Resolution of purine and pyrimidine cyclic-di-nucleotide monophosphates in lung endothelium, relevant for innate immune signaling

Purine cyclic nucleotide monophosphates (cAMP and cGMP) are well-studied second messenger signals. More recently, non-canonical pyrimidine cyclic nucleotides monophosphates (cUMP and cCMP) have been discovered and shown to play essential second messenger signaling roles, sensing infectious and/or inflammatory stimuli and triggering innate immune responses. Similarly, cyclic-di-nucleotide monophosphates (c-di-NMPs) have been resolved, and they too serve as signaling molecules linked to innate immunity. More specifically, the cyclic GMP-AMP synthase(s) (i.e., cGAS) senses cytosolic DNA, which leads to production of 2’3’-cGAMP that activates the transmembrane protein stimulator of interferon no-genes, or STING, inducing the production of type I interferons. The 2’-5’-oligoadenylate family of proteins sense double stranded RNA and produce c-di-AMP, which activates the RNase L leading to degradation of viral RNA. At present, the molecular diversity of c-di-NMPs present within mammalian cells is poorly understood. Here, we tested the hypothesis that lung microvascular endothelial cells possess diverse c-di-NMPs under basal conditions. To test this hypothesis, c-di-NMPs were extracted from flash frozen cell pellets using a 2:2:1 H 2 O/MeOH/ACN protocol, followed by analysis on an Agilent 1200 Series HPLC coupled to a Thermo Q Exactive Plus mass spectrometer and a HILICON iHILIC-Fusion (P) (2.1 x 150mm) column using a 3.0 µL injection volume for each. One full positive ionization scan from 200–800 m/z was performed at 17,500 resolution, followed by targeted MS2 scans of cGAMP (675.1045 m/z), c-di-AMP (659.1123 m/z), c-di-GMP (691.1021 m/z), and c-di-UMP (613.0578 m/z), and one full negative ionization scan from 200–800 m/z. Detection and peak analysis of c-di-NMPs were performed using Thermo XCalibur software. Pulmonary microvascular endothelial cells possessed detectable levels of cGAMP (~0.05 nmol/106 cells) and c-di-AMP (~0.05 nmol/106 cells) under basal conditions. We also detected c-di-GMP at similar basal concentrations (~0.03 nmol/106 cells). cAMP and cGMP were measured from the same samples; cNMPs were present at higher concentrations, where cAMP was ~2 nmol/106 cells and cGMP was ~0.2 nmol/106 cells. Thus, the c-di-NMPs were present at concentrations representing ~2% of the cAMP abundance and at ~20% of the cGMP levels. Remarkably, we detected the presence of c-di-UMP at a similar concentration range (~0.035 nmol/106 cells). c-di-UMP has not been previously described in any mammalian cell. The basal c-di-UMP concentrations were ~35% of cUMP levels. We next tested whether infection with exoenzyme Y competent P. aeruginosa impacts c-di-NMP abundance. Infection increased cGMP, cUMP, and to a lesser extent, cCMP, inside the cell and cGMP and cUMP outside the cell, due to activity of the promiscuous nucleotidyl cyclase exoenzyme Y. Whereas the intracellular concentrations of c-di-AMP and cGAMP were unchanged following infection, c-di-GMP levels were increased and c-di-UMP concentrations decreased to nearly undetectable levels. It is unclear whether c-di-UMP can be released from endothelial cells during infection and accumulate in the extracellular space, similar to cGMP and cUMP. Thus, we report the presence of the novel c-di-UMP in endothelium, and determine that intracellular concentrations of c-di-UMP and c-di-GMP are responsive to bacterial infection. Studies are ongoing to better understand the role of c-di-NMPs, and especially c-di-UMP, in endothelial innate immunity. Supported by HL140182, HL167997, and HL148069. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.