Low expression of TNFAIP3 enhances the function of dendritic cells in AMD via MAPK signaling pathway

doi:10.21203/rs.3.rs-1508180/v1

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Low expression of TNFAIP3 enhances the function of dendritic cells in AMD via MAPK signaling pathway

https://doi.org/10.21203/rs.3.rs-1508180/v1

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Purpose TNFAIP3 is an important negative regulator of inflammation and immunity. In this study, we explore the role of TNFAIP3 in wAMD.

Method We used real-time quantitative PCR to detect the expression level of TNFAIP3 in PBMCs and DCs of wAMD and normal people. Subsequently, we transfected DCs with adenovirus to silent TNFaip3, and detected the levels of IL-1β, IL-6 and IL-10 secreted by DCs by ELISA. The phosphorylation levels of JNK, p38 and ERK 1/2 were detected by flow cytometry.

Result Compared with normal people, the expression level of TNFAIP3 in PBMCs and DCs of wAMD patients was significantly decreased. Silenced TNFAIP3 could increase the levels of IL-1β and IL-6 secreted by DCs, decrease the level of IL-10, and also increase the levels of JNK and p38 phosphorylation.

Conclusion This study showed that the expression level of TNFAIP3 was significantly decreased in wAMD patients, and the decrease of TNFAIP3 could lead to the change of inflammatory cytokines secreted by DCs through the MAPK pathway.

Age-related macular degeneration (AMD) is a severe macular disease that is the major cause of irreversible blindness worldwide.¹ In particular, wet AMD produces severe retinal pigmented epithelial cell degeneration, photoreceptor cell loss, and/or choroid neoangiogenesis.² Although the exact cause of AMD is not entirely clear, the disease is associated with genetic and environmental risk factors. In addition, advanced age, gender, race, smoking, obesity, hypertension, diet and other factors are also related to the occurrence and development of AMD.^{3, 4} Studies have shown that the polymorphism of the TNFAIP3 gene is closely related to the development of AMD.⁵

Zinc-finger protein TNFAIP3 is an ubiquitin-editing protein that regulates the inflammatory signaling pathway and negatively regulates the NF-κB pathway.⁶ Recent studies have found that TNFAIP3 has been able to regulate a series of activation, maturation and function of dendritic cells (DCs). Mice with TNFAIP3 gene defects in DCs cells are more sensitive to the stimulation of external microorganisms, with significantly increased secretion levels of such as TNF-α, IL-1 and IL-6, moreover, these can significantly stimulate the activation and proliferation of T cells and promote the differentiation of Th1 and Th17 cells.⁷ As the most powerful professional antigen-presenting cells in the body, DCS plays a key role in the local immune response to acute and chronic tissue injury, and is related to the pathogenesis of AMD.⁸ Then, there is no research on whether TNFAIP3 affects the development of AMD through DCs. Therefore, this study clarifies its possible mechanisms in AMD mainly through the influence of TNFAIP3 on DCs.

1.1 Study subjects

We selected 15 patients with wet AMD (wAMD) and 16 age-and gender-matched normal people. Inclusion criteria: (1) AMD patients meet the diagnostic criteria in the Guidelines for the Diagnosis of Age-Related Macular Degenesis;⁹ (2) Eye examination and physical indicators in the control group were normal, with no family history of eye disease. Exclusion criteria: (1) patients with diabetes and severe cardiovascular and cerebrovascular diseases; (2) patients with cardiac, liver, renal impairments; (3) patients with other retinal diseases, glaucoma, etc; (4) patients with related eye treatment in nearly 1 month. This study was approved by the Ethics Committee of the Affiliated Hospital of Southwest Medical University.

