Bone Marrow-derived Mesenchymal Stem Cells Affect Immunologic Profiling of Interleukin-17-secreting Cells in a Chemical Burn Mouse Model

Purpose

This study investigated interleukin (IL)-17-secreting cell involvement in sterile inflammation, and evaluated the effect of mesenchymal stem cells (MSCs) on IL-17-secreting cell immunologic profiling.

Methods

Twenty mice were sacrificed at time points of 6 hours, 1 day, 1 week, and 3 weeks (each group, n = 5) after the cornea was chemically injured with 0.5N NaOH; IL-17 changes in the cornea were evaluated using enzyme-linked immunosorbent assay. Further, IL-17 secreting cells were assessed in the cervical lymph nodes by a flow cytometer. Rat MSCs were applied intraperitoneally in a burn model (n = 10), IL-17-secreting T helper 17 (Th17) cell and non-Th17 cell changes were checked using a flow cytometer in both cornea and cervical lymph nodes at 1week, and compared with those in the positive control (n = 10).

Results

IL-17 was highest in the cornea at 1 week, while, in the cervical lymph nodes, IL-17-secreting cells showed early increase at 6 hours, and maintained the increase through 1 day to 1 week, and levels returned to the basal level at 3 weeks. Specifically, the non-Th17 cells secreted IL-17 earlier than the Th17 cells. When the MSCs were applied, IL-17 secretion was reduced in CD3(+)CD4(-)CD8(-), CD3(+)CD4(+)CD8(-), and CD3(+) CD4(-)CD8(+) cells of the cervical lymph nodes by 53.7%, 43.8%, and 50.8%, respectively. However, in the cornea, IL-17 secretion of CD3(+)CD4(-)CD8(-) cells was completely blocked.

Conclusions

The results indicated that both IL-17-secreting non-Th17 and Th17 cells were involved in the chemical burn model, and MSCs appeared to mainly modulate non-Th17 cells and also partially suppress the Th17 cells.     

Th17/Treg细胞与自身免疫性葡萄膜炎的研究进展

葡萄膜炎是一组常见的由自身免疫紊乱导致的眼部炎症性致盲性眼病,现代研究认为主要是由CD4⁺T 细胞介导产生。CD4⁺T 细胞主要分为Th1,Th2,Th17和 Treg 细胞(CD4⁺CD25⁺Foxp3⁺调节性T细胞)四个亚群。以往研究主要集中在Th1,Th2细胞亚群,近年来研究表明,Th17 细胞是诱导自身免疫性葡萄膜炎发病的主要原因,而Treg细胞是负向调控葡萄膜炎的主要因素。因此,Th17和 Treg 细胞在葡萄膜炎的发病及病程演变过程中发挥重要作用,本文就Th17 细胞、Treg 细胞与自身免疫性葡萄膜炎的相关研究进展进行综述。     

Adalimumab specifically induces CD3(+) CD4(+) CD25(high) Foxp3(+) CD127(-) T-regulatory cells and decreases vascular endothelial growth factor plasma levels in refractory immuno-mediated uveitis: a non-randomized pilot intervention study

Aim

To explore immunoregulatory and anti-inflammatory pathways specifically targeted by a subcutaneous anti-TNFαdrug—adalimumab—which might be relevant for controlling refractory uveitis.

Design

Non-randomized pilot intervention study on the effects of adalimumab on Treg populations and plasma VEGF levels in refractory uveitis patients. Inflammatory and immunological parameters were measured in 12 patients before therapy, and 1 and 6 months after therapy, and analyzed in the context of ophthalmological outcomes. The results were compared with those obtained in 10 systemic prednisone-treated uveitis patients.

Results

After 1 month of treatment, all patients responded, with 67% of adalimumab group and 80% of the corticosteroid group achieving inactivity (P=0.5). Unlike steroid-treated patients, a significant increase in T-regulatory CD4⁺ CD25^high Foxp3⁺ CD127⁻ cells was observed in adalimumab patients after 1 month of treatment, and maintained after 6 months (P=0.003). A significant adalimumab-specific drop in plasma VEGF was observed after 1 and 6 months of treatment (P=0.019). In every single patient, Tregs but not VEGF correlated with disease activity.

