Journal of Ophthalmic Inflammation and Infection ( IF 2.9 ) Pub Date : 2020-12-18 , DOI: 10.1186/s12348-020-00229-9 Marta Catarina Esteves Guedes , Catarina Gregório Martins , Maria Jorge Arroz , Miguel Angelo-Dias , Luís Miguel Borrego , Rui Daniel Proença
Uveitis is an intraocular inflammation with several infectious or non-infectious etiologies. The characterization of local and systemic immune profiles in infectious viral uveitis could help recognize different clinical entities and contribute to a more targeted treatment approach.
One previous study addressing cytokine and chemokine profiles in aqueous humor (AqH) of infectious and non-infectious uveitis patients found that interleukin (IL)-1β and IL-10 levels were increased in viral uveitis whereas IL-17 was elevated in toxoplasmic uveitis [1]. IL-10 is of particular interest when studying cytokine profiles in viral infections since it has been shown to suppress the host cellular immune response and favor viral replication, increasing the susceptibility to infection [2] and viral persistence [3].
In this study, we aimed to characterize cytokine profiles in the peripheral blood and AqH of patients with herpetic uveitis (HU) and compare them with healthy controls.
Patients
For this study, both patients and controls were recruited from the Ophthalmology Department of Egas Moniz Hospital, West Lisbon Hospital Center, between October 2014 and October 2016.
Patients presenting with active uveitis from a presumed viral/herpetic etiology were included in the uveitis group. The diagnosis of active uveitis followed the clinical criteria based on inflammatory cell reaction in the anterior chamber or vitreous as per standardization of uveitis nomenclature (SUN) and National Eye Institute (NEI) grading systems [4].
At the time of sampling, all patients had active disease and both blood and AqH samples were collected at presentation. Intraocular samples were examined for the presence of cytomegalovirus (CMV), herpes simplex virus (HSV)-1 and 2, and varicella zoster virus (VZV) by real-time polymerase chain reaction (PCR) analysis as previously described [5].
Controls were selected among healthy subjects undergoing cataract or refractive surgery, with no known history of intraocular inflammation.
The study protocol was approved by the Ethics Committee of Egas Moniz Hospital, West Lisbon Hospital Center, and informed consent was obtained from each patient.
Sample collection
The AqH samples were collected with a 30-gauge needle under topical anesthesia and sterile conditions by slit lamp with the aid of one drop of povidone iodine before and after puncturing the anterior chamber. The AqH samples of control subjects were collected with a 30-gauge needle before starting surgery. Undiluted aqueous samples of at least 0.1 mL were collected from each subject and immediately sent to the laboratory for analysis.
Peripheral blood samples were also collected in order to obtain serum.
Quantification of serum cytokine expression by multiplexed flow cytometry
A multiplex bead-based immunoassay (BD CBA Flex Set, BD Biosciences, San Jose, CA, USA) was used to determine serum and AqH levels of TNF-α, IFN-ɣ, IL-17A and IL-10. A similar single-plex bead-based immunoassay was used for TGF-β.
The protocol was performed following the instructions of the manufacturer. In brief, standards and serum samples were incubated with specific capture beads for 1 h at room temperature. After adding the detection reagent, the mixtures were incubated for 2 h at room temperature in the dark. After a final wash, beads were acquired in a BD FACS Canto II, previously set up for the BD CBA Flex Set. For each cytokine, at least 300 beads were acquired per sample. The FCAP Array Software (BD Biosciences) was used for data analysis. Standard curves covered a 0–2500 pg/mL concentration range and the minimum detection levels were: 0.13 pg/mL for IL10; 0.3 pg/mL for IL17A; 1.8 pg/mL for IFN-γ and 0.7 pg/mL for TNF-α.
For TGF-β, analyzed separately, samples were previously activated with the Sample Activation Kit 1 (R&D, Minneapolis, MN, USA) according to the recommended procedure. After activation, samples were incubated with capture beads for 2 h, washed and incubated with detection reagent. Acquisition and analysis were performed as described above. For TGF-β, standard curves covered a 0–10,000 pg/mL concentration range, and minimum detection level was 14.9 pg/mL.
Statistical analysis
The Mann-Whitney U test was used to compare each 2 independent groups. A P value of < 0.05 was considered for statistical significance. Data were analyzed using GraphPad Prism, version 8 for Windows (GraphPad Software, La Jolla, California).
Four patients with presumed HU and 8 controls were included. Table 1 summarizes the demographic and clinical features for the HU group. One patient with a panuveitis associated with acute retinal necrosis was also included. All HU patients underwent anterior chamber puncture and AqH sampling as previously described. Results of AqH by real-time PCR confirmed a VZV infection in all the cases tested.
Regarding serum cytokines, there were no significant differences observed between patients and controls.