1.2 Cell separation and culture

10ml of the blood of AMD patients and normal people were extracted into the heparin anticoagulant centrifuge tube and immediately mixed to avoid blood coagulation. Human peripheral blood mononuclear cells were obtained by centrifugation at a temperature of 4℃ and at a centrifugal force of 800g for 30 minutes after adding the human lymphocyte separation fluid. Then a 20ul monocyte magnetic beads (CD14 ⁺ magnetic beads (Miltenyi Biotec, Germany)) was added and incubated in a 4℃ freezer for 15min. The CD14⁺T cells were obtained by centrifugation of PBMCs with CD14⁺ magnetic beads on MS column for 1000g at room temperature for 5min. The CD14⁺T cells were added to the 1640 medium containing 1% glutamine, 1% penicillin streptomycin and 10% fetal bovine serum (Gibco BRL, USA). GM-CSF (100 ng/mL, ACRO Biosystems, Newark, USA) and People Regroup IL-4 (50 ng/mL, ACRO Biosystems, Newark, USA) were added into the medium to induce the formation of dendritic cells (DCs). After 6 days of cell culture, adding LPS (100ng/ml, Sigma, St. Louis, Missouri, USA) to cultivate for 24h to promote its transformation into mature dendritic cells.

1.3 Extraction and real-time quantitative PCR of RNA

According to the instructions, total RNA of PBMCs and DCs was extracted using Trizol lysate (Invitrogen, Carlsbad, California, USA) and cDNA was synthesized by reverse transcription (Takara, Tokyo, Japan). Synthetic conditions for real-time quantitative PCR were 95 °C pre-degeneration 30 seconds, 95 °C degeneration 5 seconds, 60 °C annealing 34 seconds, and 95 °C extended 15 seconds for a total of 40 cycles. In this experiment, the sequences of TNFAIP3and β-actin were as follows: TNFAIP3 forward: 5'-TGGCTGAACAAGTCCTTCCT-3′, reverse: 5'-CTTCAGGTCACCAAGGGTA-3′; β-actin forward: 5'-CGAGAAGATGACCCAGATCATG-3', reverse: 5'-CAGAGGCGTACAGGGATAGCA-3'. The data analysis is calculated using the 2^{- △ △ CT}method.

1.4 Transfection of dendritic cells

Mononuclear-induced dendritic cell culture until the third day, inoculated to 24 orifice plates with a cell concentration of 1 × 10⁵/ ml, and added the Human TNFAIP3 gene interferes with the adenovirus (Hanbio, Shanghai, China) with virus volume MOI of 10, 30, 100, 300, 500, another group of target cells was added with the same titer and volume of control virus vector, and changed the fluid after 2 hours. The expression of GFP green fluorescence was observed under the fluorescence microscope 48 hours after transfection.

1.5-Flow Cytometry

To detect the mechanism of TNFAIP3 action in DCs, 48 hours after we transfected DCs with adenovirus, subsequently added the LPS (100 ng / ml) action for 30 minutes, and finally detected the phosphorylation levels of JNK, ERK1/2 and p38 (BD Biosciences, San Diego, California, USA) via flow cytometry.

1.6 ELISA

Test the levels of IL-1β, IL-6 and IL-10 in the cell supernatant in accordance with the ELISA Kit Instructions (R&D Systems, Minneapolis, Minnesota).

1.7 Statistical analysis

All data were analyzed by SPSS17.0 software statistical analysis, expressed in the form of Mean土SD, normality tests using Shapiro-Wilk normality test, independent Samples T-test, and p <0.05 was seen as having a significant statistical difference.

2.1 TNFAIP3 mRNA expression decreased in PBMCs of wet AMD patients

We first measured the expression of TNFAIP3 in normal control groups and in patients with wet AMD. The results of real-time quantitative PCR showed that the TNFAIP3 mRNA in PBMCs of wet AMD patients was significantly lower than normal people (Figure 1).

2.2 TNFAIP3 mRNA expression decreased in DCs in wet AMD

DCs is a component of PBMCs, which is believed to be the main source cell of TNFAIP3. Previous studies have found that TNFAIP3 can regulate its immunogenicity in DCs, but its role in DCs with wet AMD is unclear. Therefore, in the next step, we will detect the expression of TNFAIP3 mRNA in DCs. The results showed that the expression level of TNFAIP3 mRNA in DCs in patients with wet AMD was significantly lower than in normal people (Figure 2).

