Nghiên cứu vai trò điều hoà của gen mã hoá protein a20 và cơ chế phân tử tham gia kiểm soát quá trình sinh lý tế bào tua tt tiếng anh

However, until now, there is no publication about the role and molecular mechanism of A20 in regulating DCs physiological functions when exposed to parasite-derived antigens.. Therefore,

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

Nguyen Thu Thuy

INVESTIGATION ON THE REGULATORY ROLE OF A20

GENE AND MOLECULAR MECHANISMS INVOLVED IN CONTROLLING DENDRITIC CELL PHYSIOLOGY

Major: Biotechnology Code: 9 42 02 01

SUMMARY OF BIOTECHNOLOGY DOCTORAL THESIS

Hanoi - 2020

This work was completed: Graduate University Science and Technology - Vietnam

Academy of Science and Technology

Supervisor 1: Dr Nguyen Thi Xuan, PhD – Institute of Genetics

Supervisor 2: Dr Hoang Van Tong, PhD – Vietnam Military Medical University

INTRODUCTION

1 The urgency of the thesis

A20 (or tumor necrosis factor α-induced protein 3 - TNFAIP3) is considered as a negative regulator of nuclear factor NF-κB-dependent functions of many different cell types in response to lipopolysaccharide (LPS) and inflammatory cytokines In mice,

deficiency of A20 leads to hyperactivation of immune cells and

autoimmune diseases A20-deficient mice display severe inflammation, mortality cachexia and premature mortality Mice

lacking A20 in dendritic cells fail to LPS tolerance as they die within

6 hours after injection of LPS In humans, A20 also plays an important role in inhibiting inflammatory diseases and cancers An inactivated expression of A20 is found frequently in leukemia However, the effect of A20 on the pathogenesis of diseases is not understood

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) originated from a common bone marrow progenitor and involved in the induction of T cell-mediated adaptive immunity The two major subsets of DCs including plasmacytoid DCs (pDCs) and conventional DCs (cDCs, subdivided into CD8DCs and CD11bDCs) express different toll-like receptors (TLRs), therefore play distinct roles in immunity regarding their interaction with pathogens The cDCs exhibit antigen recognition, cytokine production, and antigen presentation, at levels higher than pDCs and are potent inducers of effector T cells in response to infection, whereas pDCs tend to mediate immune tolerance rather than immunity pDCs originated from lymphoid precursors are CD11clow B220high expressing cells and the main producers of type I interferons (IFNs) in response to

pathogens In addition, cDCs exhibit a high phagocytic capacity, whereas pDCs are only poorly phagocytic and absent in peripheral tissues in noninflammatory conditions Recruitment of pDCs is frequently seen in viral infections and autoimmune diseases such as systemic lupus erythematosus

Several previous studies on dendritic cells have shown that A20 inhibited the ability to produce some cytokines However, until now, there is no publication about the role and molecular mechanism of A20 in regulating DCs physiological functions when exposed to parasite-derived antigens Therefore, this study was conducted to investigate the regulatory role of A20 for physiological functions

when inducing by certain antigens such as profilin from Toxoplasma

gondii or LPS and some cytokines, with the subject name:

"Investigation on the regulatory role of A20 and molecular mechanisms involved in controlling dendritic cell physiology”

2 The objectives of the thesis

1 Investigate the role of A20 in regulating four physiological functions of DCs including maturation, cytokine secretion, migration and apoptosis

2 Investigate the effect of A20 on the regulating three molecular signals including NF-κB, STAT1 and STAT3 related to the physiological process of DCs

3 Main contents of the thesis

1 The role of A20 in regulating DCs physiological functions:

1.1 Differentiate DCs by FLT3 to obtain 3 collections: pDC, CD8DC and CD11bDC Treat these 3 subtypes with profilin, observe and compare the functions of maturity, migration, cytokine secretion

and apoptosis between A20-inactive DCs group and control group

1.2 Differentiate DCs with GM-CSF to obtain CD11bDC Expose this DCs with LPS to observe the role of the A20 for the above physiological functions

