A Role for Chlorinated Nucleosides in the Perturbation of Macrophage Function and Promotion of Inflammation

Abstract

During inflammation, myeloperoxidase released from activated phagocytes generates the highly reactive oxidant hypochlorous acid (HOCl). This oxidant plays an important role in the immune response but can also promote tissue damage and has been strongly linked with the development of numerous inflammatory diseases. HOCl reacts with cellular DNA forming chlorinated nucleobases, which induce strand breaks, mutations, and cross-links. Although it has been shown that chlorinated nucleosides are present within inflammatory pathologies and diseased tissue, whether or not these species are biomarkers formed as a byproduct of chronic inflammation or play a role in the disease progression has not been ascertained. In this study, we show that exposure of J774A.1 macrophage-like cells to chlorinated ribose and deoxyribose nucleosides results in the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA), and 8-chloro-guanosine (8ClG) into the cellular RNA and 5-chloro-deoxycytidine (5CldC) but not 8-chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA) into cellular DNA. Evidence was obtained for the clearance of 5ClC from the RNA, with a loss of 8ClA and 8ClG observed to a lesser extent, whereas an increase in the level of 5CldC in DNA was seen on further incubation of treated cells in the absence of chlorinated nucleosides. Importantly, exposure of the macrophages to chlorinated nucleosides, particularly 8ClG and 5ClC, resulted in the increased expression of interleukin-1β, and other pro-inflammatory cytokines and chemokines. With 5ClC, this inflammatory response was associated with the increased nuclear translocation of the NF-κB subunit, p65, rather than inflammasome activation. This alteration in gene expression appeared to be unrelated to the extent of incorporation of the chlorinated nucleosides into RNA or DNA and was not associated with any significant changes in cell viability or proliferation. Taken together, these results highlight a potential biological role for chlorinated nucleosides to promote inflammatory disease, in addition to their utility as biomarkers.

8-Chloroadenosine Alters the Metabolic Profile and Downregulates Antioxidant and DNA Damage Repair Pathways in Macrophages

“…Initial studies were performed to assess the effect of prolonged exposure (up to 72 h) of J774A.1 cells to different chlorinated ribose (8ClA, 8ClG, 5ClC) and deoxyribose (8CldA, 8CldG, 5CldC) nucleosides (50 μM, 0.16 pmol/cell) on the cell viability by measuring alterations in the metabolic activity of the cells compared to nontreated cells. There was no significant change in metabolic activity on treatment of the cells with any of the chlorinated nucleosides for 4 or 24 h compared to the nontreated cells (Figure A) in agreement with previous studies . However, a significant, time-dependent, loss in metabolic activity was observed on increasing the incubation time of the cells to 48 and 72 h with 8ClA but not the other chlorinated nucleosides (Figure A).…”

“…The mechanism involved appears to be independent of ATP depletion but may be related to the accumulation of 8Cl-ATP or the activation of other cellular stress responses. We showed previously that 8ClA incorporates into the RNA of J774A.1 cells under comparable experimental conditions, which may influence the expression of genes relating to cell survival and stress responses. The incorporation of 8Cl-ATP into RNA of malignant cells is reported to promote apoptosis by transcription inhibition of short-lived antiapoptotic proteins. , However, no evidence was obtained for any alteration in the mRNA expression of BCL2L1, an antiapoptotic protein that prevents the release of cytochrome c by stabilizing the mitochondrial membrane …”

“…In these experiments, the cells were exposed to each chlorinated nucleoside for 24 h, where no significant change in metabolic activity or cell viability was observed. Initial studies focused on the expression of genes associated with DNA repair and apoptosis in light of previous studies showing that 8ClA, together with the other chlorinated nucleosides, incorporate into the cellular RNA and DNA of this cell type under analogous treatment conditions …”

“…In addition, whether the observed changes in gene expression in the macrophages are associated with incorporation of 8Cl-ATP into RNA with subsequent effects on transcription, translation, or mRNA stability is not known. We have shown previously that exposure of macrophages to 8ClA results in an increase in the incorporation of this analog into cellular RNA, though the specific RNA pool affected was not examined …”

“…Exposure of cells to chlorinated nucleosides in vitro results in their incorporation into cellular RNA or DNA in a manner dependent on the nucleoside structure as well as the specific cell type under investigation. ,− This has a range of resulting cellular consequences, which are again dependent on the structure of the chlorinated nucleoside. Chlorinated nucleosides are mutagenic ,, rendering some chlorinated nucleosides (e.g., 5CldC) useful as radiosensitizers within therapeutic settings, , whereas their formation and presence during chronic inflammatory conditions has been proposed as a potential pathway to the initiation of cancer. , More recently, it has been suggested that the formation of 5ClC and 8ClG could help to propagate inflammation through the ability of these chlorinated nucleosides to increase the expression of key pro-inflammatory cytokines interleukin 1β and interleukin 18 in macrophages …”