p16-senescent fibroblasts in culture spread senescence to other cells through membrane vesicles without the classical inflammatory response
p16-senescent fibroblasts in culture spread senescence to other cells through membrane vesicles without the classical inflammatory response
On October 8, Ana O'Loghlen's group in Madrid published in the journal Aging experiments on cultures of human fibroblasts, cells that make up connective tissue. Fibroblasts driven into senescence by the protein p16 do not secrete the familiar inflammatory factors, so it remained unclear how they affect their surroundings. Their membrane vesicles arrested division in other fibroblasts and induced signs of DNA damage in them.
A senescent cell has stopped dividing in response to damage or stress but continues to influence its neighbors. It secretes a set of factors known as the SASP. This set typically includes inflammatory proteins such as interleukins, which recruit the immune system to clear the cell. It also contains extracellular vesicles, small membrane-bound particles that carry proteins and nucleic acids.
In 2011, Judith Campisi's laboratory artificially activated the protein p16 in human cells: p16 inhibits cell division and serves as a marker of senescent cells, which become more numerous with age. The cells stopped dividing but did not secrete inflammatory factors and had no effect on epithelial cells, a tissue that covers body surfaces. The laboratory concluded that the SASP is a damage response that can be separated from growth arrest. In 2019, O'Loghlen's group showed that both soluble factors and vesicles transmit senescence to neighboring cells. The group has now tested whether vesicles from p16-senescent cells also induce senescence in their neighbors.
The experiments are described in a preprint on bioRxiv, an early version of the work released before peer review. Three types of fibroblasts were used: control cells, cells made senescent by the oncogene RAS, and cells made senescent by p16. RAS is a mutant gene that constitutively signals the cell to divide; in response, the cell irreversibly arrests and enters senescence. RAS-senescent cells served as a model of conventional senescence and produced an inflammatory SASP; p16-senescent cells did not produce one but released more vesicles than controls.
Vesicles isolated by centrifugation were added to dividing fibroblasts for three days. Vesicles from p16-senescent cells arrested division in the recipient cells, raised their endogenous p16 levels, and increased γH2AX, a marker of DNA damage. According to the paper, they did not trigger production of the interleukins IL-6 and IL-8, whereas vesicles from RAS-senescent cells, according to the preprint, did trigger IL-8.
Vesicles also transmitted senescence in two other cellular models. In one, cells expressed progerin, a protein that causes progeria, a disease of accelerated aging. In the other, the function of telomeres, the protective caps at chromosome ends, was disrupted. A mixture of vesicles from fibroblasts of four donors aged 67 to 81 raised β-galactosidase activity, a marker of cellular senescence, in fibroblasts from donors aged 1 to 3.
With age, the immune system becomes less effective at clearing senescent cells. NK cells (natural killer cells) from donors around age 70 were less effective at killing senescent fibroblasts in culture than the same cells from young donors around age 21. The preprint authors propose that p16-senescent cells, which accumulate with age, produce a weak or absent inflammatory signal that would normally recruit immune clearance, and that they therefore spread senescence through vesicles instead. Damaged neighbors arrest without inflammation, and, in the authors' view, tissue renewal suffers as a result.
Senescent cells with a suppressed SASP have also been described in the blind mole rat Spalax, a long-lived subterranean rodent: in its senescent fibroblasts, the expression of genes for interleukins and other SASP factors was undetectable or lower than in mouse and human cells. The authors of that study attribute this to efficient DNA repair: the damage that would trigger the SASP does not accumulate.