Role of FGF10/FGFR2b Signaling in Homeostasis and Regeneration of Adult Lacrimal Gland and Corneal Epithelium Proliferation - PubMed (original) (raw)

Role of FGF10/FGFR2b Signaling in Homeostasis and Regeneration of Adult Lacrimal Gland and Corneal Epithelium Proliferation

Emma N Finburgh et al. Invest Ophthalmol Vis Sci. 2023.

Abstract

Purpose: Fibroblast growth factor 10 (FGF10) is involved in eye, meibomian, and lacrimal gland (LG) development, but its function in adult eye structures remains unknown. This study aimed to characterize the role of FGF10 in homeostasis and regeneration of adult LG and corneal epithelium proliferation.

Methods: Quantitative reverse transcription PCR was used for analysis of FGF10 expression in both early postnatal and adult mouse LG, and RNA sequencing was used to analyze gene expression during LG inflammation. FGF10 was injected into the LG of two mouse models of Sjögren's syndrome and healthy controls. Flow cytometry, BrdU cell proliferation assay, immunostaining, and hematoxylin and eosin staining were used to evaluate the effects of FGF10 injection on inflammation and cell proliferation in vivo. Mouse and human epithelial cell cultures were treated with FGF10 in vitro, and cell viability was assessed using WST-8 and adenosine triphosphate (ATP) quantification assays.

Results: The level of Fgf10 mRNA expression was lower in adult LG compared to early postnatal LG and was downregulated in chronic inflammation. FGF10 injection into diseased LGs significantly increased cell proliferation and decreased the number of B cells. Mouse and human corneal epithelial cell cultures treated with FGF10 showed significantly higher cell viability and greater cell proliferation.

Conclusions: FGF10 appears to promote regeneration in damaged adult LGs. These findings have therapeutic potential for developing new treatments for dry eye disease targeting the ability of the cornea and LG to regenerate.

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Conflict of interest statement

Disclosure: E.N. Finburgh, None; O. Mauduit, None; T. Noguchi, None; J.J. Bu, None; A.A. Abbas, None; D.F. Hakim, None; S. Bellusci, None; R. Meech, None; H.P. Makarenkova, None; N.A. Afshari, None

Figures

Figure 1.

Figure 1.

FGF10 injection increased proliferation rates. (A) Fgf10 expression decreased in adult LGs, as shown by qRT-PCR using two different primer sets. (B, C) Immunostaining (BrdU in green, heparan sulfate [HS] in red, and DAPI in blue) of LGs injected with BSA (B) or with FGF10 (C). (D) Quantification of BrdU-positive cells in control (BSA-injected) and FGF10-treated LGs. One gland of each mouse was injected with BSA (control) and another gland was injected with Fgf10 (n = 7; **P < 0.01, nonparametric unpaired _t_-test).

Figure 2.

Figure 2.

FGFR2b is necessary for LG regeneration. (A) To block FGFR2b signaling, we used DOX-inducible R26rtTA:tet(O)sFgfr2b:Pax6-LacZ transgenic mice, which, after DOX treatment, express soluble FGFR2b, attenuating normal FGFR2b function. Pax6-LacZ mice were used as controls. Mice received a DOX diet for 45 days, and the LGs were injured with IL-1α and analyzed five days after injury. (B, C) Injured LGs of control mice showed the presence of well-defined acinar and ductal compartments. (D, E) Injured LGs of mice expressing soluble FGFR2b had a severe reduction of the acinar compartment, but ducts were still present. (F) Percentage of acinar structures within the LG section was assessed by evaluation of the area represented by acini compared to the total area of the whole LG section. Five mice (10 LGs) per each group were used. ****P < 0.0001 using nonparametric unpaired _t_-tests (Mann–Whitney).

Figure 3.

Figure 3.

FGF10 injection into the LGs increased cell proliferation and reduced LG inflammation in the mouse models of pSS, the NOD.B10.H2b and TSP - _1_–/– mice. (A–C) mRNA expression fold changes of Fgf10 ligand (A) and its receptors Fgfr1 (B) and Fgfr2 (C) in NOD.B10.H2b mice compared to BALB/c at 2, 4, and 6 months of age with adjusted P values (FDR). (D) One gland of each mouse was injected with BSA (control) and another gland was injected with FGF10. Injection of FGF10 increased BrdU incorporation (n = 10; **P < 0.01, non-parametric paired _t_-test [Wilcoxon]). (E) Injection of FGF10 decreased inflammation (focus scores) in the LGs of the NOD.B10.H2b mice (NOD, 4-month-old males). Three slides were analyzed for each gland (n = 5; ***P < 0.001, non-parametric unpaired _t_-test [Mann–Whitney]). (F–H) FGF10 injections into LGs of TSP - _1_–/– and NOD.B10.H2b mice reduced B-cell infiltration as shown by immunostaining with B220 antibody (F, G; five mice per group) and by flow cytometry using CD19 antibody (H). In the flow cytometry experiments, one gland of each mouse was injected with BSA (control) and another gland was injected with FGF10 (n = 6; *P < 0.05, parametric paired _t-_test).

Figure 4.

Figure 4.

FGF10 increased the viability of primary mouse corneal epithelial cells. (A) Mouse corneal epithelial cells were seeded in multi-well dishes, treated with FGF10 (30 ng/mL) or BSA (control), and cultured for 1, 3, and 7 days. Cells were processed for viability assay (tetrazolium salt, WST- 8). Dots represent the number of repeats (n = 9) for each condition/time. Statistical significance was assessed with unpaired _t_-tests. All bar plots are mean ± SD. *P < 0.05; ****P < 0.0001. (B) FGF10, B27, and HCGS improved cell viability in human corneal epithelial cell cultures. The graph shows normalized raw data, means, and standard deviations for each corneal epithelial cell culture condition using the ATP quantification cell viability assay. **P < 0.01 compared to EpiLife without supplements control; ++P < 0.01; NS, not significant compared to the control with the same supplement but without Fgf10.

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