Ultrasound selectively stimulates senescent cells, promoting their removal by macrophage recruitment In Woo Kim 1,2,# , Su Hyun Lee , Hyung Min Kim , Ki Joo Pahk , So Yeon Kim 1,2,* 1,# 3 2,3 ¹Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea, 2 Division of Bio-Medical Science and Technology, KIST school, Korea University of Science and Technology (UST), Seoul 02792, Republic of Korea, ³Center for Bionics, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea #
These authors contributed equally. * Correspondence to: So Yeon Kim (e-mail: soyeonkim@kist.re.kr; telephone 82-2-958-5914; fax 82-2-958-5909), Hwarangno 14-gil 5, Seongbuk-gu, Seoul, Republic of Korea 02792 Abstract Aim Senescent cells play critical roles in aging and age-associated diseases and Can we utilize ultrasound therefore, various techniques have been developed for selective removal of senescent cells. As such, we have provided a new method to selectively stimulation to enhance stimulate senescent cells by utilizing low-intensity ultrasound treatment. We secretion of inflammation
found that ultrasound treatment enhanced cytokine secretion involved in related cytokines in macrophage recruitment in senescent cells, while no significant change was observed in normal cells. In fact, the migration rate of monocyte, macrophage senescent cells, such that (Mφ) and M1 to the senescent cells, as well as the phagocytosis activity of these activated immune cells by immune cells increased by ultrasound treatment. Due to the advantages of cytokines promote removal of ultrasound treatment, such as non-invasiveness, deep penetration capability and senescent cells? easiness of clinical
application, we expect that our method can be applied to treat • Ultrasound stimulation selectively promoted SA β-gal activity in late cells, various senescent-associated diseases, in combination with other established producing blue staining by x-gal. therapy. • Ultrasound stimulation significantly promoted inflammation-related cytokines, Material and methods • Cytokine array revealed that stimulation of late cells with ultrasound increased such as IL-6, IL-1β, GM-CSF and CCL3. secretion of various cytokines including G-CSF and GM-CSF, which attracts Background immune cells. • Supernatant of
late cells stimulated by ultrasound promoted the migration of THP-1 monocyte, Mφ, M1, and M2 macrophages. • These results suggest that ultrasound could recruit immune cells via Senescent cells cause age-related diseases upregulation of inflammation related cytokines expression and secretion. Ultrasound promotes inflammation cytokines Causes Senescent cells Effects expression via ROS dependent p38-NF-kB pathway Telomere erosion Atherosclerosis DNA damage Fibrosis Oxidative stress Arthritis Sarcopenia Tumor suppressor loss Phenotypes SA-β-gal SASP Metabolic changes • The ultrasound setup was
constructed as shown in the figure. The cell analysis was conducted a day or three days after ultrasound stimulation. Cell cycle arrest • Ultrasound exposure intensity was optimized to as shown above. • We defined late cell as HS68 between 38 and 43 passages, and the early cell as between 16 and 20 passages (see below for proliferation rate). • Cellular senescence is defined as a state of permanent and irreversible cell cycle arrest. It is caused by a variety of external stimulations or repeated divisions. Senescent cell affects surrounding cells and ultimately causes detrimental effects.
Results and discussion • Senescent cells can be characterized by enhanced senescent associated (SA) –β-gal activity and inflammation cytokine secretions called senescence- associated secretory phenotype (SASP). Ultrasound does not affect on replicative senescence Senescent cells are eliminated by immune cells • Flow cytometry analysis with ROS sensitive fluorophore revealed that stimulation of late cells with ultrasound significantly increased ROS generation. • β-gal activity was increased by ultrasound stimulation in late HS68 cells, while • Senescent cells are naturally removed NAC (ROS
scavenger) significantly decreased the β-gal activity in ultrasound- because they have various detrimental stimulated late cells. effects on our bodies. • SASP recruits immune cells, resulting • Stimulation of late cells with ultrasound enhanced the phosphorylation levels of both p38 and NFκB. in removal of senescent cells. • Pretreatment of Bay 11-7802(NF-κB inhibitor) significantly inhibited upregulation of IL-6, IL-1β and GM-CSF. • The migration of immune cells was increased by ultrasound stimulation in late cells, but pretreatment of Bay 11-7082 significantly decreased the immune Oishi,
Yumiko, et al, NPJ aging and mechanisms of disease, 2016 cells migration. • These results suggest that ultrasound stimulation promotes immune cells migration by upregulation of inflammation cytokine expression via ROS-p38- Ultrasound is used in various medical applications NFκB pathway. • Ultrasound stimulation did not caused any significant change in cell viability and cell proliferation. Conclusions • Ultrasound stimulation had no significant effect on the cell cycles and its dependent protein expression of p16 and p21. In this work, we demonstrated that ultrasound could selectively
stimulate senescent cells, enhancing immune cell recruiting for their removal. The effect Ultrasound selectively increases inflammation of ultrasound stimulation on cell viability was not significant, and neither cell related cytokines secretion in senescent cells, proliferation nor cell cycle was affected. On the other hand, both β-gal activity and inflammation cytokines were increased by ultrasound stimulation in late cells. leading to enhanced macrophage migration. Especially, ultrasound increased immune cell attraction markers, GM-CSF, CCL2 • Ultrasound is used for surgery, cancer
