8-Oxoguanine DNA Glycosylase 1 Recruits Transcription Factor STAT1 to Promote the Inflammatory Responses in Sickle Cell Disease

A key intermediate in the inflammatory pathway is 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent oxidation DNA product of reactive oxygen species (ROS). In the process of repair and release of 8-oxoG from the genome, 8-Oxoguanine-DNA glycosylase-1 (OGG1) has been reported to increase expression of pro-inflammatory cytokines. In our previous study, we showed that 8-oxoG and OGG1 were highly expressed in peripheral CD14+ monocytes from patients with sickle cell disease (SCD) and peritoneal macrophages from Townes HbSS mice. Macrophages incubated with plasma from SCD-HbSS patients showed increased expression of pro-inflammatory cytokines IL-1ꞵ, IL-6, TNF-α, which was largely blocked by pretreatment with SU0268, a selective OGG1 inhibitor, further confirmed using OGG1-unique siRNAs.

Objective: To investigate the mechanism of how OGG1 regulates expression of the pro-inflammatory cytokines utilizing mass-spectrometry analysis.

Methods:

Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD-HbSS enrolled under protocols NCT03049475 and NCT00047996). CD14+ monocytes were isolated from PBMCs of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). Human THP-1 monocytic cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. To acquire macrophages from monocytes, THP1 cells were incubated with 50 ng/ml PMA (Sigma-Aldrich, MO USA) for 24h. One transfection reagent and two siRNAs from an evaluation of four different transfection reagents and four OGG1-unique siRNAs were chosen for the study. After differentiation, macrophages derived from THP1 cells were transfected with OGG1 siRNA for 48h and then incubated with HbAA or HbSS steady state or crisis plasma, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, and TNF-α mRNA. To identify the binding partner of OGG1, a mass-spectrometry method was employed to analyze the product immunoprecipitated by OGG1. The identified proteins were further filtered based on the Human Transcription Factor database. Differential expressed proteins were selected based on t-test and fold change along with volcano plot. Null expression PSM was adjusted by adding negligible value. All analyses were conducted with R (v4.3.2). The interaction between OGG1 and STAT1 was confirmed by co-immunoprecipitation, and the level of tyrosine phosphorylation of STAT1 (Tyr-p-STAT1) measured by Western blotting. To confirm the effect of STAT1 on immune response, THP1 derived macrophages were treated with fludarabine (STAT1 inhibitor) or transfected with two STAT1-unique siRNAs, and then incubated with plasma from HbSS patients, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA. The putative binding sites of STAT1 on IL-1ꞵ, IL-6, and TNF-α promoters were predicted by JASPAR database.

Results:

We first confirmed that siRNA knockdown of OGG1 blocked the increased expression of IL-1ꞵ, IL-6, and TNF-α in macrophages treated with plasma from HbSS patients (steady and crisis status). We next sought to identify the binding partners of OGG1 in the initiation of expression of these inflammatory cytokines. Mass-spectrometry analysis of OGG1 immunoprecipitation product identified more than one thousand proteins, of which 81 were transcription factors. Signal transducer and activator of transcription 1 (STAT1) ranked as the second highest occurrence. We confirmed the interaction between OGG1 and STAT1 via co-immunoprecipitation. Interestingly, the activity of STAT1 (Tyr-p-STAT1) in HbSS CD14+ monocytes is higher than that in HbAA CD14+ monocytes, suggesting STAT1 as a potential therapeutic target of inflammation in SCD. To investigate the role of STAT1 in regulating cytokine expression, we treated THP1 derived macrophages with fludarabine or STAT1 siRNA. Plasma from HbSS patients increased the expression of OGG1, IL-1ꞵ, IL-6, and TNF-α, and the increase was largely blocked by fludarabine or STAT1 siRNA. A search of JASPAR database revealed three, three, and one putative binding sites of STAT1 on the promoters of IL-1ꞵ, TNF-α, and IL-6, respectively, which suggests a direct regulatory role of STAT1 on these pro-inflammatory cytokines.

Conclusion:

OGG1 recruits STAT1 to regulate the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.