Thymic rejuvenation via FOXN1-reprogrammed embryonic fibroblasts (FREFs) to counteract age-related inflammation
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
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Research Article Aging Immunology

Thymic rejuvenation via FOXN1-reprogrammed embryonic fibroblasts (FREFs) to counteract age-related inflammation

Abstract

Age-associated systemic, chronic inflammation is partially attributed to increased self-autoreactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), whereas exogenous FOXN1 in TECs can partially rescue age-related thymic involution. TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule, and intrathymic injection of naturally young TECs can lead to middle-aged thymus regrowth. Therefore, as a thymic rejuvenation strategy, we extended these 2 findings by combining them with 2 types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these FREFs directly into the aged murine thymus. We found substantial regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and autoreactive T cell–mediated inflammation in old mice. Therefore, this approach has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to substantially, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in elderly people.

Authors

Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su

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Figure 1

Preparation and characterization of MEFs and FREFs.

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Preparation and characterization of MEFs and FREFs. Mouse embryonic fibr...
Mouse embryonic fibroblasts (MEFs) were isolated via trypsinized digestion from E13.5 embryonic mice and cultured in plates with or without 4-hydroxy tamoxifen (xTM). (A) Representative live images from confocal microscopy show MEFs expressing GFP, which represents exogenous FoxN1 (right) and was driven by either endogenous FOXN1-carried Cre-recombinase at 3′UTR (FTg:FoxN1Cre; top) or R26-carried CreERT treated with TM (FTg:R26CreERT xTM; bottom) and panels without GFP (left) owing to either no Cre transgene or no active Cre. Scale bar: 20 μm. (B) Representative flow cytometric dot plots (EpCAM vs. GFP: top; and MHC-II vs. GFP: bottom), in which MEFs expressing GFP (FTg:R26CreERT xTM and FTg:FoxN1Cre) are termed “FREFs” (in red, middle and right panels), compared with MEFs that did not express GFP (FTg:R26CreERT owing to Cre inactivated without TM treatment, left panels). (C) Summarized gene (FoxN1) expression (via reverse transcription PCR; RT-PCR) in cells of 4 groups: (a) FTg-only (without Cre), (b) FTg:FoxN1Cre (Cre expression was endogenously turned on in FoxN1+ cells), (c) FTg:R26CreERT xTM (Cre was activated via TM induction), and (d) a newborn thymus control group. One-way ANOVA Newman-Keuls multiple-comparisons test was used. (D) Summarized gene (Dll4) and (E) (Ccl25) expression (via RT-PCR) in cells of 4 groups as in C (these are 2-and-2 group comparisons). Student’s t test was used to determine statistical significance. P values are shown between every 2 groups, and all P values represent the mean ± SD. Each symbol represents cells from an individual embryonic sample. Statistical significance levels were set at *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.