Investigating Asymmetric Dimethylarginine (ADMA) and Calcium-sensing receptor (CaSR) signalling in the macrophage

Abstract

Macrophages play a significant role in maintaining physiological homeostasis and orchestrating immune responses. In vascular diseases like atherosclerosis, they actively participate in plaque formation and progression by accumulating lipids and modulating inflammatory processes. A G-protein coupled receptor (GPCR) known as the calcium-sensing receptor (CaSR) is activated by a plethora of ligands and couples to distinct signalling cascades. Initially recognised for its role in regulating calcium (Ca2+) levels in calciotropic tissues, the CaSR has since been revealed to exhibit tissue/cell-specific effects. In macrophages, CaSR expression is confirmed where it influences immunological processes. Our research group demonstrated that asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, acts as a CaSR ligand in adipocytes, exerting NOS- independent effects. Based on this, it was hypothesised that a similar signalling pathway operates in macrophages, modulating inflammation and can be targeted in disease. In this study, immunocytochemistry confirmed the expression of both CaSR and the ADMA-metabolising enzyme, dimethylarginine dimethylaminohydorlase-2 (DDAH-2), in the RAW264.7 macrophage cell line used in this investigation, indicating their functional presence within these cells. Furthermore, their mRNA expression was unaffected by the pro-inflammatory stimuli, lipopolysaccharides (LPS). Next, the immunological impact of the DDAH-2/ADMA metabolic pathway in inflammation was explored using RNA-sequencing of LPS-stimulated murine macrophages with a macrophage-specific DDAH-2 knockout. This revealed that DDAH-2 plays a crucial role in modulating several genes associated with the LPS- response, exerting an anti-inflammatory effect. Specifically, DDAH-2 attenuates LPS-stimulated effects in macrophages, including antigen presentation, phagocytosis and chemotaxis. Additionally, DDAH-2 was found to influence the expression of a number of CaSR target genes that were previously identified in the literature. Furthermore, qPCR investigations on the role of ADMA in the expression of selected genes that displayed differential expression upon DDAH-2 deletion showed that ADMA regulates Il17a expression, highlighting a pro-inflammatory ii effect of ADMA in these cells. This study further revealed that ADMA does not influence the expression of any of the other selected genes for validation, including those recognsised as CaSR targets. This demonstrates that DDAH-2 has ADMA-independent effects in activated macrophages. This thesis also delved into the impact of CaSR on macrophage function, revealing its pro-inflammatory role. Specifically, in non-stimulated macrophages, activation of CaSR by its positive allosteric modulator (PAM), cinacalcet, induced IL-6 expression. ADMA treatment in these cells did not elicit a similar response, indicating the absence of ADMA- CaSR crosstalk in IL-6 regulation. This study further demonstrated the pro- inflammatory effect of ADMA in non-stimulated macrophages where ADMA induced a dose-dependent induction of IL-1β and TNF-α which was independent of CaSR signalling. By developing a novel macrophage-specific CaSR knockout mouse model, this thesis validated the utility of this model for future investigations into macrophage CaSR. This validation was achieved through confirmed significant deletion of CaSR and the viability of these mice. Furthermore, this work demonstrated that macrophage CaSR deficiency resulted in the upregulation of TNF-α in non- stimulated macrophages, highlighting CaSR’s role in regulating both pro- and anti- inflammatory effects in macrophages. Additionally, the absence of the CaSR in macrophages resulted in an age- and sex-dependent reduction in weight gain observed in female mice, an observation that requires exploration in future studies. Taken together, this thesis demonstrates DDAH-2, ADMA, and CaSR as regulators of the macrophage response. Nevertheless, it did not reveal a signalling crosstalk between ADMA and CaSR within this cell type. Future research should focus on elucidating the mechanisms underlying the observed effects, where they can be explored as potential drug targets against pathologies where macrophages are implicated.