2.3 Expression level of TNFAIP3 in adenovirus transfected DCs and DCs after transfection.

To study the biological function in DCs, we transfected DCs with adenovirus with GFP fluorescence. The results showed that at the MOI value of 500, most of the DCs were observed with green fluorescence (Figure 3A). TNFAIP3 mRNA expression was significantly lower after being transinfected with an adenovirus compared to normal controls (Figure 3B).

2.4 Effect of TNFAIP3-shRNA on the secreted cytokines in DCs

To investigate whether TNFAIP3 has an impact on DCs-related inflammatory factors, we measure the levels of IL-1β, IL-6 and IL-10 by using ELISA. Relevant data showed that after transfection with TNFAIP3-shRNA, the secretion of IL-1β and IL-6 increased, while the expression of IL-10 decreased (Figure 4).

2.5 Effect of TNFAIP3 on the MAPK signaling pathway

To find out how TNFAIP3 affects DCs, we conducted the following experiments. After 6 days of cell culture, LPS was added for half an hour, and then the phosphorylation levels of p38, JNK and ERK1/2 were detected by flow cytometry. Results are shown, (Figure 5)

This study showed that TNFAIP3 expression was decreased in peripheral blood mononuclear cells and DCs in wAMD compared with healthy controls. Next, we transfected DCs with adenovirus containing TNFAIP3 gene silencing vector. The results showed that TNFAIP3 silencing causes increased levels of proinflammatory cytokine IL-1β, IL-6 and reduced expression of IL-10, and this role of TNFAIP3 may be associated with the activation of MAPK.

The cause of wAMD has not yet been determined and may be related to genetic, chronic photodamage, nutritional disorders, etc. There is currently evidence that it is a chronic nonspecific inflammation of the immune-related.^9-11 TNFAIP3 is an inductive expression protein detected in the response of umbilical vein endothelial cells treated with TNF-α. It plays an important role in the regulation of innate immunity and adoptive immunity, and plays an important role in the regulation of immune homeostasis. To study the role of TNFAIP3 in wAMD, we first detected the expression of TNFAIP3 mRNA in PBMCs and DCs in patients with wAMD. The results showed that lower TNFAIP3 expression in PBMCs and DCs in wAMD patients compared with healthy controls. This is consistent with many immune diseases.^12-14 However, our results are inconsistent with an earlier study that showed they used RT-PCR to detect the expression of TNFAIP3 mRNA in PBMCs of chronic hepatitis B patients and healthy controls, and the level of TNFAIP3 mRNA in patients with chronic hepatitis B was significantly higher than that in healthy controls.¹⁵ The reason for the difference in TNFAIP3 expression between the two diseases is unknown, but it may be due to the significant difference in the mechanism that caused these two diseases. wAMD is a fundus disease with a multifactor composite effect related to age, while chronic hepatitis B is caused by infection with hepatitis B virus. The process of hepatitis B virus infection is complex. Hepatitis B virus protein has immunomodulating ability and can start the molecular mechanism of “escape” host immune surveillance.

Many studies have been published on the way TNFAIP3 works, but less on its impact on DCs. To address this problem, we transfected dendritic cells with an adenovirus containing the silencing vector of the TNFAIP3 gene. The results showed that TNFAIP3 silencing caused increased levels of pro-inflammatory cytokines IL-1β, IL-6 and decreased expression of anti-inflammatory factors IL-10. Our findings are consistent with earlier studies.¹⁶ However, it should be noted that earlier studies used a different method, in which the expression of TNFAIP3 was reduced by siRNA transfection, and significantly enhanced expression of pro-inflammatory cytokine IL-6 after silent TNFAIP3, suggesting that TNFAIP3 plays a negative regulation role in the immune response, which also shows that no matter what transfection method is used, it does not affect the negative regulation function of TNFAIP3.

