Mesenchymal Stem Cells Extract (MSCsE)-Based Therapy Alleviates Xerostomia and Keratoconjunctivitis Sicca in Sjogren's Syndrome-Like Disease

Abstract

Sjogren's syndrome (SS) is an autoimmune disease that manifests primarily in salivary and lacrimal glands leading to dry mouth and eyes. Unfortunately, there is no cure for SS due to its complex etiopathogenesis. Mesenchymal stem cells (MSCs) were successfully tested for SS, but some risks and limitations remained for their clinical use. This study combined cell- and biologic-based therapies by utilizing the MSCs extract (MSCsE) to treat SS-like disease in NOD mice. We found that MSCsE and MSCs therapies were successful and comparable in preserving salivary and lacrimal glands function in NOD mice when compared to control group. Cells positive for AQP5, AQP4, α-SMA, CK5, and c-Kit were preserved. Gene expression of AQP5, EGF, FGF2, BMP7, LYZ1 and IL-10 were upregulated, and downregulated for TNF-α, TGF-β1, MMP2, CASP3, and IL-1β. The proliferation rate of the glands and serum levels of EGF were also higher. Cornea integrity and epithelial thickness were maintained due to tear flow rate preservation. Peripheral tolerance was re-established, as indicated by lower lymphocytic infiltration and anti-SS-A antibodies, less BAFF secretion, higher serum IL-10 levels and FoxP3⁺ T_reg cells, and selective inhibition of B220⁺ B cells. These promising results opened new venues for a safer and more convenient combined biologic- and cell-based therapy.

Keywords: Sjogren’s syndrome (ss); autoimmune diseases; biologic therapy; bone marrow; cell extract; lacrimal gland; mesenchymal stem cells (MSCs); non-obese diabetic mice (NOD); salivary glands; submandibular glands.

Figure 1

Salivary and lacrimal glands function represented as SFR (Salivary Flow Rate) and TFR (Tear Flow Rate), respectively. SFR and TFR were assessed pre-treatment at week 0 (8-week-old) then 4, 8, 12, and 16 weeks post-treatment. (A) SFR was determined by volume of saliva/min/gm body weight (multiplied by 10 for simplicity). Control group showed a continuous decrease of SFR (lost almost 47% of SFR at week 16 in comparison to the highest reached level, week 4) whereas MSCs-/MSCsE-treated groups maintained a significantly higher SFR (maintained almost 75–100% of SFR at week 4) than the control, their results were comparable to each other, and to the ICR group, (n = 5–12). (B) TFR was determined by length of wetted phenol red thread in mm/min. Control group showed a continuous decrease of TFR; whereas, MSCs-/MSCsE-treated groups maintained significantly higher TFRs that are comparable to each other and to the wild type ICR group. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, n = 3–6. All data were presented as mean ± S.D. Control: saline-treated; MSCs: Mesenchymal stem cells; MSCsE: Mesenchymal stem cells extract.

Figure 2

Special cell subpopulations in submandibular (SMG) and lacrimal glands (LG) were evaluated by immunofluorescence staining at 16 weeks post-treatment. (A,C) SMG/LG immunofluorescence staining, respectively, positive for AQP5 (marker for water channel protein expressed by acinar cells in SMG and acinar/ductal cells in LG), α-SMA (marker for myoepithelial cells), AQP4 (marker for acinar and ductal cells), CK5 (marker for ductal/progenitor cells), and c-Kit (marker for stem/progenitor cells) were tested in frozen sections, scale bar = 148 μm. (B,D) Quantification of protein immunofluorescence expression levels in submandibular/lacrimal glands, respectively, from 4–6 random fields/glands by Image J software. MSCs-/MSCsE-treated groups showed higher intensities for all the tested markers when compared with the control group. All images were randomly taken at 200× magnification. *p ≤ 0.05; **p ≤ 0.01, n = 3–6. All data were presented as mean ± S.D. Control: saline-treated; MSCs: Mesenchymal stem cells; MSCsE: Mesenchymal stem cells extract.