2 Study the role of A20 regulating physiological processes through molecular signaling in DCs, including the phosphorylation of IκB-α, STAT1 and STAT3

Chapter 1 OVERVIEW 1.1 Protein A20

Protein A20 is a tumor-stimulating factor necrosis factor α-induced protein 3 (TNFAIP3) that has anti-inflammatory properties and reverses the physiological activity of immune cells by blocking the phosphorylation of several molecular signals such as NF-κB transcription factor (nuclear factor kappa-light-chain-enhancer of activated B cells) and STAT signal (signal transducer and activator of

transcription) Genetic studies show that the A20 mutation alters the

biological function of the A20 protein and is one of the causes of certain conditions as autoimmune diseases, infections and cancers The length of A20 is 790 amino acids and its molecular weight is

89614 Da Protein A20 is coded by A20 (TNFAIP3), its expression is

induced by TNF (tumor necrosis factor) TNFAIP3 encodes a cytoplasmatic zinc finger protein that inhibits NF-кB activation and TNF-mediated apoptosis Studies in knockout mice show that TNFAIP3 is important for limiting inflammation by terminating TNF-induced NF-κB responses TNFAIP3 is a gene with the length

of 15869 bp comprising 9 exons and 8 introns Exon 1, the 5' part of exon 2 and the 3' part of exon 9 are non-coding Length of the transcript is 4446 bp, the coding sequence is CDS 67-2439

A20 plays an important role in regulating the immune system by affecting different immune cells such as DCs, B cells, T cells and

macrophages Therefore, inactivation of the A20 may be a strategy to

improve DCs-based disease prevention and treatment efficacy for

cancer and infectious diseases The A20 is a sensitive locus for

autoimmune diseases, including rheumatoid arthritis, idiopathic arthritis, lupus erythematosus, colitis, psoriasis, type I diabetes, and multiple diseases sclerosis

1.2 Dendritic cells (DCs)

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) to induce immune responses DCs combine and transmit information from the outside environment to the cells of the immune system DCs is not only important in generating innate and adaptive immune responses but also modulates the type of T-cell mediated immune responses Recently, modern medicine has applied Immunotherapy based on DCs to fight cancer and infectious diseases DCs are easily exposed to a variety of exogenous antigens because of DCs available in lymphoid organs such as the spleen and lymph nodes, skin epithelium, gastrointestinal tract, respiratory tract and in the interstitial fluid of most muscles parenchymal organs An important morphological feature of DCs is the presence of a membrane spread out from the main cell body, similar to dendrites on neurons, which should be named dendritic cells, derived from the word "Dendron" meaning tree, in Greek

DCs is divided into two subtypes: lymphoma DCs derived from lymphoid organs (DC plasmacytoid - pDC) and myeloma DCs (classical DC - cDC) The cDC myeloid cells are divided into two subtypes, CD8DC and CD11bDC, which exhibit different levels of

TLR receptors, thus playing a different role in their ability to interact with pathogens

DCs have two main functions: antigen presentation and immunomodulation DCs physiological processes play an important role in the functioning of the immune system, including differentiation, maturation, migration, phagocytosis, cytokine secretion and apoptosis

In the world, DC-vaccine therapy has been tried and tested for melanoma, prostate and kidney cancer patients In Vietnam, the stem cell laboratory of the University of Science, National University

of HCM City has been conducting researches on the use of therapy in the treatment of breast cancer However, this is only a testing step in the laboratory, it can not be applied to actual treatment

DC-in patients Significant advances DC-in understandDC-ing DCs biology have paved the way for the development of medical treatment regimens Therefore, it is expected that this will be an effective cancer treatment