The classic way of TNFAIP3 is to regulate the activity of the transcription factor NF-κB signaling system by the deubiquitination function of TRAF6 and the E3 ubiquitin ligase function, so as to play a negative regulation role on the innate immune response. Recent researches have shown that in animal models of acute liver failure in male Wistar rats, TNFAIP3 inhibited the activation of the NF-κB signaling pathway, thereby enhancing liver cells proliferation and cell cycle processes while inhibiting apoptosis of liver cells.¹⁷ In Patients with colitis, TNFAIP3 may reduce inflammation by inhibiting NF-κB, phosphorylating STAT3 and IL-17 to inhibit the progression of colitis.¹⁸ There are also experimental results showing that increased TNFAIP3 in human umbilical vein endothelial cells and SW480 cell lines can inhibit the IKK-α /NF-κB signaling pathway, causing inflammatory cytokines TNF-α and I L-1β downregulation.¹⁹ The function of the TNFAIP3 is all achieved through the NF-κB signaling path. However, it has also been shown that TNFAIP3 can regulate the production of the cytokine IL-1 by inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway.²⁰ Activation of the MAPK signaling pathway can stimulate the production of pro-inflammatory or anti-inflammatory cytokines such as IL-10, IL-6 and IL-1β.^{21, 22} Because the role of TNFAIP3 in controlling the DCs signaling pathway is unclear, we conducted extensive gene silencing experiments to address this problem. TNFAIP3 silence leads to an increase in JNK and p38 activation but does not affect ERK1/2, which is similar to results in other studies.²³ But as they differ in the study of mesenchymal stem cells, the p38 MAPK signaling pathway is suppressed after knocking off the TNFAIP3, thereby promoting the production of TNF-α and inhibiting the production of IL-10.²⁴ The reason for this difference may be due to the different types of cells used, and mesenchymal stem cells are a very special cell type with the ability to self-update and various differentiation.

It is noted that there are still some limitations in our study. Our results show reduced TNFAIP3 expression in PBMCs and DCs in wAMD patients, and it is unclear whether reduced TNFAIP3 expression also exists in other macular degeneration, such as PCV, macular neovascularization and so on. Our in vitro experiments show that TNFAIP3 has a regulatory effect on DCS, but it is unclear whether it can obtain a similar effect in vivo, which needs further animal model verification. If the development of wAMD is related to the decrease in the expression of TNFAIP3, then using medication directly or indirectly to restore TNFAIP3 to normal levels, which may provide a new treatment option for t wAMD.

In conclusion, our results show that the reduced expression of TNFAIP3 in wAMD and that silenced TNFAIP3 can negatively regulate the ability of DCs to produce inflammatory cytokines through the MAPK signaling pathway.

Acknowledgements

We would like to thank all donors participating in the present study.

Author’ contributions statement

Yue He and Jie Chen conceived and designed research. Zhiru Zhang, Tianyu Yao and Li Huang performed experiments. Yue He and Wei Yang analyzed data and interpreted results of experiments. Wei Yang and Li Huang prepared figures. Yue He drafted manuscript. All authors edited and revised manuscript and approved final version of manuscript.

Declaration of Competing Interest

he authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by the Scientific Research Topics of Sichuan Health and Family Planning Commission [16PJ560]; Affiliated Hospital of Luzhou Medical College scientific research project [2015-PT-017]; Youth Innovation in Medical Research in Sichuan Province [Q15014]; Doctoral Research Initiation Fund of Affiliated Hospital of Southwest Medical University.

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No competing interests reported.

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Low expression of TNFAIP3 enhances the function of dendritic cells in AMD via MAPK signaling pathway

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Version 1

Abstract

Figures

Introduction

1. Object and Methods

1.1 Study subjects

1.2 Cell separation and culture

1.3 Extraction and real-time quantitative PCR of RNA

1.4 Transfection of dendritic cells

1.5-Flow Cytometry

1.6 ELISA

1.7 Statistical analysis

2. Results

2.1 TNFAIP3 mRNA expression decreased in PBMCs of wet AMD patients

2.2 TNFAIP3 mRNA expression decreased in DCs in wet AMD

2.3 Expression level of TNFAIP3 in adenovirus transfected DCs and DCs after transfection.

2.4 Effect of TNFAIP3-shRNA on the secreted cytokines in DCs

2.5 Effect of TNFAIP3 on the MAPK signaling pathway

3. Discussion

Declarations

References

Additional Declarations

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