Conclusions

In refractory uveitis patients treated with adalimumab, clinical efficacy may be mediated through upregulation of Tregs in addition to modulation of VEGF-mediated inflammatory pathways. These biological properties, which were not observed in patients treated with corticosteroids, may reflect the specificity of TNF-αtargeting.     

Effect of CD4+CD25+ regulatory T cells in the development of anterior chamber-associated immune deviation

AIM: To investigate whether CD4+CD25+ regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID). METHODS: The dynamic changes in the frequency of CD4+CD25+ T cells, CD4+CD25+ FoxP3+ T cells and CD4+CD25+ PD-1+ T cells from spleens of mice with ACAID were analyzed by flow cytometry. Foxp3 mRNA expression in purified CD4+CD25+ T cells was analyzed using real-time PCR. The suppressive effect of purified CD4+CD25+ T cells on the proliferation of CD4+CD25– T cells was evaluated by [3H] thymidine incorporation. A blocking experiment was performed to further address the role of CD4+CD25+ T cells in ACAID. The expression of IL-10 in purified CD4+CD25+ T cells was evaluated by ELISA. RESULTS: Increased frequencies of CD4+CD25+ T cells, CD4+CD25+ FoxP3+ T cells and CD4+CD25+ PD-1+ T cells were observed in ACAID. The CD4+CD25+ T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4+CD25– T cells. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID. Furthermore, purified CD4+CD25+ T cells from ACAID mice secreted IL-10. CONCLUSION: Our results demonstrate that Treg cells are induced in the mice undergoing ACAID. These Treg cells may play a role in the development of ACAID.     

Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2) on untransfected human corneal endothelial cells (HCECs) in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L) altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01) and the length of F-actin, reduced the mean optical density (P<0.01) of the collagen type IV in extracellular matrix (ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.     

Role of Regulatory T Cells in Tubercular Uveitis

Purpose: To study the role of regulatory T cells (Tregs) in patients with tubercular uveitis.

Methods: Frequencies of peripheral Tregs, Th1, Th17 cells, and intracellular cytokines were determined in 17 tubercular uveitis patients and 18 disease controls. Function of Tregs, Th1, and Th17 cells was assessed in vitro. Simultaneously, ocular levels of IFN-γ, IL-17A, IL-4, and IL-10 were also measured.

Results: Frequencies of peripheral Tregs in tubercular uveitis subjects were significantly lower compared with disease controls. Furthermore, expression of TGF-β and IL-2Rα, but not CTLA4, was reduced in Tregs of the tubercular uveitis group. The tubercular uveitis group demonstrated heightened Th1, Th17 responses following in vitro stimulation with phorbol myristate acetate (PMA)/ionomycin. Interestingly, Treg suppression assay did not show a significant difference between the two groups. Ocular levels of IFN-γ, IL-17A, and IL-10 were also elevated in tubercular uveitis group.

Conclusions: Low Treg frequency and hyporesponsive function contribute to proinflammatory responses manifesting at ocular level in tubercular uveitis.     

Effects of 530 nm monochromatic light on basic fibroblast growth factor and transforming growth factor-β1 expression in Müller cells

AIM: To expose rat retinal Müller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) expression.METHODS: Three groups of rat retinal Müller cells cultured in vitro under a 530 nm monochromatic light were divided into 6, 12 and 24h experimental groups , while cells incubated under dark conditions served as the control group. The bFGF and TGF-β1 mRNA expression, protein levels and fluorescence intensity of the Müller cells were analyzed.RESULTS:ThebFGF mRNA expression and protein levels were significantly upregulated in Müller cells in all three experimental groups compared with the control group (P<0.05), while that of TGF-β1 was downregulated (P<0.05). Also, bFGF expression was positively correlated, but TGF-β1 expression was negatively correlated with illumination time. The largest changes for both cytokines were seen in the 24h group. The changes in bFGF and TGF-β1 fluorescence intensity were highest in the 24h group, and significant differences were observed among the experimental groups (P<0.05).CONCLUSION:The expressions ofbFGF and TGF-β1 changed in a time-dependent manner in Müller cells exposed to 530 nm monochromatic light with 250 lx illumination intensity. Müller cells might play a role in the development of myopia by increasing bFGF expression or decreasing TGF-β1 expression. Changes in cytokine expression in retinal Müller cells may affect monochromatic light-induced myopia.     