Toxoplasma gondii-derived profilin (TgPRF) is a TLR 11/12

activating ligand of immune cells including DCs in mice and recognised by TLR5 in humans TgPRF contributes to actin-

dependent gliding motility and cellular invasion for T gondii Depletion of DCs renders mice susceptible to T gondii infection A similar study on pDCs shows that infection with T gondii up-

regulates expressions of MHC class II and costimulatory molecules

as well as cell migration to induce proliferation of naive CD4+ T cells

and these cells involve in controlling T gondii infection in the initial

stages

Chapter 2 MATERIAL AND METHODS

2.1 Materials

Mice: BALB/c mice are purchased from Taconic Farms (Hudson,

NY, USA) and housed in a specific pathogen-free facility at the Institute of Genome Research Animal care and experimental procedures are performed according to the Vietnamese law for the welfare of animals and are approved by the institutional review board

of the Institute of Genome Research

Bone marrow-derived DCs: This study used dendritic cells, which

are isolated from mouse bone marrow (BM) and induced

differentiation into DCs in vitro, using Fms-related tyrosine kinase 3

ligand (FLT3) to differentiate to 3 subtypes, including CD8+DC, CD11b+DC and pDC or granulocyte-macrophage colony-stimulating factor (GM-CSF) to differentiate to CD11b+DC, depend on the purpose

2.2 Chemicals, culture media, antibiotics, and kits

The chemicals used in the study included TRIzol ™ Plus RNA Purification Kit; Lipofectamine RNAiMAX Transfection Reagent; IL-6, IL-10, IL-12p70, TNF-α, INF- γ Mouse ELISA Kit; mouse antibodies IgG isotype control, anti-mouse CD11c, anti-mouse CD86, anti-mouse CD40 and anti-mouse IA/IE provided by international standards brands such as Thermo, Sigma, Invitrogen

2.3 Research equipment

The equipment used for the research included Biosafety cabinets class

II, Fluorescence microscopes, Flow cytometry, Western blot kits, ELISA readers and other specialized equipment at Genome Research Institute - Vietnam Academy of Science and Technology and Vietnam Military Medical University

2.4 Research methods

 Bone marrow-derived DCs: Bone marrow-derived DCs

(BMDCs) were obtained from the bone marrow of 6-12 week old BALB/c mice Cells were cultured for 8 days in RPMI- 1640 (Gibco) Cultures were supplemented with FLT3 (200 ng/ml) or GM-

CSF (35 ng/mL) depend on the purpose

 Transfection of DCs with siRNA: BMDCs were transfected with

siRNA targeting A20 (pre-designed siRNA, Applied Biosystems)

with the help of Lipofectamine RNAiMAX Reagent (Invitrogen) to deliver siRNA into BMDCs 48 hours post-transfection, cells were

stimulated with or without LPS and used for further experiments

 Cytokine quantification in cell supernatants: BMDCs were

transfected with A20 siRNA and followed by stimulating with LPS

for 24 h Cell culture supernatant was collected and stored at -80°C until use for ELISA For analysis of IL-6, IL-10, IL-12p40, TNF-α và IFN-γ TNF-α, IL-6 and IL-10 concentrations, commercially available ELISA kits were used according to the manufacturer’s instructions

 Immunostaining and flow cytometry: Cells were incubated in

FACS buffer containing fluorochrome-conjugated antibodies The following antibodies (eBioscience) were used for staining: mouse IgG isotype control, anti-mouse CD11c, anti-mouse MHC II, anti-mouse CD86, anti-mouse CD40 and anti-mouse I-A/I-E and analysed with flow cytometry (FACSAria Fusion, BD Biosciences)

The apoptosis of DCs was evaluated by the expressions of biological markers including Annexin V, 7-AAD, and caspase-3 using flow cytometry

 Migration assay: Migration was assessed in triplicate in a

multiwell chamber with a pore diameter size of 8 µm (BD Falcon)

The cell suspension was placed in the upper chamber to migrate into the lower chamber in which either CCL21 (PeproTech) or medium

alone as a control for spontaneous migration were included

 Western blotting: The possible signalling pathways are analysed

by Western blotting using anti-GAPDH, anti-p-IκB-α,

anti-p-Ser727-STAT-1 and anti-p-STAT-3 (Santa Cruz)

 Bioinformatics methods: Data are provided as means ± SEM, n represents the number of independent experiments

