Page 43 - M. Immunology

Mechanism of SOCS1 action in the promotion of glucocorticoid-induced M2 polarization
: Role of p38-GILZ and HIF-1α as a target of SOCS1

Hana Jeong and Choong-Eun Lee
Department of Biological Science, Sungkyunkwan University, Suwon, South Korea

BACKGROUND AIM
Macrophages are critical role players in the host defense, exhibiting pro- To identify the mechanism of SOCS1 action on M2 type macrophage
inflammatory (M1) and anti-inflammatory (M2) functions in different phases of polarization induced by glucocorticoid (GC) using monocytic cells transduced
infection to maintain immune homeostasis. As mediators of inflammation, the with SOCS1 or shSOCS1
regulatory role of SOCS proteins on the macrophage differentiation into M1
vs M2 has been studiedin diverse systems.

METHODS
• Cell culture and generation of SOCS over-expressing or knock-down cells
pMSCV. pAdEasy-1 constructs containing SOCS1, shSOCS1, shHIF-1α or control vectors were transfected into complementing HEK 293 cells using Lipofectamine 3000. Recombinant
viruses were purified from the HEK 293ft culture and used for infection into THP1 cells.
• Induction of inflammatory cytokine production dexamethasone treatment
THP-1 monocytic cells were pre-differentiated for 16 h by 200 nM PMA treatment without βME, which were then stimulated with 40 ng/ml dexamethasone(Dex) to induce M2 differentiation.
• Determination of intracellular ROS by FACS
Cells were stimulated with dexamethasone and 1μM H 2 DCF-DA was added to cultures 20~30 min before the end of the incubation. Fluorescence was measured with excitation at 480 nm
and emission at 530 nm.
• Measurement of cytokines by ELISA
IL-10 for M2 cytokine were measured by using human IL-10 ELISA kits.
• Analysis of signaling mediators by immunoblotting
Cell extracts were subjected to blotting with antibodies to STAT, HIF-1α, MAPK, MKP1, Trx, SOD, GILZ, Arg1 and SOCS1. The blots were developed using an ECL detection kits.
• Analysis mRNA expression level by qRT-PCR
After stimulation with Dex, cells were harvested and total RNA extracted. Expression levels of the different genes such as IL-10, CD163 and Arg1. qRT-PCR amplification was performed
using SYBR green mix.
RESULTS
A B A

C
D Arg1
4
B
3 C
2
1
0
NT 24h 48h
Fig 1. Dex-induced M2 polarization involves ROS- and p38-dependent pathways.
: Effect of NAC and SB203580 on the induction of ROS, IL-10, Arg1, CD163 and GILZ
A B
Fig 3. SOCS1-induced promotion of M2 polarization involves HIF-1α down-regulation.
SOCS1 enhances HIF-1α ubiquitination (A) and forced HIF-1α up-regulation suppresses SOCS1-mediated M2 induction
with p38/GILZ and IL-10 regulation (B, C).

A B C

Fig 4. HIF-1α knock-down promoted Dex-induced M2 polarization mediated by p38-dependent pathways.
Fig 2. SOCS1 promotes Dex-induced M2 polarization with the regulation of p38, GILZ and HIF-1α.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

The promotive function of SOCS1 1. Marie B, Benoît D and Jean-Louis M. (2008) Macrophage This study is supported by KRF Grant
on Dex-induced M2 polarization polarization in bacterial infections. J Immunol. 181:3733- #2018R1A2B6002201
via p38-dependent GILZ and HIF-1α regulation 3739. H Jeong is also supported by GPF
2. Quax RA, Laura M, Koper JA, Hazes JM, Lamberts SWJ, #2017H1A2A1043022
van Rossum EFC, and Feelders RA. (2013) Glucocorticoid
Dex sensitivity in health and disease. Nat Rev Endocrinol. 9,
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