil extracellular traps (NETs) being one of the predominant activities of neutrophils has become its key defense mechanism owing to its extensive role in inflammation and infection. However, the mechanisms regulating NET formation or NETosis still remains to be better understood. Our earlier whole genome transcriptomic data revealed two G-protein couple receptors (GPCRs) - complement component 5a receptor 1 (C5aR1) and leukotriene B4 receptor 1 (LTB4R1) were downregulated in term low birth weight (tLBW) newborns with deficient NET formation abilities. Neutrophils employ C5aR1 and LTB4R1 for mediating their immune responses, inflammation and antimicrobial activity. Hence, this study was aimed to explore the role of two GPCRs, C5aR1 and LTB4R1 including their downstream signaling molecules in NETs induction and regulation.

METHODS

The validation of the transcriptomic data for C5aR1 and LTB4R1 was done using quantitative real time PCR. Pharmacological inhibition of C5aR1 and LTB4R1 using W-54011 and LY223982 on neutrophils of adults and newborns' was done to study their impact on NETosis. Extracellular DNA release, Reactive oxygen species (ROS) generation, expression of NET proteins, and signaling molecules downstream to C5aR1 and LTB4R1 were quantified using plate reader based assay, immunofluorescence, and western blotting. Myeloperoxidase (MPO)-DNA quantified by flow cytometry. Knockdown studies using siRNA against C5aR1 and LTB4R1 were done in HL-60 cells derived surrogate neutrophils and expression of downstream molecules of the two GPCRs, C5aR1 and LTB4R1 signaling axis along with NET proteins was quantified by western blotting.

RESULTS

The expression of C5aR1 and LTB4R1, extracellular DNA, ROS and NET associated proteins (NE, CitH3, PAD4 and MPO) was notably increased upon NET induction in healthy adults and normal birth weight (NBW) newborns' neutrophils. Pharmacological inhibition of these two GPCRs led to substantial reduction in NETosis, extracellular DNA, ROS generation, and expression of NET associated proteins like CitH3, NE, PAD4, MPO along with downstream signaling molecules Rap1a, B-Raf and pERK. Our observations suggest a precise role of C5aR1 and LTB4R1 on induction of NETs via Rap1a/B-Raf/ERK signaling axis.

CONCLUSION

The C5aR1 and LTB4R1 signaling via Rap1a/B-Raf/ERK axis acts as a signal-relay mechanism to regulate NET formation in neutrophils. Further, C5aR1 and LTB4R1 signaling cascade along with NET-associated proteins are remarkably downregulated in tLBW newborns' neutrophils leading to impaired NETosis in them. Therefore, C5aR1 and LTB4R1 and their signaling molecules could provide an effective therapeutic target for compromised NETosis like tLBW newborns.