Chapter 3 RESULTS AND DISCUSSION

3.1 Roles of A20 in controlling dendritic cell physiology

3.1.1 Effect of transfection of DCs with A20-siRNA

After silencing A20 in DCs, the expression level of A20 in

inactivated and control cells are checked and compared by Western blot The results are shown in Fig 3.1

Fig 3.1 Effect of transfection of DCs with A20-siRNA

Results on the Western blot images showed that after the introduction

of A20-siRNA into DCs, A20 expression was inactivated almost

completely compared to the control group (control siRNA) The control bands (GAPDH) are strongly clear and have the same size in

each well This result confirmed that after the introduction of

A20-siRNA into DCs, A20 expression was inactivated

3.1.2 A20 inhibits pDC maturation

To ask whether A20 influences in expressions of surface markers on CD8DCs, CD11bDCs and pDCs Cells were transfected with control

or A20 siRNA and followed by TgPRP treatment DC subsets were

collected and stained for IgG isotype control, MHC class II, costimulatory molecule CD86 and CD40 Challenge with TgPRP, increased percentages of MHCII+, CD86+ and CD40+ expressing CD8DCs and pDCs, and did not affect the expression of these markers on CD11bDCs

Fig 3.2 Effect of A20 on maturation of pDCs

In the absence of A20, expressions of MHCII and CD40, but not

CD86 on TgPRP-mature pDCs were significantly enhanced, whereas expressions of MHCII, CD86 and CD40 on both CD8DCs and CD11bDCs were unaltered (Fig 3.2), indicating that A20 prevented expressions of MHC II and CD40 markers on pDCs This result is also appropriate with studies by Pepper et al showing that pDCs is a

prominent DC subtype, related to the early stage of T gondii

infection The ability to present parasitic antigens and the production

of cytokines plays a very important role in controlling infections

In the study of Xuan et al, after exposure CD11bDC to LPS and

comparing the results between the A20-inactivated group and the

control group, the expression of the MHC II, CD86 and CD40

markers were significantly enhanced in the A20 inactivation

subtypes Thus, this experiment has shown that A20 protein plays a role in inhibiting maturation in CD11bDC However, in this study, after differentiating DCs with FLT3 to obtain CD8DC, CD11bDC and pDC and then exposing 3 DC subtypes with profilin, A20 protein only inhibits the maturation of pDC but does not affect the maturation of CD8DC and CD11bDC Thus, exposing to DCs with different antigens, the regulatory role of the A20 protein for the expression of mature markers is different

3.1.3 Roles of A20 in cytokine secretion in DCs

3.1.3.1 A20 inhibits cytokine secretion in pDCs and CD11bDCs when exposed with TgPRP

We next examined cytokine productions secreted by CD8DCs, CD11bDCs and pDCs when exposed with TgPRP Challenge of control DC subsets with TgPRP increased cytokine productions IL-6, IL-10, IL-12p40, TNF-α and IFN-γ secreted by pDCs and slightly enhanced levels of IL-6 and TNF-α in CD11bDCs Results are illustrated in Fig 3.3 and 3.4

Fig 3.3 Effect of A20 on cytokine productions by CD11bDC

when exposed with TgPRP

All the cytokines were measured but IL-10, IL-12p40 and IFN-γ could not be detected in CD11bDCs and therefore only IL-6 and TNF-α are presented in Fig 3.3 Consistently, recent studies report that cytokines IL-12p40 and IFN-γ are not produced by CD11b+DCs

during T gondii infection

Fig 3.4 Effect of A20 on cytokine productions by pDCs

when exposed with TgPRP

To investigate the role of A20 in the regulation of cytokine secretion

by DC subsets, we observed that treatment of the cells with A20 siRNA resulted in the enhanced release of IL-6 and TNF-α by both pDCs and CD11bDCs and levels of IL-12p40 and IFN-γ by pDCs only (Fig 3.4) In addition, IL-10, IL-12p40 and IFN-γ by TgPRF-treated CD11bDCs were measured and not detected The evidence indicated that A20 inhibited inflammatory reaction in pDCs and

partially in CD11bDCs when exposed to TgPRP