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

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING 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: 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 Committee member 1: … Committee member 2: … Committee member 3: … This work will be defended at Graduate University Science and Technology Vietnam Academy of Science and Technology INTRODUCTION The urgency of the thesis A20 (or tumor necrosis factor α-induced protein - 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 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” The objectives of the thesis Investigate the role of A20 in regulating four physiological functions of DCs including maturation, cytokine secretion, migration and apoptosis Investigate the effect of A20 on the regulating three molecular signals including NF-κB, STAT1 and STAT3 related to the physiological process of DCs Main contents of the thesis The role of A20 in regulating DCs physiological functions: 1.1 Differentiate DCs by FLT3 to obtain collections: pDC, CD8DC and CD11bDC Treat these 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 Study the role of A20 regulating physiological processes through molecular signaling in DCs, including the phosphorylation of IκB-α, STAT1 and STAT3 Chapter OVERVIEW 1.1 Protein A20 Protein A20 is a tumor-stimulating factor necrosis factor α-induced protein (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 exons and introns Exon 1, the 5' part of exon and the 3' part of exon 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 DCtherapy in the treatment of breast cancer However, this is only a testing step in the laboratory, it can not be applied to actual treatment in patients Significant advances in understanding 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 actindependent 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 upregulates 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 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 ligand (FLT3) to differentiate to 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 days in RPMI- 1640 (Gibco) Cultures were supplemented with FLT3 (200 ng/ml) or GMCSF (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 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, antimouse 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 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-Ser727STAT-1 and anti-p-STAT-3 (Santa Cruz)  Bioinformatics methods: Data are provided as means ± SEM, n represents the number of independent experiments Chapter 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 A20siRNA into DCs, A20 expression was inactivated 3.1.2 A20 inhibits pDC maturation 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 TgPRFtreated CD11bDCs were measured and not detected The evidence indicated that A20 inhibited inflammatory reaction in pDCs and partially in CD11bDCs when exposed to TgPRP 11 3.1.3.2 A20 inhibits cytokine secretion in CD11bDCs when exposed with LPS Fig 3.5 Effect of A20 on cytokine productions by CD11bDCs when exposed with LPS Compared to the inactive control group, inactivation of A20 in CD11bDC leads to increased production of IL-10 and TNF-α but did not affect IL-6 secretion This result is also consistent with previous studies showing that IL-6 is not related to the regulatory role of the A20 in DCs and that its level in mouse serum is even lower in mice that are deficient A20 in DCs compared with controls In this second experimental group, inactivation of A20 in CD11bDCs resulted in increased production of IL-10 and TNF-α compared to the inactive control group but did not affect IL-6 secretion However, in the first experimental group, when exposed to profilin, in CD11bDCs group, inactivated A20 increased IL-6 and TNF-α secretion Thus, the use of different antigens to stimulate maturation leads to different cytokine secretion or it can be said to lead to different maturation manifestations 3.1.4 Roles of A20 in migration in DCs 3.1.4.1 A20 inhibits migration in pDCs when exposed with TgPRP 12 Fig 3.6 Effect of A20 on CD8DCs, CD11bDCs and pDCs migration when exposed with TgPRP A recent study shows the role of A20 in suppressing migration of cancer cells Similar to our results attained from the expression of maturation markers, challenge of DC subsets with TgPRP also led to enhanced migration of CD8DCs and pDCs, but not CD11bDCs (Fig 3.6A– C) In the absence of A20, migration of mature pDCs only was further enhanced (Fig 3.6C), indicating that the migration of TgPRPmatured pDCs was inhibited by the presence of A20 3.1.4.2 A20 inhibits migration in CD11bDCs when exposed with LPS Fig 3.7 Effect of A20 on CD11bDCs migration when exposed with LPS In the CD11bDC subtype, A20 inactivation enhances mobility when dealing with LPS However, this result does not exactly match the previous experiment when conducting exposure to profilin This result again confirms that when stimulating maturation by different antigens leads to different maturation manifestations 13 3.1.5 Roles of A20 in apoptosis of Cd11bDCs Fig 3.8 Effect of A20 protein on apoptosis through the number of cells that react positively with marker Annexin V After exposure to LPS and some cytokines such as IL-10, IL-2, TNF-α and INF-γ, compared to the control cell group, the A20 inactivated group did not show a difference in percentage of cell responded positive to Annexin V antibody and negative for 7-AAD Similarly, when analyzing apoptosis by the ratio of positive cells to Caspase-3, the results showed no difference between the A20 inactivated group and the control group Thus, in these two experiments, A20 protein did not affect apoptosis of DCs However, in other studies, T Das's team and Z Jin's team, both showed that A20 plays an inhibitory role through apoptosis in many types of immune cells Regarding the role of some cytokines in apoptosis, through both survey methods, the percentage of cells positive for Annexin V, negative for 7AAD and the percentage of cells positive for Caspase-3, The results showed that compared to the control group, the cell group was treated with TNF- α (10 ng/ml), INF-γ (10 ng/ml) and IL-2 (30 ng/ml) There was no apparent difference in apoptosis percentage Meanwhile, IL-10 at