Investigators have demonstrated the potential to reduce inflammatory responses in diseases such as rheumatoid arthritis using a small molecule agonist of the chemokine receptor CXCR3. Researchers at Newcastle University and Northumbria University in the U.K. have found that a previously reported CXCR3-specific agonist effectively desensitizes T cell chemokine receptors and stops the migration of activated T cells to chemokines present in the synovial fluid of patients with active RA. Importantly, the compound had no effect on the mobilization of regulatory T cells.
Newcastle’s John A. Kirby, Ph.D., and colleagues, report their findings in PNAS, in a paper titled “Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.”
Inflammatory diseases such as rheumatoid arthritis are mobilization of T lymphocytes in diseases such as rheumatoid arthritis is regulated via stimulation of the cells’ chemokine receptors. The human chemokine system is also complex, comprising some 44 ligands and 21 G protein-coupled receptors (GPCRs) that have so far been identified.
The situation is complicated further because most chemokines can activate more than one receptor, and a number of the receptors have more than one chemokine ligand. In fact, over 12 chemokines have been identified in synovial fluid samples from patients with rheumatoid arthritis, the researchers write. Although a number of chemokine receptor/ligand-neutralizing antibodies and small molecule receptor antagonists have been developed as therapeutic approaches to inflammatory diseases, it isn’t yet clear whether these will be effective in a clinical setting.
The Newcastle University resaerchers’ current work focused on the potential to dampen responses triggered by the chemokine receptor CXCR3. Activation of CXCR3 by CXCL9, CXCL10, and CXCL11 represents a proinflammatory signal that recruits activated CXCR3-expressing T cells. Indeed cells expressing CXCR3 have been implicated in a range of diseases including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis
Dr. Kirby’s team and others have previously generated nonglycosaminoglycan-binding chemokines that still act as agonists to their receptors, but also mediate a powerful anti-inflammatory effects by a mechanism believed to involve chemokine receptor desensitization.
One report identified a 589 Da small molecule known as PS372424, as an agonist that is specific to the human form of CXCR3, and the Newcastle team has now further evaluated the potential of the compound to desensitize CXCR3 and other chemokine receptors on activated human T cells.
Their initial studies showed that treating activated T cells with PS372424 induced high levels of ERK phosphorylation and rapid internalization of cell-surface CXCR3. Moreover, the agonist significantly inhibited T cell migration towards its CXCL11, CXCL12, or CCL5, whereas the previously reported CXCR3 antagonist NBI-74330 only prevented T cell migration towards CXCL11.
Further analysis of chemokine receptors expressed by resting and activated human T cells and human regulatory T cells (Tregs) showed that while CCR5 is primarily expressed by activated T cells and Tregs, CXCR3 is almost exclusively expressed by activated T cells, and CXCR4 is expressed at roughly the same levels by each of T cell subpopulations. Rheumatoid arthritis synovial fluid (RASF) from patients with active inflammation induced significant migration of each T-cell population, and PS37242 specifically blocked migration of activated T cells towards RASF. In contrast chemotactic migration of the three T cell populations towards RASF couldn’t be reduced by treatment with either the CXC3 neutralizing antibody or a CXCR4 antagonist AMD3100.
PS372424 is specific for human CXCR3, so to carry out in vivo studies the researchers established a murine model of human T cell migration, using animals humanized using peripheral blood mononuclear cells (PBMCs). An air-pouch model of inflammation was established in these animals, by injecting human CXCL11 into air pouches, and this induced significant recruitment of human CD45+ cells. Notably, this cell recruitment was markedly reduced following intravenous administration of PS372424, without any apparent adverse effects on the animals, even after five days of daily treatment.
Intravenous PS372424 also reduced to background levels the recruitment of human lymphocytes to air pouches filled with either CXCL12, CCL5, or, notably, human RASF. In contrast, neither the CXCR4 antagonist AMD3100, the CXCR3-blocking antibody, nor the small-molecule CXCR3-antagonist NBI-74330 could inhibit human cells in response to RASF. “This result reflects the situation during active human disease, where inflammation is produced by a myriad of chemokines,” the authors write. “Importantly, PS372424 inhibited the migration of activated T cells toward RASF but allowed normal chemotactic recruitment of resting and regulatory T cells, which both express little CXCR3, suggesting a potential to modify the composition of the inflammatory infiltrate in vivo.”
Moreover, they report, NBI-74330 wasn’t capable of modifying human cell migration toward pouches filled with CCL5. This compound doesn’t have any agonist activity, and the experimental results thus indicate that PS372424 may exert its heterologous anti-inflammatory effects by signaling through CXCR3 rather than just by blocking formation of the CXCR3/CCR5 heterodimer.
In fact, analysis of cell-surface chemokine receptor expression by human T cells recovered from the spleen of PS372424-treated humanized mice identified a significant reduction in both CXCR3 and CCR5, which FRET analysis had demonstrated form heterodimers on the cell surface. “Chemokine receptor heterodimerization suggests a mechanism by which specific activation of one receptor can induce desensitization of another,” the investigators suggest.
PS372424 may therefore desensitize multiple chemokine receptors expressed by activated T cells, “but did not affect the migration of potentially beneficial Tregs,” they conclude. “These data make a clear argument for exploration of agonist-induced chemokine receptor desensitization strategies for the treatment of important inflammatory diseases.”