In AqH samples however, patients showed increased concentrations of IL10 (p = 0.018), TNF-α (p = 0.018) and IFN-ɣ (p = 0.024).
Interestingly, the levels of serum and AqH cytokines differ within the two groups. While controls presented higher levels of IL10, IFN-ɣ and TGF-β in serum samples compared to those found in AqH (respectively, p = 0.001; p = 0.002 and p = 0.001), in the patients’ group, only TGF-β showed higher serum concentrations when compared to AqH (p = 0.029), with comparable values for the other cytokines tested.
Table 2 shows the results and comparison of cytokine levels in the serum and AqH of both groups.
Despite including just a few HU cases, our preliminary results show an elevation of intraocular TNF-α and IFN-ɣ levels which is likely associated with active disease and anterior chamber inflammation.
The increase in IL-10 levels also found in patients’ AqH samples is, as described in a previous study [1], probably related to the viral etiology, in this case herpetic. However, since all the patients included tested positive for VZV infection, it is possible that other herpetic etiologies, such as HSV-1 and 2 or CMV, show different serum and intraocular cytokine profiles.
The immunosuppressive ability of IL-10 to impair T-cell responses leading to persistent viral infection has already been demonstrated in mice [6, 7], reinforcing its possible use as a biomarker for viral infection or even as a therapeutic target since it has been shown that in vivo administration of an antibody against the murine IL-10 receptor completely prevented the viral persistence by restoring T-cell function [3].
In conclusion, although more studies are needed to confirm our findings, elevated intraocular IL-10, TNF-α and IFN-ɣ levels seem to be associated to VZV-associated uveitis.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
- AqH:
-
Aqueous humor
- CMV:
-
Cytomegalovirus
- HSV:
-
Herpes simplex virus
- HU:
-
Herpetic uveitis
- IFN-ɣ:
-
Interferon-Ɣ
- IL:
-
Interleukin
- PCR:
-
Polymerase chain reaction
- TGF-β:
-
Transforming Growth Factor β
- TNF-α:
-
Tumor Necrosis Factor α
- VZV:
-
Varicella zoster virus
- 1.
Sauer A, Villard O, Creuzot-Garcher C, Chiquet C, Berrod JP, Speeg-Schatz C et al (2015) Intraocular levels of interleukin 17A (IL-17A) and IL-10 as respective determinant markers of toxoplasmosis and viral uveitis. Clin Vaccine Immunol 22(1):72–78
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This research project was funded by the Portuguese Ophthalmology Society.
Affiliations
Ophthalmology Department, Western Lisbon Hospital Center- Egas Moniz Hospital, Rua da Junqueira, 126, 1349-019, Lisbon, Portugal
Marta Catarina Esteves Guedes
CEDOC, NOVA Medical School, Nova University of Lisbon, Campo dos Mártires da Pátria, 1169-056, Lisbon, Portugal
Catarina Gregório Martins, Miguel Angelo-Dias & Luís Miguel Borrego
Comprehensive Health Research Centre (CHRC), NOVA Medical School, Nova University of Lisbon, Campo dos Mártires da Pátria, 1169-056, Lisbon, Portugal
Catarina Gregório Martins, Miguel Angelo-Dias & Luís Miguel Borrego
Western Lisbon Hospital Center- São Francisco Xavier Hospital, Estr. Forte do Alto Duque, 1449-005, Lisbon, Portugal
Maria Jorge Arroz
Faculty of Medicine, Coimbra Hospital and Universitary Center, Praceta Professor Mota Pinto, 3004-561, Coimbra, Portugal
Rui Daniel Proença
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Contributions
MG was involved in patients and controls recruitment, data collection, review of literature, and drafting of the manuscript. MJA, CGM and LMB were involved in the data collection and flow cytometric analysis as well as results interpretation and final editing of the manuscript. MAD was responsible for the statistical analysis. RDP was involved in supervision, fine tuning, and final editing of the manuscript. The author(s) read and approved the final manuscript.
Corresponding author
Correspondence to Marta Catarina Esteves Guedes.
