Optimisation of a screening platform for determining IL-6 inflammatory signalling in the senescence-associated secretory phenotype (SASP)

Abstract

Cellular senescence has been shown to be sufficient for the development of multiple age-related pathologies. Senescent cells adopt a secretory phenotype (the SASP) which comprises a large number of pro-inflammatory cytokines, chemokines and proteases. The SASP itself is thought to be causative in many pathologies of age-related diseases, and there is growing interest in developing seno-modifying agents that can suppress the SASP. However, in order to identify new agents, it is necessary to conduct moderate to high throughput screening with robust assays for the required outcome. Here, we describe optimisation and validation of a cell-based biosensor HEK cell line for measurement of IL-6 concentrations within the range secreted into conditioned medium by primary senescent fibroblasts, adapted for a 384 well plate format suitable for library screening applications. We further show that the assay can measure changes in IL-6 secretion dependent on cell population age, and that the assay is responsive to mTOR inhibition in the senescent cells, which reduces the SASP, including IL-6. Hence, we propose that this optimised biosensor, which we term HEK-SASP, may prove of value in studies requiring robust, renewable and relatively inexpensive assays for measuring SASP factors. Electronic supplementary material The online version of this article (10.1007/s10522-019-09796-4) contains supplementary material, which is available to authorized users.

Intercellular Transfer of Mitochondria between Senescent Cells through Cytoskeleton‐Supported Intercellular Bridges Requires mTOR and CDC42 Signalling

“…Cells were determined to be replicatively senescent when populations fulfilled each of the following criteria: failure to increase the cell number within >2 weeks, a cumulative population doubling number of >85, and positive SA- β -gal staining according to the manufacturer's instructions (Cell Signaling Technology #9860S). The secretion of IL-6, a canonical SASP factor, and upregulation of p21 CDKN1 (see Supplementary Figures S1 , S2 , and S3 ) were assessed as additional readouts of senescence (see [ 23 ] for western blotting conditions and [ 20 ] for ELISA protocol).…”

From causes of aging to death from COVID-19

“…Potential suppression of cytokine storm and hyperinflammation (Figure 1). As we discussed in the section "Cytokine storm is a hyperfunction", cytokine storm and hyper-inflammation is a main cause of death in COVID-19 pneumonia [36-40, 42, 45, 135, 141-143] Rapamycin, an antiinflammatory agent, inhibits hyper-functions, cellular senescence and decrease secretion of cytokines ( [74,81,144]. Rapamycin inhibits the Jak2/Stat4 signaling pathway [145] and reduces IFγ and TNF-α levels [112].…”

Skeletal Muscle Gene Expression in Long-Term Endurance and Resistance Trained Elderly

“…secretome listed in the KEGG pathway were downregulated in trained muscles, in particular the pathognomonic cytokine IL-6 [98]. Moreover, as discussed above, components of the autophagy process were upregulated by exercise training.…”