(H-289) A Glycogen Synthase Kinase-3 Inhibitor Attenuates cGAMP Induced CXCL10 Production by Peripheral Blood Mononuclear Cells

During the innate immune response, immune cells, e.g., monocytes/macrophages, sense pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) which leads to cytokine, including a subset of cytokines termed chemokines, expression. One important chemokine often generated in this process is CXCL10 (a.k.a IP-10) (1). While the expression of CXCL10 is critical for an efficient host response, aberrant CXCL10 activity has been implicated in a host of pathologies [e.g. cytokine storms, arthritis, severe COVID-19 (2–4)]. Consequently, inhibiting aberrant CXCL10 expression is an emerging therapeutic target [e.g.(4)]. Our group has identified a novel small molecular compound, namely COB-187, that appears to be a highly potent and selective inhibitor of glycogen synthase kinase-3 (GSK-3) (5). We have previously shown that COB-187 attenuates LPS (a bacterial PAMP)-induced cytokine expression (6). In particular, we found that COB-187 dramatically reduces LPS induction of CXCL10 (6). Another important PAMP/DAMP process is immune recognition of dsDNA. A key part of this process can be cyclic GMP–AMP synthase (cGAS) recognition of dsDNA leading to the generation of 2′3′ cyclic GMP–AMP (cGAMP) (7–10). cGAMP in turn can activate the STING pathway ultimately leading to the generation of CXCL10 (9, 11). Further, exogenous cGAMP can activate plasma membrane STING (12) or intracellular STING by entering the cytoplasm via SLC19A1 (13). Combining all of these observations led us to test the hypothesis that COB-187 inhibits cGAMP-induced CXCL10 expression.    

Frozen suspensions of deidentified peripheral blood mononuclear cells (PBMCs) were obtained from Cellular Technology Limited (Shaker Heights, OH) and maintained in liquid nitrogen until used for an experiment. For an experiment, the PBMCs were thawed and washed. Aliquots of PBMCs were placed into wells of a multi-well plate in regular media or media containing: 20 mg/ml of cGAMP (InvivoGen; San Diego, CA); or 20 mg/ml cGAMP with either 0.5% DMSO (carrier control for COB-187) or 0.5% DMSO along with various concentrations of COB-187. Once treated, PBMCs were incubated at standard culture conditions for 6 hours. Subsequently, the supernatants and PBMCs were harvested and the level of CXCL10 protein in the supernatants determined via enzyme linked immunosorbent assays (ELISAs) and the PBMC mRNA analyzed via RT-PCR (6).  

Results: We found that the CXCL10 protein concentration in supernatants from PBMCs treated with cGAMP is significantly higher than the concentration found in supernatants from untreated PBMCs. Further, we found that COB-187 potently attenuates cGAMP-induced expression of CXCL10 protein with an IC₅₀ of ~1 mM. Similar results were obtained at the mRNA level; cGAMP-induced CXCL10 mRNA expression by PBMCs and COB-187 dramatically attenuated this induction.

Conclusion: COB-187, a potent and selective inhibitor of GSK-3 (5), dramatically reduces cGAMP induction of CXCL10 protein and mRNA expression in PBMCs. 

Discussion: Treatment of PBMCs with cGAMP leads to CXCL10 expression which is inhibited by COB-187. Several reports suggest that cGAMP may directly induce CXCL10 and/or indirectly induce CXCL10 via secondary signaling (e.g. through interferon protein expression) (11, 14–16). Our laboratory is currently investigating the details of cGAMP induction of CXCL10 in PBMCs and COB-187 attenuation of this induction. 

This work was funded by the National Institutes of Health (R15GM110602) and the National Science Foundation (CDS&E Grant 1953311).

1.         AD Luster, et al., Nature. 315, 672–676 (1985).
2.         F Coperchini, et al., Front Immunol. 12, 668507 (2021).
3.         MA Murayama, et al., Front Immunol. 14, 1100869 (2023).
4.         DC Fajgenbaum, CH June, N Engl J Med. 383, 2255–2273 (2020).
5.         MS Noori et al., Am J Phys-Cell Phys. 317, C1289-C1303 (2019).
6.         MS Noori, et al., Eur J Pharmacol. 883, 173340 (2020).
7.         L Sun, et al., Science. 339, 786–791 (2013).
8.         J Wu et al., Science. 339, 826–830 (2013).
9.         EJ Diner et al., Cell Rep. 3, 1355–1361 (2013).
10.       P Gao et al., Cell. 153, 1094–1107 (2013).
11.       N Congy-Jolivet et al., EBioMedicine. 80, 104047 (2022).
12.       X Li et al., J Clin Invest. 132, doi:10.1172/JCI144339.(2022).
13.       RD Luteijn et al., Nature. 573, 434–438 (2019).
14.       J Brownell et al., J Virol. 88, 1582–1590 (2014).
15.       K Motani, et al., J Immunol. 194, 4914–4923 (2015).
16.       PS Sung et al., Sci Rep. 7, 6387 (2017).