Analysis of Th17-associated cytokines in tears of patients with dry eye syndrome

Purpose

To determine the levels of Th17-associated cytokines, particularly interleukin (IL)-17 and IL-22 in tears of patients with dry eye syndrome.

Methods

Tear samples were collected from 20 healthy volunteers, 20 dry eye (DE) patients with non-Sjögren''s syndrome (NSSDE) and 20 DE patients with Sjögren''s syndrome (SSDE). Symptom questionnaire was self-administered and multiple dry eye disease (DED)-related clinical tests were performed. The levels of IL-17 and IL-22 in tears were measured by enzyme-linked immunosorbent assay.

Results

The levels of IL-17 and IL-22 were significantly increased in tears of DE patients compared with those of controls and also higher in SSDE patients compared with those of NSSDE patients (P<0.05). Moreover, the levels of IL-17 and IL-22 were positively correlated with questionnaire score and keratopathy score but negatively correlated with tear film break-up time and Schirmer I test in both NSSDE and SSDE patients (P<0.05).

Conclusions

The levels of IL-17 and IL-22 in tears were significantly increased in DE patients, which were associated with the disease severity. Therefore, Th17 cell-associated cytokines, particularly IL-17 and IL-22, may have important roles in the immunopathogenesis of the DED.     

Blockade of interleukin-6 signaling suppresses experimental autoimmune uveoretinitis by the inhibition of inflammatory Th17 responses

The aim of this study was to investigate the effect of anti-mouse IL-6 receptor monoclonal antibody (MR16-1) treatment on CD4 T cell differentiation and compared it to the effect of anti-TNF mAb treatment with using a murine model of experimental autoimmune uveoretinitis (EAU). C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) to induce ocular inflammation treatment with control IgG or MR16-1 or anti-TNF mAb. Helper T cells differentiation was analyzed during the development of EAU. Immunization with IRBP increased the frequency of Th17 cells rather than Th1 cells in the early stage of EAU. Treatment with MR16-1 on the same day as immunization (day 0) or one day after (day 1) suppressed ocular inflammation in EAU mice. Treatment with MR16-1 on day 0 inhibited the induction of Th17 cells in vivo, and inhibited not only IRBP-responsive Th17 cells but also their Th1 counterparts and induced IRBP-responsive regulatory T (Treg) cells in vitro. The administration of anti-TNF mAb had no significant protective effect in EAU mice. The protective effect of anti-IL-6R mAb treatment, but not anti-TNF mAb treatment on EAU correlated with the inhibition of Th17 differentiation. This finding suggests that IL-6 blockade may have a therapeutic effect on human ocular inflammation which is mediated via mechanisms distinct from those of TNF blockade. IL-6 blockade may thus represent an alternative therapy for patients with ocular inflammation who are refractory to anti-TNF mAb therapy.     

TGF-β1 in retinal ganglion cells in rats with chronic ocular hypertension: its expression and anti-apoptotic effect

AIM: To investigate the anti-apoptotic effect of transforming growth factor beta-1 (TGF-β1) on chronic ocular hypertension. METHODS: The expression of TGF-β1 in retinal ganglion cells (RCGs) was measured using the immunohistochemiscal S-P method and real-time PCR in the normally control group, the ocular hypertension group (experimental group A), the ocular hypertension plus antibody intervention group (experimental group B) and the ocular hypertension plus antigen intervention group (experimental group C) at 1, 2, 3 and 4 weeks postoperatively. The count of apoptotic RCGs was measured using the TUNEL method. RESULTS: The expression of TGF-β1 was significantly higher in experimental group C than that in other three groups (P＜0.05). The expression was the lowest in experimental group B (4.17%). A statistically significant difference was noted between the four groups (P<0.01). The count of apoptotic RCGs was statistically significantly lower in experimental group C than that in the experimental groups A and B (P<0.01). A statistically significant difference was noted in the count of apoptotic RCGs between these three experimental groups (P<0.01). CONCLUSION: TGF-β1 can inhibit the apoptosis of RCGs in rats with chronic ocular hypertension.     

