IFNγ and TNFα optimize salivary gland mesenchymal stromal cells: an alternative to marrow- and adipose-MSCs for radiation xerostomia

Michele C Larsen; Ilya Gurevic; Liliana Berube; Addie Vande Loo; Elizabeth Hanson; Ryan Adam; Valeria Manfrè; Maxwell Parker; Cristina Paz; Jacques Galipeau; Randall J Kimple; Grace Blitzer; Sara S McCoy

doi:10.1016/j.reth.2025.11.004

IFNγ and TNFα optimize salivary gland mesenchymal stromal cells: an alternative to marrow- and adipose-MSCs for radiation xerostomia

Regen Ther. 2025 Nov 14:30:1086-1100. doi: 10.1016/j.reth.2025.11.004. eCollection 2025 Dec.

Authors

Michele C Larsen¹, Ilya Gurevic¹, Liliana Berube^{2

3}, Addie Vande Loo¹, Elizabeth Hanson¹, Ryan Adam¹, Valeria Manfrè^{1

4}, Maxwell Parker¹, Cristina Paz², Jacques Galipeau⁵, Randall J Kimple^{2

5}, Grace Blitzer², Sara S McCoy¹

Affiliations

¹ Department of Medicine, School of Medicine and Public Health, Division of Rheumatology University of Wisconsin, Madison, WI, USA.
² Department of Human Oncology, School of Medicine and Public Health, Madison, WI, USA.
³ Department of Medical Physics, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.
⁴ Department of Medicine, University of Udine, Academic Hospital "Santa Maria Della Misericordia,", Udine, Italy.
⁵ Department of Medicine, University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI, USA.

Abstract

Objectives: Local mesenchymal stromal cell (MSC) administration is a promising therapy for xerostomia. MSCs deploy their advantageous effects through their trophic secretome and immunomodulatory capabilities. These functions are enhanced with IFNγ pre-licensing, but the effects of TNFα pre-licensing are unknown. Our objective was to compare MSCs by tissue source (MSC(BM), MSC(AD), and salivary gland-derived [MSC(SG)]) and by cytokine pre-licensing conditions.

Methods: We used single cell and bulk RNA sequencing and ELISA to determine key trophic and immunomodulatory features differing between human MSC(BM), MSC(AD), and MSC(SG). We used ELISA and flow cytometry of T-cell co-culture to define the effect of IFNγ and/or TNFα on MSC trophic secretome and immunomodulatory capacity. Finally, we studied salivary flow and glandular recovery with MSC injection in radiation-induced xerostomia mice.

Results: Bulk RNA sequencing (RNAseq) of MSC(BM), MSC(AD), and MSC(SG) revealed that they shared 85 % of transcripts. Key differences included extracellular matrix production and response to cytokines in MSC(SG). Single cell RNA sequencing showed MSC(SG) treated with IFNγ and TNFα transcriptionally diverged from other treatment conditions. Regardless of MSC source, dual stimulation of MSCs with IFNγ and TNFα produced an average of more than a 20-fold increase in R-Spondin 3 compared to vehicle conditions. Additionally, IFNγ and TNFα pre-licensing optimized immunomodulatory marker expression more than IFNγ alone. Intercellular adhesion molecule 1 increased 12-fold more, programmed death ligand 1 increased 1.4-fold more, and indoleamine 2,3 dioxygenase increased 2-fold more with IFNγ/TNFα pre-licensing than IFNγ alone. Both cytokine stimulation conditions resulted in a 1.2-fold decrease in T-cell proliferation. Gland structure, aquaporin 5, and salivary flow are preserved in irradiated mice treated with MSC(SG) pre-licensed with IFNγ/TNFα.

Conclusion: MSC(SG) pre-licensed with both IFNγ and TNFα deploy advantageous functional cell attributes for salivary gland regenerative medicine.

Keywords: Adipose; Bone marrow; Cytokine; IFNγ; Mesenchymal stromal cell; Radiation; Salivary gland; Secretome; TNFα; Xerostomia.

Associated data

Dryad/10.5061/dryad.9ghx3ffv6

Abstract

Associated data

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