NINJ1 impairs the anti-inflammatory function of hUC-MSCs with synergistic IFN-γ and TNF-α stimulation.
10.1016/j.cjtee.2025.04.003
- Author:
Wang HU
1
;
Guomei YANG
2
;
Luoquan AO
1
;
Peixin SHEN
1
;
Mengwei YAO
1
;
Yuchuan YUAN
1
;
Jiaoyue LONG
3
;
Zhan LI
4
;
Xiang XU
5
Author Information
1. Department of Stem Cell & Regenerative Medicine, State Key Laboratory of Trauma and Chemical Poisoning, Daping Hospital, Army Medical University, Chongqing, 400042, China.
2. Central Laboratory, Daping Hospital, Army Medical University, Chongqing, 400042, China.
3. Research Center, Chongqing Kangcell Biotechnology Co., Ltd, Chongqing, 400000, China.
4. Central Laboratory, Daping Hospital, Army Medical University, Chongqing, 400042, China. Electronic address: Zhan_Li@tmmu.edu.cn.
5. Department of Stem Cell & Regenerative Medicine, State Key Laboratory of Trauma and Chemical Poisoning, Daping Hospital, Army Medical University, Chongqing, 400042, China; Yunnan Key Laboratory of Stem Cell and Regenerative Medicine, Science and Technology Achievement Incubation Center, Kunming Medical University, Kunming, 650500, Yunnan province, China; Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Army Medical University, Chongqing, 400038, China; Chongqing Key Laboratory of Precision Diagnosis and Treatment for Kidney Diseases, Department of Stem Cell and Regenerative Medicine, Daping Hospital, Army Medical University, Chongqing, 400042, China. Electronic address: xiangxu@tmmu.edu.cn.
- Publication Type:Journal Article
- Keywords:
IFN-γ;
NINJ1;
Sepsis;
TNF-α;
hUC-MSCs
- MeSH:
Mesenchymal Stem Cells/drug effects*;
Animals;
Interferon-gamma/pharmacology*;
Tumor Necrosis Factor-alpha/pharmacology*;
Humans;
Mice;
Umbilical Cord/cytology*;
Cells, Cultured;
Apoptosis;
Male
- From:
Chinese Journal of Traumatology
2025;28(4):276-287
- CountryChina
- Language:English
-
Abstract:
PURPOSE:To investigate the regulatory role of nerve injury-induced protein 1 (NINJ1) in the anti-inflammatory function of human umbilical cord mesenchymal stem cells (hUC-MSCs) co-stimulated by interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α).
METHODS:hUC-MSCs were expanded in vitro using standard protocols, with stem cell characteristics confirmed by flow cytometry and multilineage differentiation assays. The immunomodulatory properties and cellular activity of cytokine-co-pretreated hUC-MSCs were systematically evaluated via quantitative reverse transcription RT-qPCR, lymphocyte proliferation suppression assays, and Cell Counting Kit-8 viability tests. Transcriptome sequencing, Western blotting and small interfering RNA interference were integrated to analyze the regulatory mechanisms of NINJ1 expression. Functional roles of NINJ1 in pretreated hUC-MSCs were elucidated through gene silencing combined with lactate dehydrogenase release assays, Annexin V/Propidium Iodide apoptosis analysis, macrophage co-culture models, and cytokine Enzyme-Linked Immunosorbent Assay. Therapeutic efficacy was validated in a cecal ligation and puncture-induced septic mouse model: 80 mice were randomly allocated into 4 experimental groups (n=20/group): sham group (laparotomy without cecal ligation); phosphate-buffered saline-treated group (cecal ligation and puncture (CLP) + 0.1 mL phosphate-buffered saline); hUC-MSCs (small interfering RNA (siRNA)-interferon-gamma and tumor necrosis factor-alpha co-stimulation (IT))-treated group (CLP + hUC-MSCs transfected with scrambled siRNA); and hUC-MSCs (siNINJ1-IT)-treated group (CLP + hUC-MSCs with NINJ1-targeting siRNA).
RESULTS:hUC-MSCs demonstrated compliance with International Society for Cellular Therapy criteria, confirming their stem cell identity. IFN-γ/TNF-α co-pretreatment enhanced the immunosuppressive capacity of hUC-MSCs, accompanied by the reduction of cellular viability, while concurrently upregulating pro-inflammatory cytokines such as interleukin-6 and interleukin-1β. This co-stimulation significantly elevated NINJ1 expression in hUC-MSCs, whereas genetic silencing of NINJ1 effectively suppressed pro-inflammatory cytokine production and attenuated damage-associated molecular patterns release through inhibition of programmed plasma membrane rupture. Furthermore, the NINJ1 interference potentiated the ability of cytokine-pretreated hUC-MSCs to suppress LPS-induced pro-inflammatory responses in RAW264.7 macrophages. In cecal ligation and puncture-induced sepsis model, NINJ1-silenced hUC-MSCs exhibited enhanced therapeutic efficacy, manifested by reduced systemic inflammation and multi-organ damage.
CONCLUSION:Our findings shed new light on the immunomodulatory functions of cytokine-primed MSCs, offering groundbreaking insights for developing MSC-based therapies against inflammatory diseases via interfering the expression of NINJ1.