Effects of amniotic membrane transplantation on cytokines expression in chemically burned rat corneas

AIM: To investigate the effect of amniotic membrane transplantation (AMT) on the expressions of inflammatory-related, angiogenic-related and growth-related cytokines in rat corneas after chemical injury. METHODS: Alkali wounds were inflicted on the central corneas of rats by applying a round filter paper soaked in 1mol/L NaOH for 40 seconds. One week after alkali burn, 12 rats were randomly divided into 2 groups: the AMT group and the control group, and AMT was performed on the rats in the AMT group. Corneal opacity and neovascularization were observed by slit-lamp microscopy. The protein levels of interleukin (IL)-2, interferon (IFN)-γ, IL-10 and transforming growth factor (TGF)-β were determined by enzyme-linked immunosorbent assay 2 weeks after AMT. The mRNA levels of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were evaluated by real-time quantitative PCR. RESULTS: In the AMT group, the corneal opacity was improved (P=0.011) and the area of corneal neovascularization was significantly decreased (P=0.005) compared with the control group. The amount of IL-2 and IFN-γ secreted by Th1 cells were decreased after AMT, whereas the amount of IL-10 and TGF-β secreted by Th2 cells were increased (P<0.05). The level of MMP-2 was significantly down-regulated (P=0.013) at the mRNA level in the AMT group, while the expression of EGF was significantly higher (P=0.022) compared with controls. CONCLUTION: AMT may suppress corneal neovascularization after chemical injury by modulating the expressions of soluble factors.     

Th1、Th17细胞相关因子在实验性自身免疫性葡萄膜炎大鼠中的表达及作用

目的　探讨辅助性Ｔ细胞（Ｔｈｅｌｐｅｒｃｅｌｌｓ,Ｔｈ）１、Ｔｈ１７细胞相关因子在实验性自身免疫性葡萄膜炎（ｅｘｐｅｒｉｍｅｎｔａｌａｕｔｏｉｍｍｕｎｅｕｖｅｉｔｉｓ,ＥＡＵ）中的表达及作用。方法　取清洁级纯系健康雌性Ｌｅｗｉｓ大鼠４０只随机分为ＥＡＵ组（３２只）和对照组（８只）,ＥＡＵ组用光感受器间维生素Ａ类结合蛋白诱导大鼠ＥＡＵ模型,进行临床症状评分,免疫组织化学方法检测造模后视网膜内干扰素（ｉｎｆｅｒｆｅｒｏｎ,ＩＦＮ）-γ、诱导型一氧化氮合酶（ｉｎｄｕｃｉｂｌｅｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ｉＮＯＳ）、白细胞介素（ｉｎｔｅｒｌｅｕｋｉｎｅ,ＩＬ）-１７、ＩＬ-６的表达。ＥＬＩＳＡ法对比分析房水中各细胞因子的变化情况。结果　ＥＡＵ模型建立成功;造模后第１４天,视网膜损害以外层为主,视网膜内有大量炎细胞浸润,从而导致视网膜内结构紊乱,同时在视网膜的视锥、视杆细胞层和神经节细胞层ｉＮＯＳ、ＩＬ-１７、ＩＬ-６、ＩＦＮ-γ表达,细胞阳性率分别为２９％、４８％、５２％、７３％。在ＥＡＵ发病过程中,ＩＬ-６于造模后第７天迅速升高,第１０天达到高峰;ＩＬ-１７于造模后第１４天达到最高值,变化趋势与炎症进程一致;ＩＦＮ-γ在炎症后期仍有升高,于造模后第１６天达到最高值;ＩＬ-６、ＩＬ-１７、ＩＦＮ-γ与对照组相比,各时间点表达差异均有统计学意义（均为Ｐ＜０．０５）。ｉＮＯＳ在炎症进程中表达有所增加,但与对照组相比,各时间点的表达差异均无统计学意义（均为Ｐ＞０．０５）。结论　Ｔｈ１、Ｔｈ１７细胞相关因子调节网络共同参与实验性自身免疫性葡萄膜炎的发生发展。     

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-β1 (TGF-β1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 in vitro were induced by TGF-β1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the α-smooth muscular actin (α-SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the α-SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-β1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-β1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both α-SMA and CTGF, while to some extent inhibited that of collagen I. TGF-β1 significantly promoted the proteins expressions of α-SMA, CTGF and collagen I. After OTFS treated by both TGF-β1 and Y-27632, of α-SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the α-SMA, CTGF and collagen I mRNA in 30, 150, 750μmol/L Y-27632 group were statistically significant, compared with those in control group, respectively (α-SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I, P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and α-SMA whatever OTFS induced by TGF-β1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.     