Ethics approval and consent to participate
The study protocol was approved by the Ethics Committee of Egas Moniz Hospital, West Lisbon Hospital Center, and informed consent was obtained from each patient.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Guedes, M.C.E., Martins, C.G., Arroz, M.J. et al. Cytokine profiles in the peripheral blood and aqueous humor of patients with herpetic uveitis. J Ophthal Inflamm Infect 10, 37 (2020). https://doi.org/10.1186/s12348-020-00229-9
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DOI: https://doi.org/10.1186/s12348-020-00229-9
中文翻译：
疱疹性葡萄膜炎患者外周血和房水中的细胞因子谱
葡萄膜炎是具有几种感染性或非感染性病因的眼内炎症。感染性病毒性葡萄膜炎的局部和全身免疫特征的表征可以帮助识别不同的临床实体，并有助于更具针对性的治疗方法。
先前针对感染性和非感染性葡萄膜炎患者房水（AqH）细胞因子和趋化因子分布的一项研究发现，病毒性葡萄膜炎中白介素（IL）-1β和IL-10水平升高，而弓形体性葡萄膜炎中IL-17升高[ 1]。在研究病毒感染中的细胞因子谱时，IL-10特别令人感兴趣，因为它已显示出抑制宿主细胞免疫反应并促进病毒复制，增加感染敏感性和病毒持久性的作用[3]。
在这项研究中，我们旨在表征疱疹性葡萄膜炎（HU）患者外周血和AqH中的细胞因子谱，并将其与健康对照进行比较。
耐心
在这项研究中，患者和对照组均于2014年10月至2016年10月间从西里斯本医院中心Egas Moniz医院眼科招募。
根据推测的病毒/疱疹病因而出现活动性葡萄膜炎的患者包括在葡萄膜炎组中。根据葡萄膜炎命名法（SUN）和美国国立眼科学院（NEI）分级系统的标准化，活动性葡萄膜炎的诊断遵循临床标准，该标准基于前房或玻璃体中的炎症细胞反应。
在取样时，所有患者均患有活动性疾病，并且在就诊时均采集了血液和AqH样品。如前所述，通过实时聚合酶链反应（PCR）分析检查眼内样品中是否存在巨细胞病毒（CMV），单纯疱疹病毒（HSV）-1和2，水痘带状疱疹病毒（VZV）[5]。
从接受白内障或屈光手术的健康受试者中选择对照，没有已知的眼内炎症史。
研究方案已由西里斯本医院中心Egas Moniz医院伦理委员会批准，并从每位患者获得了知情同意。
样品采集
在穿刺前房之前和之后，在局部麻醉和无菌条件下，通过裂隙灯在局部麻醉和无菌条件下，用30号针头收集AqH样品。开始手术前，用30号针头收集对照受试者的AqH样品。从每个受试者中收集至少0.1 mL的未稀释水溶液样品，并立即送至实验室进行分析。
还采集外周血样品以获得血清。
多重流式细胞术定量血清细胞因子表达
使用基于多重珠的免疫测定法（BD CBA Flex Set，BD Biosciences，美国加利福尼亚州圣何塞）来确定血清和TNF-α，IFN-γ，IL-17A和IL-10的AqH水平。相似的基于单重珠的免疫测定用于TGF-β。
该协议是按照制造商的说明进行的。简而言之，将标准品和血清样品与特异性捕获珠在室温下孵育1小时。加入检测试剂后，将混合物在室温黑暗中孵育2小时。最后清洗后，在先前为BD CBA Flex Set设置的BD FACS Canto II中采集珠子。对于每种细胞因子，每个样品至少采集300个珠子。使用FCAP阵列软件（BD Biosciences）进行数据分析。标准曲线涵盖了0-2500 pg / mL的浓度范围，最低检测水平为：IL10为0.13 pg / mL；IL17A为0.3 pg / mL；IFN-γ为1.8 pg / mL，TNF-α为0.7 pg / mL。
对于TGF-β，需单独分析，样品先根据推荐程序使用样品激活试剂盒1（R＆D，明尼阿波利斯，明尼苏达州，美国）激活。活化后，将样品与捕获珠孵育2小时，洗涤并与检测试剂孵育。如上所述进行采集和分析。对于TGF-β，标准曲线涵盖了0–10,000 pg / mL的浓度范围，最低检测水平为14.9 pg / mL。
统计分析
使用Mann-Whitney U检验比较每个2个独立组。甲P的<0.05值被认为是统计显着性。使用适用于Windows的GraphPad Prism版本8（加利福尼亚州拉荷亚的GraphPad软件）分析数据。
包括4名假定的HU患者和8名对照。表1总结了HU组的人口统计学和临床特征。还包括一名伴有急性视网膜坏死的胰腺炎患者。如前所述，所有HU患者均接受了前房穿刺和AqH采样。实时PCR的AqH结果在所有测试的病例中均证实了VZV感染。
关于血清细胞因子，在患者和对照之间没有观察到显着差异。
然而，在AqH样品中，患者的IL10（p = 0.018），TNF-α（p = 0.018）和IFN-γ（p = 0.024）浓度升高。
有趣的是，两组中血清和AqH细胞因子的水平不同。与对照组相比，对照组的血清中IL10，IFN-ɣ和TGF-β的水平更高（分别为p = 0.001；p = 0.002和p = 0.001），而在患者组中，只有TGF-β与AqH相比，血清浓度更高（p = 0.029），与其他测试的细胞因子值相当。
表2显示了两组的血清和AqH中细胞因子水平的结果和比较。