ablation, and palliative treatment, as and CCL3, promoting immune cell migration. Lastly, we identified that ultrasound regulates inflammation cytokines via ROS dependent-p38-NF-kB pathway. well as therapeutic imaging and drug delivery. Although further experiments, such as direct monitoring of senescent cell removal by phagocytosis is necessary, our strategy can be applied to treat various age- Ultrasound can regulate cytokine expression related diseases with simple ultrasound stimulation in vivo. References • McHugh, Journal of Cell Biology, 2018 • Amaya-Montoya, Advances in Therapy, 2020 •
Oishi, NPJ aging and mechanisms of disease, 2016 • Lu, Journal of Orthopaedic Research, 2016 Acknowledgements Lu et al, Journal of Orthopaedic Research, 2016 • Previous studies have reported that ultrasound can regulate inflammation related cytokine expression. • This work was supported the KIST grant (2E30350). [A. Aging-1] Ultrasound selectively stimulates senescent cells, promoting their removal by macrophage recruitment In Woo Kim¹˙²˙#, Su Hyun Lee¹˙#, Hyung Min Kim²˙³˙*, Ki Joo Pahk³˙*, So Yeon Kim¹˙²˙* ¹Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and
Technology, Seoul 02792, Republic of Korea, ²Division of Bio-Medical Science and Technology, KIST school, Korea University of Science and Technology (UST), Seoul 02792, Republic of Korea, ³Center for Bionics, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea Senescent cells play critical roles in aging and age-associated diseases and therefore, various techniques have been developed for selective removal of senescent cells. As such, we have provided a new method to selectively stimulate senescent cells by utilizing low-intensity ultrasound
treatment. We found that ultrasound treatment enhanced cytokine secretion involved in macrophage recruitment in senescent cells, while no significant change was observed in normal cells. In fact, the migration rate of monocyte, macrophage (Mφ) and M1 to the senescent cells, as well as the phagocytosis activity of these immune cells increased by ultrasound treatment. Due to the advantages of ultrasound treatment, such as non-invasiveness, deep penetration capability and easiness of clinical application, we expect that our method can be applied to treat various senescent-associated diseases, in
combination with other established therapy. ATM mediated-p53 signaling pathway forms a novel axis for senescence control Su Young Hwang and Joon Tae Park* Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, Korea Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway
was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative Figure 2. p53 as a potential target for ATM-mediated senescence amelioration. effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic (A) p53 co-immunoprecipitation with ATM antibody. (B) Western blot analysis to determine effects of reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, nutlin3 on p53 activation in the absence or presence of KU-60019. (C) Effects of
nutlin3 on cellular our data indicated that p53 pathway functions as potential target for ATM-mediated senescence proliferation (**P < 0.01, ##P < 0.01, Mann-Whitney test). Mean ± S.D., N = 3. (D) Effects of nutlin3 at amelioration. indicated concentrations on ROS. Flow cytometric analysis of mitochondrial ROS using MitoSOX (*P Keywords: Mitochondria, ATM inhibition, p53, senescence alleviation, metabolic < 0.05, **P < 0.01, Mann-Whitney test). Mean ± S.D., N = 3. reprogrammer Senescence is defined as a state where normal somatic cells lose their replicative capacity after prolonged division
or from a variety of stresses (Hayflick, 1965; Ziegler et al., 2015). Senescence has been considered an irreversible phenomenon because senescent cells exhibit significant changes in the shape and function of cellular components (Hwang et al., 2009). Among these cellular organelles, the most pronounced changes occur in mitochondria (López-Otín et al., 2013). Mitochondria undergo structural alteration accompanied by the gradual increase in the mitochondrial mass and size (Lee et al., 2002; Passos et al., 2007). This increase is mainly due to a feedback Figure 3. p53 inhibition via a chemical
and genetic approach yields senescence amelioration. response to compensate for mitochondrial dysfunction (Westermann, 2012). Furthermore, (A) Effects of pifithrin-α on p53 inactivation as determined by western blot analysis. (B) Flow dysfunctional mitochondria are the major sites of ROS production as well as the major targets of cytometric analysis of mitochondrial ROS using MitoSOX (**P < 0.01, Mann-Whitney test). Mean ± ROS-induced damage (Houtkooper et al., 2011; James et al., 2015). These damage resulted in the S.D., N = 3. (C) Flow cytometric analysis of autofluorescence. (**P < 0.01,
Mann-Whitney test). Mean overall deterioration of the electron transport complex (ETC), which leads to a decrease in oxidative ± S.D., N = 3. (D) Soft agar assay (scale bar 20 μm). (E) Effects of p53 inhibition with p53 shRNA on phosphorylation (OXPHOS) with a decrease in ATP production (Houtkooper et al., 2011; James et ROS (**P < 0.01, Mann-Whitney test). Mean ± S.D., N = 3. (F) Flow cytometric analysis of al., 2015). Thus, senescent cells shows higher dependence on glycolysis for an energy source (Lin autofluorescence. (**P < 0.01, Mann-Whitney test). Mean ± S.D., N = 3. et al., 2001).
Given the fact that changes in energy metab
