Human CCL2/MCP-1 ELISA Kit

Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1 beta processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14(+) monocytes and Kupffer cells primarily mediates IL-1 beta release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1 beta release, reducing hepatocyte caspase 3/7 activity, IL-1 beta-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.

The Siponimod (Mayzent) is a newly developed drug, similar to Fingolimod (FTY720) but with fewer side effects, approved by the Food and Drug Administration for the treatment of multiple sclerosis (MS). The therapeutic effect of siponimod and FTY720 in MS relies on their inhibitory effect on the sphingosine 1-phosphate (S1P) signaling. These drugs bind to the S1P receptors and block the CCL2 chemokine pathway that is responsible for the exit of the immune cells from the lymphoid organs, and circulation, thus preventing immune cell-dependent injury to the nervous system. We recently found that FTY720 beside its effect on the S1P pathway also blocks the RhoA pathway, which is involved in the actin cytoskeleton-related function of macrophages, such as expression/recycling of fractalkine (CX3CL1) receptors (CX3CR1), which direct macrophages to the transplanted organs during the development of the long-term (chronic) rejection. Here we tested the effects of siponimod on the RhoA pathway and the expression of the S1P1 and CX3CR1 receptors in mouse RAW 264.7 macrophages. We found that siponimod downregulates the expression of RhoA protein and decreases the cell surface expression of S1P1 and CX3CR1 receptors. This newly discovered crosstalk between S1P and RhoA/CX3CR1 pathways may help in the development of novel anti-chronic rejection therapies in clinical transplantation.