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. 2016 Oct 1;57(13):5161-5170.
doi: 10.1167/iovs.15-17824.

Adipose-Derived Mesenchymal Stem Cells Reduce Lymphocytic Infiltration in a Rabbit Model of Induced Autoimmune Dacryoadenitis

Xue Li  1 , Xiaoxiao Lu  1 , Deming Sun  2 , Xilian Wang  3 , Liyuan Yang  1 , Shaozhen Zhao  1 , Hong Nian  1 , Ruihua Wei  1
Affiliations

Adipose-Derived Mesenchymal Stem Cells Reduce Lymphocytic Infiltration in a Rabbit Model of Induced Autoimmune Dacryoadenitis

Xue Li et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: To investigate the immunoregulatory roles of adipose-derived mesenchymal stem cells (ADSCs) in autoimmune dacryoadenitis.

Methods: Rabbits were treated with ADSCs or phosphate-buffered solution on days 1, 3, 5, 7, and 9 after injection of activated peripheral blood lymphocytes, and clinical scores were determined by assessing tear production, break-up time, and fluorescein and hematoxylin and eosin staining. Inflammatory response was determined by measuring the expression of different mediators of inflammation in the lacrimal glands. The Th1/Th17-mediated autoreactive responses were evaluated by determining the proliferative response and the expression of cytokine genes and the lineage-determining transcription factors. The frequency of regulatory T cells (Tregs) was also examined.

Results: The ADSC-treated rabbits showed decreased autoimmune responses, and the secretory function of their lacrimal gland was restored significantly. Treatment with ADSCs downregulated the Th1 and Th17 responses but enhanced Tregs function. In addition, ADSC treatment noticeably suppressed the expression of matrix metalloproteinase (MMP)-9, MPP-2, IL-1β, and IL-6, whereas it enhanced the expression of the anti-inflammatory cytokine IL-10.

Conclusions: Our results demonstrated that ADSC administration efficiently ameliorates autoimmune dacryoadenitis mainly via modulating Th1/Th17 responses.

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Figures

Figure 1
Figure 1
Characterization of ADSCs. (A, B) Morphology of ADSCs at passage 3. Most cells remained fibroblastic (spindle shaped) ADSCs upon subculturing to P3. (C) Adipogenic differentiation assay. Intracellular accumulated lipid droplets (red color in the image) in adipogenic ADSCs as revealed by Oil Red O staining. (D) Osteogenic differentiation assay. Vast extracellular calcium deposits (orange-red in the image) in osteogenic ADSCs as revealed by alizarin red S staining. (E) Reverse transcription–PCR analysis for MSC surface markers and hematopoietic markers. Total RNA was isolated from ADSCs (P3) and was subjected to RT-PCR. It showed that ADSCs were positive for the MSC markers CD29, CD44, CD73, and CD90, and negative for hematopoietic markers CD34 and CD45.
Figure 2
Figure 2
Adipose-derived MSCs alleviated clinical symptoms of autoimmune dacryoadenitis. Rabbits with autoimmune dacryoadenitis were injected intravenously with PBS (untreated group) or with 1 × 107 rabbit ADSCs (ADSC-treated group) every other day starting on day 1 after transfer of activated PBLs. (A) Tear production. Schirmer test was performed at 0, 2, 4, and 6 weeks after first ADSC administration. (B) Tear break-up time demonstrates tear instability. Slit-lamp examination was performed in control, untreated, and ADSC-treated group. (C) Images and scores of fluorescein staining. Detection of ocular surface defects due to deficiency in preocular tear film protection was evaluated with fluorescein staining (top), and the intensity of staining of the cornea was scored (bottom). (D–J) Histologic analysis of lacrimal glands in rabbits. (D–I) The representative photographs (H&E staining) of lacrimal glands in untreated, ADSC-treated, and control rabbits are shown. (D, E) Substantial lymphocytic infiltration in the lacrimal glands of untreated group. Arrows indicate lymphocytic foci in the typical periductal and perivascular distribution (>2 foci per 4 mm2 of this representative LG section. The area (∼6 mm2) of the section (×40) examined was measured from the photographs, by using CellSen software. (F, G) Reduced lymphocyte infiltration in the lacrimal glands of the ADSC-treated group (<1 focus per 4 mm2 of this representative LG section). The area (∼9 mm2) of the section (×40) examined was measured from the photographs, by using CellSen software. (H, I) Occasional small lymphocytic aggregates in normal control group. (J) The numbers of lymphocytic foci in lacrimal glands were evaluated as described in Materials and Methods. The number of foci per 4 mm2 is shown. Data are presented as mean ± SD (A, B, C, J), n = 3. *P < 0.05, **P < 0.01. Data are representative of three independent experiments (A–J), six rabbits per group in each experiment.
Figure 3
Figure 3
Adipose-derived MSC treatment inhibited the proliferation of pathogenic T cells in vitro and in vivo. (A) Adipose-derived MSCs' effect on T-cell proliferation. Splenic lymphocytes labeled with 5 μM CFSE were cultured with irradiated pLGECs in the presence of ADSCs at different MSC: T-cell ratios of 1:5, 1:10, and 1:30. After 5 days, the lymphocytes were harvested and subjected to FACS analysis. (B) The proliferation index of PBLs from ADSC-treated or untreated rabbits was determined by BrdU assay. Data are representative of three independent experiments, and bar graphs show mean ± SD. *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Adipose-derived MSC treatment regulated the expression of pro- and anti-inflammatory cytokines and chemokines. Lacrimal glands were obtained from untreated and ADSC-treated rabbits at 6 weeks after first ADSC administration, and IL-1β, IL-6, IL-10, IL-18, TNF-a, TGF-β, MMP-2, and MMP-9 mRNA expression was measured by qPCR. Data are representative of three independent experiments and bar graphs show mean ± SD, n = 3. *P < 0.05, **P < 0.01.
Figure 5
Figure 5
Adipose-derived MSC treatment regulated Th1/Th17/Tregs in vivo and in vitro. (A) Lacrimal glands were removed from untreated and ADSC-treated rabbits, and IL-17, IFN-γ, T-bet, and RORC mRNA expression was analyzed by real-time qPCR. (B) Adipose-derived MSCs cocultured with splenic lymphocytes at the ratio of 1:5 were collected. The mRNA expression levels of IL-10, IFN-γ, IL-17, TNF-α, and TGF-β were measured by qPCR. (C) Percentage of CD4+Foxp3+ cells among splenic lymphocytes isolated from ADSC-treated and untreated rabbit spleen. (D) Percentage of CD4+Foxp3+ cells among lymphocytes isolated from LGs of ADSC-treated and untreated rabbits. (E) Adipose-derived MSCs cocultured with splenic lymphocytes at the ratio of 1:5 were measured by flow cytometry for the percentages of CD4+Foxp3+ cells at day 5. (F, G) Quantitative PCR analysis of Foxp3 mRNA expression in LGs and spleens of ADSC-treated and untreated rabbits. (H, I) Immunoblot of LGs and spleens of ADSC-treated and untreated rabbits. Tissue lysates were immunoblotted with indicated antibodies. Left: Levels of Foxp3 and β-actin. Right: Relative Foxp3 level = Foxp3 level/β-actin level. Data are representative of three independent experiments and bar graphs show mean ± SD, n = 3. *P < 0.05, **P < 0.01.
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