Page 1 - A. Aging

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).