尽管仅包括少数HU病例，我们的初步结果显示眼内TNF-α和IFN-γ水平升高，这可能与活动性疾病和前房炎症有关。
如先前的研究[1]所述，在患者的AqH样品中也发现IL-10水平升高，可能与病毒病因有关，在本例中为疱疹。但是，由于所有患者均包括VZV感染检测呈阳性，因此其他疱疹病因，例如HSV-1和2或CMV，可能显示出不同的血清和眼内细胞因子谱。
已经在小鼠中证明了IL-10抑制T细胞反应导致持续性病毒感染的免疫抑制能力[6，7]，因为它已经被证实可以用作病毒感染的生物标志物甚至治疗靶标。研究表明，体内给予抗鼠IL-10受体的抗体可通过恢复T细胞功能完全阻止病毒的持久性[3]。
总之，尽管需要更多的研究来证实我们的发现，但眼内IL-10，TNF-α和IFN-γ水平升高似乎与VZV相关的葡萄膜炎有关。
在当前研究中使用和/或分析的数据集可应合理要求从相应的作者处获得。
- AqH：
-
水幽默
- CMV：
-
巨细胞病毒
- HSV：
-
单纯疱疹病毒
- HU：
-
疱疹性葡萄膜炎
- IFN-ɣ：
-
干扰素
- IL：
-
白介素
- PCR：
-
聚合酶链反应
- TGF-β：
-
转化生长因子β
- TNF-α：
-
肿瘤坏死因子α
- VZV：
-
水痘带状疱疹病毒
- 1。
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Sloan DD，Jerome KR（2007）单纯疱疹病毒重塑T细胞受体信号传导，导致p38依赖的白介素10选择性合成。J病毒81（22）：12504–12514
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Brooks DG，Trifilo MJ，Edelmann KH，Teyton L，McGavern DB，Oldstone MB（2006）Interleukin-10确定了体内的病毒清除率或持久性。Nat Med 12（11）：1301-1309
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Kongyai N，Sirirungsi W，Pathanapitoon K，Tananuvat N，Kunavisarut P，Leechanachai P等人（2012）泰国原因不明的前葡萄膜炎的病毒原因。眼睛26（4）：529–534
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Jenkins C，Garcia W，Godwin MJ，Spencer JV，Stern JL，Abendroth A等人（2008）在感染的潜伏期中由人巨细胞病毒表达的人白介素10病毒同系物的免疫调节特性。J Virol 82（7）：3736–3750
CAS文章Google学术搜索
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该研究项目由葡萄牙眼科协会资助。
隶属关系
西里斯本医院中心眼科-Egas Moniz医院，Rua da Junqueira，126，1349-019，葡萄牙里斯本
玛塔·卡塔琳娜·埃斯特维斯·盖德斯
CEDOC，里斯本新星大学NOVA医学学校，Campo dosMártiresdaPátria，1169-056，葡萄牙里斯本
CatarinaGregórioMartins，Miguel Angelo-Dias和LuísMiguel Borrego
里斯本新星大学诺瓦医学院综合健康研究中心（CHRC），Campo dosMártiresdaPátria，1169-056，葡萄牙里斯本
CatarinaGregórioMartins，Miguel Angelo-Dias和LuísMiguel Borrego
西里斯本医院中心-圣弗朗西斯科Xavier医院，Estr。福尔托·杜·阿尔托·杜克（Forte do Alto Duque），1449-005，葡萄牙里斯本
玛丽亚·豪尔赫·阿罗兹
科英布拉医院和大学中心医学院，Praceta Mota Pinto教授，3004-561，葡萄牙科英布拉
Rui DanielProença
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会费
MG参与了患者和对照的招募，数据收集，文献回顾和手稿的起草。MJA，CGM和LMB参与了数据收集和流式细胞仪分析以及结果解释和手稿的最终编辑。MAD负责统计分析。RDP参与了手稿的监督，微调和最终编辑。作者阅读并批准了最终手稿。
通讯作者
对应于Marta Catarina Esteves Guedes。
道德规范的批准和同意参加
研究方案已由西里斯本医院中心Egas Moniz医院伦理委员会批准，并从每位患者获得了知情同意。
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不适用。
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作者宣称他们没有竞争利益。
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Guedes，MCE，Martins，CG，Arroz，MJ等。疱疹性葡萄膜炎患者外周血和房水中的细胞因子谱。ĴOphthal Inflamm传染 10， 37（2020）。https://doi.org/10.1186/s12348-020-00229-9
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DOI ：https ：//doi.org/10.1186/s12348-020-00229-9
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