Signaling mechanisms linking SAMHD1-deficiency to the type I interferonopathy

Abstract

SAMHD1 is an enzyme which has dual enzymatic activities: deoxynucleoside triphosphohydrolase (dNTPase) and phosphorolytic 3′-5′ exoribonuclease. Even though SAMHD1 was identified initially as the human ortholog of the mouse IFN-induced gene Mg11, studies about SAMHD1 have focused overwhelmingly on the inhibitory mechanism of SAMHD1 against HIV-1 replication. SAMHD1 was demonstrated to restrict HIV-1 replication by reducing cellular dNTP concentrations below the levels required for retroviral reverse transcription in dNTPase dependent manner. In addition, it is also suggested that SAMHD1 can bind to and degrade HIV-1 RNA to restrict the replication of HIV-1 through the RNase activity. A disturbance of the type I interferon homeostasis is central to the pathogenesis of the autoimmune diseases. The autoimmune disorder Aicardi-Goutières syndrome (AGS) is characterized by a constitutive type I interferon response clinically overlapping with congenital infection and systemic lupus erythematosus (SLE). All the genes that are mutated in patients with AGS encode enzymes (TREX1, RNASEH2, ADAR, SAMHD and IFIH1) that are associated with nucleic acid metabolism, leading to the hypothesis that the inappropriate accumulation of endogenous nucleic acid species resulting from the dysfunction of AGS-related enzymes triggers the chronic type I interferon response. The mechanisms by which malfunctions of TREX1, RNASEH2, ADAR1 and IFIH1 occur AGS are considerably investigated and suggested. However, how SAMHD1-deficiency causes the type I interferon response in patients with AGS remains unknown, even though mutations in SAMHD1 cause AGS. In addition, Samhd1-deficient mice did not exhibit any distinct clinical phenotypes. Therefore, it is very important to identify the mechanism by which SAMHD1-deficiency results in AGS in human patients. Here, I generated SAMHD1-deficient THP-1 cell lines and showed that those cell lines recapitulate AGS phenotypes which include the activation of type I interferon response and delayed cell cycle progression. I further showed that SAMHD1 proteins purified from undifferentiated THP-1 cells possessed RNase activity. Then, RNA derived from SAMHD1-deficient cells, but not that from wild-type cells neither DNA derived from wild-type and SAMHD1-deficient cells, significantly activated IFN-α expression. In addition, the reconstitution of wild-type SAMHD1 and SAMHD1D137N, which possess RNase activity, only repressed the IFN- induction in SAMHD1-deficient cells. These results suggest that cytosolic RNA species accumulated in the absence of SAMHD1 act as a major immunogenic source for the type I interferon response. I also proposed that innate sensing of the endogenous retroelements-derived transcripts accumulated in SAMHD1-deficient cells results in significant type I interferon response and this accounts for the cause of SAMHD1-related AGS. This was supported with my data showing significant portion of the retroelement RNAs identified in SAMHD1 CLIP-seq and upregulated in SAMHD1-deficient cells. Even though many nucleic acid sensing pathways were already identified, this IFN signature occurred independently of all previously known nucleic acid sensing pathways that ultimately converge on activation of TBK1. Only IRF3 was indispensable for the spontaneous IFN signature in SAMHD1-deficient cells. Therefore, I sought to identify the RNA sensing pathway associated with ISG induction in SAMHD1-deficient cells and showed that the PI3K/AKT/IRF3 signaling pathway is essential for the type I interferon response in SAMHD1-deficient THP-1 cells. AKT and IRF3 were highly activated in SAMHD1-deficient cells, as assessed by the phosphorylation levels of these molecules. Then, treatment of PI3K or AKT inhibitors dramatically reduced the type I interferon signatures in SAMHD1-deficient cells. Moreover, SAMHD1/AKT1 double knockout relieved the type I interferon signatures to the levels observed in wild-type cells. The reconstitution of wild-type SAMHD1 and SAMHD1D137N inhibited the activation of AKT in SAMHD1-deficient cells, showing that RNase activity of SAMHD1 is critical for AKT activation as well as spontaneous IFN response. In human PBMCs, siRNA-mediated SAMHD1 silencing recapitulated the phenotypes seen in SAMHD1-deficient THP-1 cells. By comparison, knockout of SAMHD1 in not only HEK293T and HeLa cells but also PMA-differentiated THP-1 cells did not result in the activation of STAT1 or the induction of ISGs, suggesting that the type I interferonopathy associated with SAMHD1-deficiency is cell type-specific. My data provide an insight not only into the pathogenesis of the type I interferonopathies but also will encourage the development and use of immunosuppressive therapies in AGS and related autoimmune diseases.