Effects of extract of Buddleja officinalis on partial inflammation of lacrimal gland in castrated rabbits with dry eye

AIM: To assess the effects of extract of Buddleja officinalis on tear secretion volume, tear film stability, expressions of TGF-β1, IL-1β, TNF-α in lacrimal gland of castrated rabbits with dry eye. METHODS: A total of 30 victory rabbits were divided averagely into normal group(A), model group(B), therapy group with low dose extract of Buddleja officinalis (C), therapy group with high dose extract of Buddleja officinalis (D) and therapy group with genistein (E). The dry eye model was established with orchiectomy on Group B, C, D, E. Group C, D, E were administered intragastrically with corresponding dose extract of Buddleja officinalis or genistein for 30 days. All rabbits were detected with SIT. TGF-β1, IL-1β, TNF-α were detected with immunohistochemistry and the ultrastructure of lacrimal gland was observed under transmission electron microscope. RESULTS: The SIT value of group C, D, E were respectively 13.167±4.957, 14.667±5.279, 8.667±0.516, obviously higher than that of group B 5.667±2.338 (P<0.01). The positive expression of IL-1β in acinar cell and glandular tube cell of group C, D were 0.470±0.048, 0.510±0.088, obviously lower than that of group B 0.770±0.118 (P<0.01). The positive expression of TNF-α of group C, D were 0.498±0.156, 0.435±0.069, obviously lower than that of group B 0.769±0.095 too (P<0.01). The positive expression of TGF-β1 of group C, D were 0.406±0.171, 0.497±0.147, obviously higher than that of group B 0.222±0.113(P<0.01). Any result of group C, D was positive compared with that of group E (P <0.05). Ultrastructure of the lacrimal gland of group C, D, E was well preserved, especially in D group it was remarkable. CONCLUSION: The extract of Buddleja officinalis can adjust lacrimal gland partial inflammation of dry eye.     

Fyn regulates eosinophil infiltration into the conjunctiva by downregulating the Th2 response

Purpose

Under certain circumstances, fyn may serve to negatively regulate the differentiation of naïve helper T (Th) cells into Th2 cells. This study aimed to investigate whether fyn negatively regulates the development of experimental immune-mediated blepharoconjunctivitis (EC), in which Th2 cells play an important role in C57BL/6 mice.

Methods

C57BL/6 background wild-type (WT) or fyn knockout (fyn-/-) mice were subcutaneously immunized with ragweed (RW) adsorbed in aluminum hydroxide. Ten days later the mice were challenged with RW in eye drops, and 24 h after challenge, eyes, blood and spleens were harvested for histology, measurement of serum IgE, and proliferation or cytokine assays, respectively. RW-primed splenocytes from WT and fyn-/- mice were cultured in the presence of RW. Seventy-two hours later, either whole splenocytes or isolated CD4+T cells were transferred into syngeneic WT mice. Four days after the transfer, the recipient mice were challenged with RW and evaluated as described above.

Results

Infiltration of eosinophils into the conjunctiva induced by active immunization was significantly increased in fyn-/- mice relative to WT mice. Total serum IgE was also significantly higher in fyn-/- mice than in WT mice. In parallel, a higher level of IL-4 production from splenocytes was induced by concanavalin A stimulation in fyn-/- mice than in WT mice. In contrast to active immunization, transfer of whole splenocytes or separated CD4+T cells derived from WT or fyn-/- mice induced similar levels of eosinophilic infiltration in WT mice.

Conclusions

Fyn regulates infiltration of eosinophils into the conjunctiva through downregulation of Th2 responses. This negative regulation is exerted only during the induction phase of EC.