Immune Clearance of Senescent Cells Via Activation of Endogenous Retroviruses and the Viral Recognition Pathway

Expression of genomic repeat element (RE) is known to induce interferon signaling termed 'viral mimicry'. RE activates double-stranded RNA (dsRNA) recognition via mitochondrial antiviral signaling protein (MAVS), leading to activation of interferon-stimulated genes (ISG) and subsequent cell death. We recently showed that the expression of RE is dysregulated during acute myeloid leukemia (AML) development and in myelodysplastic syndrome (MDS). RE expression is suppressed in leukemic stem cells (LSCs) compared to pre-leukemic stem cells and blasts. Similarly, high-grade MDS showed significant suppression of RE and immune pathways compared to low-grade MDS. These findings suggest that the viral recognition pathway plays a significant role in hematopoietic disorders.

Senescent cells activate interferon response and secrete inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). To investigate whether the inflammatory phenotype seen in senescent cells is mediated by RE expression,we analyzed the RNA sequencing data of senescent cell lines for expression of RE. We found significant upregulation of REs following multiple modalities of senescence induction. We induced senescence in Human HSPCs and 3 different cell lines (K562, HCT116 and IMR90) using an alkylating agent, mitomycin (MMC), and/or radiation, and found significant upregulation of the measured REs. dsRNA is known to trigger the type 1 interferon response through the dsRNA sensor RIG-1/MDA5. Upon recognition of dsRNA by RIG-1, MDA5 is activated, which in turn leads to aggregation of MAVS in the outer mitochondrial membrane. Following senescence, we found aggregation of MAVS in the mitochondrial fraction of murine HSPCs and HCT116 cell. Aggregated MAVS phosphorylates IRF3 and IRF7, leading to translocation of IRF3, IRF7, and NF-κB into the nucleus, where these proteins induce expression of ISGs. Western blots of MMC-treated murine HSPCs showed increase in nuclear NF-κB and p-IRF7. qRT-PCR analysis of murine HSPCs and HCT116, IMR90, and K562 cells following MMC treatment showed increased expression of ISGs and NK ligands, suggesting activation of the dsRNA recognition pathway along with the transcription of ISGs and NK ligands upon senescence induction.

We examined whether senescent cells activate NK cells. Mononuclear cells were isolated from the blood of healthy donors when incubated with HCT116 and IMR90 cells following MMC treatment, showed activation of NK cells (6-10-fold). To study NK-mediated cell killing of senescent cells, we incubated senescent IMR90 cells (target cells) with NK92Mi (human NK) cells. MMC-treated IMR90 cells were reduced by 40%. No significant reduction in mock cells was observed. This suggests that activation of ds-RNA recognition pathway in senescence (like in viral infection) leads to up-regulation of NK ligands, NK cell activation, and clearance of senescent cells by NK cells. In order to investigate if MAVS is critical for senescence mediated immune clearance, we employed cell lines with knock out or knock down of MAVS. Abrogation of the dsRNA recognition pathway diminished the interferon response, NK ligand expression and NK mediated clearance of senescent cells.

Finally, to evaluate if senescence in human HSPCs also increases RE expression in vivo, we isolated human senescent HSPCs from healthy donors' blood using senescence associated □-galactosidase (SA-β-gal) and did RNA sequencing. Human senescent HSPCs showed overexpression of RE along with pathways of inflammation. % of SA-β-gal+ cells in HSPCs from 26 donors between 20 and 71 years of age (median = 39 years) showed no significant correlation with age (P= 0.25, R² = 0.06), suggesting that senescent HSPCs do not accumulate with age; implying they are cleared as they are formed. Taken together, these findings suggest that RE expression and the subsequent activation of the dsRNA recognition pathway plays an important role in maintaining homeostasis during aging and dysregulation of this pathway could play a role in hematopoietic disorders such as AML and MDS.