Figure 3

Proliferation rates, serum EGF levels, and gene expression levels of key genes at 16 weeks post-treatment. (A) Immunofluorescence staining of submandibular (SMG) (upper panel) and lacrimal (LG) (lower panel) glands for proliferation protein Ki-67 (red), nuclei were stained with DAPI (blue), (B) Proliferation rate (Ki-67-positive cells %) for submandibular and lacrimal glands was calculated using random 200× magnified images (acquired by Volocity software) using Image J software. Two examiners independently analyzed images in a blind manner. MSCs/MSCsE treatments promoted tissue proliferation in the glands significantly higher than the control group and their rates were comparable to the ICR group. (C) Serum levels of EGF measured by ELISA. Both treatments elevated EGF levels in comparison to the control group, but only MSCsE treatment induced a significantly higher level. (D) Relative expression of key genes for tissue function, repair, regeneration, and apoptosis were analyzed by quantitative RT-PCR in lacrimal (LG) and submandibular (SG) glands. Gene expression levels in the lacrimal gland were significantly higher in MSCs-/MSCsE-treated groups than that of the control for AQP5, EGF, LYZ1, MMP2, and BMP7. Gene expression levels in the submandibular gland were significantly higher in MSCs-/MSCsE-treated groups than that of the control for AQP5, EGF, FGF2, and BMP7; and significantly lower for MMP2 and CASP3. Y-axis shows the relative expression of the gene compared to GAPDH, three experimental replicates were conducted for each sample. Scale bar = 74 μm, *p < 0.05, **p < 0.01; ***p < 0.001, n= 3–6. All data were presented as mean ± S.D. Control: saline-treated; MSCs: Mesenchymal stem cells; MSCsE: Mesenchymal stem cells extract.

Figure 4

Thickness of the central cornea (total) and the corneal epithelium at 16 weeks post-treatment. (A) H&E stained images of the cornea, arrowheads represent the epithelial thickness. (B) Analysis of the total cornea thickness. (C) Analysis of corneal epithelium thickness. Images of H&E stained sections of the cornea were obtained using Volocity software then Image J was used to assess the thickness. The MSCs-/MSCsE-treated groups showed a significantly higher corneal epithelial thickness. Scale bar = 74 μm, *p ≤ 0.05, n = 3–6. All data were presented as mean ± S.D. Control: saline-treated; MSCs: Mesenchymal stem cells; MSCsE: Mesenchymal stem cells extract.

Figure 5

Focus score, focus area and lymphocytes composition analysis in the submandibular (SMG) and lacrimal (LG) glands at 16 weeks post-treatment. (A) H&E stained images of lymphocytic infiltrates in the submandibular glands (upper panel) and lacrimal glands (lower panel). (B) Focus score analysis (number of lymphocytic infiltrates/4 mm²) using serial H&E stained sections, cut at different levels, under the light microscope. The analysis revealed a lower score for the treated groups but was not statistically significant. (C) Focus area (in µm²) was calculated by Image J software using H&E images (400×/200×) acquired by Volocity software. Treated groups showed significantly smaller focus areas. Immunohistochemical staining of lymphocytic infiltrate for B220 (a pan B cell marker in mice), BAFF (B cells activating factor), and FoxP3 (forkhead box P3, T_reg marker) in submandibular glands (D) and lacrimal glands (E). (F,G) Quantification of protein expression for BAFF and B220, respectively. The positive signals were measured using Image J software then divided by the size of the lymphocytic infiltrate (focus area). The results were represented as % of signal intensity. (H) Quantification of FoxP3⁺ T_reg cells. Positive cells were counted in each lymphocytic infiltrate then divided by the focus area (cell/ µm²) using Image J software. MSCs/MSCsE groups exhibited a significantly higher FoxP3⁺ and lower B220⁺ and BAFF⁺ cells in the lymphocytic infiltrates when compared to the control group. All images were taken at 200× magnification. Scale bar = 148 μm, *p ≤ 0.05; **p ≤ 0.01, n = 3–6. All data were presented as mean ± S.D. Control: saline-treated; MSCs: Mesenchymal stem cells; MSCsE: Mesenchymal stem cells extract.

Figure 6

Serum levels of anti-SSA/Ro autoantibodies and IL-10, and gene expression levels of anti-/pro-inflammatory cytokines/factors at 16 weeks post-treatment. (A) Serum levels of anti-SSA/Ro autoantibodies (assessed by ELISA) for MSCs-/MSCsE-treated groups exhibited significantly lower levels in comparison to the control group. (B) Serum levels of IL-10 (assessed by ELISA) for MSCs-/MSCsE-treated groups were significantly higher than the control group. (C) Gene expression levels for anti-/pro-inflammatory cytokines/factors were measured using quantitative RT-PCR in the submandibular and lacrimal glands. MSCs/MSCsE treatments upregulated IL-10 and down-regulated TNF-α gene expressions in both glands, and down-regulated TGF-β, IL-1β in lacrimal glands. Y-axis shows the relative expression of the gene compared to GAPDH, three experimental replicates were conducted for each sample. *p < 0.05; **p < 0.01; ***p < 0.001; ****p ≤ 0.0001, n= 4–6. All data were presented as mean ± S.D. Control: saline-treated; MSCs: Mesenchymal stem cells; MSCsE: Mesenchymal stem cells extract.