Leader: Silvano Sozzani (SAPIENZA); Other collaborator(s):
The accumulation of senescent cells in tissues underlies age-related disorders. Our initial effort concentrated on a reversible form of senescence endowed with an inflammatory secretome capable of recruiting and activating neutrophils, in part through the action of interleukin-8 (IL-8) and acute-phase serum amyloid A1 (SAA1). These findings may be relevant for the control of tissue local inflammation. As a second aspect of the project that was pursued, we characterized the regulation of chemotactic receptors in senescent neutrophils. Senescent cells upregulated CXCR4 and CCRL2. By FRET analysis we found that CCRL2 regulates CXCR4 signalling by the formation of heterodimers.
Brief description of the activities and of the intermediate results:The accumulation of senescent cells in tissues is a key driver of age-related disorders. Senescent cells profoundly influence the surrounding microenvironment by secreting numerous bioactive molecules and inflammatory factors. In this study, we investigated the induction of cellular senescence within the inflammatory tumor microenvironment using breast cancer cells, with a specific focus on senescent neutrophils. We identified a reversible form of tumor cell senescence that exhibits an inflammatory secretome capable of recruiting and activating neutrophils, partially mediated by CXCL8 and acute-phase serum amyloid A1. Neutrophil activation was associated with the release of neutrophil extracellular traps (NETs) and the phagocytic clearance of senescent tumor cells. Our findings revealed that, similar to macrophages and natural killer cells, neutrophils can be recruited and engaged by senescent tumor cells to facilitate their removal.
Furthermore, single-cell RNA sequencing (scRNA-seq) identified a unique phenotype of tumor-infiltrating senescent neutrophils, characterized by reprogrammed expression of chemotactic receptors. Specifically, senescent cells upregulated CXCR4 and CCRL2. Using FRET analysis, we demonstrated that CCRL2 forms heterodimers with CXCR4. Biochemical and functional assays suggest that CCRL2 expression may regulate CXCR4 functions through this mechanism.
Finally, we are currently investigating the interaction between CCRL2 and CMKLR1, a functional chemotactic receptor that shares the ligand chemerin with CCRL2.
Brief description of the activities and of the intermediate results:The accumulation of senescent cells in tissues is a key driver of age-related disorders. Senescent cells profoundly influence the surrounding microenvironment by secreting numerous bioactive molecules and inflammatory factors. In this study, we investigated the induction of cellular senescence within the inflammatory tumor microenvironment using breast cancer cells, with a specific focus on senescent neutrophils. We identified a reversible form of tumor cell senescence that exhibits an inflammatory secretome capable of recruiting and activating neutrophils, partially mediated by CXCL8 and acute-phase serum amyloid A1. Neutrophil activation was associated with the release of neutrophil extracellular traps (NETs) and the phagocytic clearance of senescent tumor cells. Our findings revealed that, similar to macrophages and natural killer cells, neutrophils can be recruited and engaged by senescent tumor cells to facilitate their removal.
Furthermore, single-cell RNA sequencing (scRNA-seq) identified a unique phenotype of tumor-infiltrating senescent neutrophils, characterized by reprogrammed expression of chemotactic receptors. Specifically, senescent cells upregulated CXCR4 and CCRL2. Using FRET analysis, we demonstrated that CCRL2 forms heterodimers with CXCR4. Biochemical and functional assays suggest that CCRL2 expression may regulate CXCR4 functions through this mechanism.
Experiments on the interaction between CCRL2 and CMKLR1, a functional chemotactic receptor that shares the ligand chemerin with CCRL2, are ongoing.
During this reporting period, we extended our investigation of senescence-associated immune responses by developing an in vivo model to track the migration of senescent cells across tissues, including lung, liver, and bone marrow.
Single-cell RNA sequencing analyses revealed marked heterogeneity among infiltrating immune populations, with distinct organ-specific profiles. In the lung, we identified a subset of SiglecF⁺ SA-β-Gal⁺ Ccrl2⁺ cells that progressively accumulated over time and exhibited prolonged survival together with immunosuppressive features.
Further analyses confirmed the involvement of CCRL2 in regulating immune cell dynamics within the senescent microenvironment. These findings expand previous observations by highlighting tissue-specific adaptations of senescence-associated immune responses.
During this reporting period, mechanistic analyses of CCRL2-dependent signalling pathways were completed.
FRET experiments confirmed the formation of CCRL2–CXCR4 heterodimers, and functional assays demonstrated that CCRL2 modulates CXCR4 activity, promoting neutrophil accumulation within the lung microenvironment. Evidence also suggests that this mechanism may contribute to the recruitment of additional immune populations, including natural killer cells.
These results support a model in which CCRL2 acts as a key regulator of chemotactic signalling in senescent contexts, influencing immune cell trafficking and local immune responses.
Overall, our findings identify the CCRL2–CXCR4 axis as a potential target for modulating immune cell recruitment and function in senescence-associated pathological conditions.
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