concentrations of 20 ng/ml and 200 ng/ml enhances the process of apoptosis This indicates that IL-10 promotes DCs apoptosis, while other 14 cytokines such as TNF-α, INF-γ, and IL-2 not enhance this process This result is also consistent with previous studies showing that IL-10 anti-inflammatory cytokines are involved in inducing apoptosis in several different cell types including DCs and macrophages When using LPS as a control, the results also showed that A20 did not affect apoptosis of CD11bDC Therefore, we did not study the effect of A20 protein on apoptosis of DC subtypes when exposed to profilin 3.2 Investigation on the regulatory role of A20 and molecular mechanisms involved in controlling dendritic cell physiology 3.2.1 A20 inhibits activations of IκB-α and STAT-1 signallings in pDCs Since A20 suppressed the maturation and activation of pDCs and partially inflammatory reaction in CD11bDCs, therefore we examined expressions of signalling molecules involved in the regulatory functions of pDCs and CD11bDCs CD11bDCs and pDCs were treated with TgPRP for one hour and total cell protein was extracted by using RIPA-1 lysis buffer 15 Fig 3.9 Effect of A20 on IκB-α, STAT1 and STAT3 signaling pathways in CD11bDCs upon exposure to profilin (n = 5) Fig 3.10 Effect of A20 on IκB-α, STAT1 and STAT3 signaling pathways in pDCs upon exposure to profilin (n = 5) As shown in Fig 3.9 and 3.10, treatment with A20 siRNA resulted in the enhanced phosphorylation of IκB-α and STAT1 in TgPRPchallenged pDCs, whereas activations of these molecules in A20silenced CD11bDCs was slightly increased, but not reaching to the significance The results showed that A20 contributed to an inhibitory effect on the activations of IκB-α and STAT-1 signallings in pDCs 3.2.2 A20 inhibits activations of IκB-α and STAT-1 signallings in CD11bDCs when exposed to LPS 16 Fig 3.11 Effect of A20 on IκB-α, STAT1 and STAT3 signaling pathways in CD11bDCs upon exposure to LPS (n=5) LPS stimulation leads to the generation of phosphorylation of IκB-α and STAT1 Compared to control cells, inactivation of the A20 significantly enhanced IκB-α and STAT1 phosphorylation The control bands (GAPDH) are strongly clear and have the same size in each well This result indicates that A20 protein negatively regulates the STAT1 pathway in Cd11bDCs However, after LPS activation, STAT3 expression was similar in both A20 and inactivated genotypes These results indicate that the A20 does not affect the STAT3 signaling pathway in the CD11bDC when treated with LPS The results of this study showed that A20 inhibits STAT1 phosphorylation in both pDC cells when exposure to profilin and CD11bDC cells when exposed to LPS/profilin However, no effect on STAT3 phosphorylation This result coincides with several recent 17 studies, the A20 has been shown to regulate the STAT1 pathway in several cell types including myeloid bone marrow cells or HCC liver cancer cells Many studies showed the correlation between A20 and STAT1 signal Since 1998, Manthey and colleagues have shown that in mononuclear leukocytes, the expression of the A20 is regulated by the STAT1 signal stream and then De Wilde's team also reported the inhibitory effect of STAT1 signaling by A20 gene on myeloid cells 3.2.4 Roles of A20 in functions of pDCs through IκB-α and STAT1 signallings 3.2.4.1 A20 inhibits expressions of maturation markers of pDCs through IκB-α and partial STAT1 signallings We next performed experiments to ask whether changes in biological properties of pDCs is mediated through IκB-α and STAT-1 activations The pharmacological inhibitors of STAT-1 signaling fludarabine and IκB-α signaling IKK inhibitor were added in pDC culture, expressions of maturation markers, cytokine productions and cell migration were examined 18 Fig 3.12 Effects of A20 on the expression of pDC marker via signal pathways IκB-α and STAT1 As shown in Fig 3.12, the inhibitory role of A20 on expressions of MHCII and CD40 on pDCs was significantly blunted in the presence of fludarabine or IKK inhibitor, although fludarabine significantly suppressed expressions of MHCII on control and A20-silenced pDCs only 3.2.4.2 A20 inhibits cytokine productions of pDCs through IκB-α and partial STAT1 signallings Fig 3.13 Effects of A20 on the cytokine productions of pDC via signal pathways IκB-α and STAT1 19 Besides, the contents of the pro-inflammatory cytokines IL-6, IL12p40, TNF-α and IFN-γ were checked when mature pDCs were treated with fludarabine or IKK inhibitor As shown in Fig 5B, the role of IKK inhibitor in inhibiting cytokine productions of IL-6, IL12p40, TNF-α and IFN-γ were observed, while fludarabine significantly reduced level of TNF-α only in both control and A20silenced mature pDCs Importantly, the suppressing effect of A20 on IFN-γ secretion only by pDCs was abolished, whereas the differences in releases of IL-6, IL-12p40 and TNF-α between control and A20silenced mature pDCs remained unaltered (Fig 3.13) 3.2.4.3 A20 inhibits migration of pDCs through IκB-α and partial STAT1 signallings Fig 3.14 Effects of A20 on the migration of pDC via signal pathways IκB-α and STAT1 Finally, the increased migration of A20-silenced pDCs was also abolished when the cell culture was added IKK inhibitor or fludarabine (Fig 3.14) Based on the results attained, we revealed that A20 prevented maturation and activation of pDCs through NF-κB and partial STAT-1 signalling pathways To our knowledge, we showed for the first time that downregulation of A20 expression resulted in an increased pDC maturation/activation 20 and inflammatory reaction in CD11bDCs in response to TgPRF More importantly, the phosphorylations of IκB-α and STAT1, but not STAT3 were significantly elevated in A20-silenced pDCs and slightly increased in A20-silenced CD11bDCs in the exposure with TgPRP (Fig 3.9 and 3.10), therefore IKK inhibitor and fludarabine were added to pDC culture to suppress the activation of IκB-α and STAT-1 signalling molecules As expected, the IKK inhibitor and fludarabine completely reversed the differences in expression of maturation markers, migration capacity and IFN-γ production only between control and A20-silenced pDCs (Fig 3.12 to 3.14) In many studies, A20 is a negative regulator of NF-κB-mediated cell functions, although the role of A20 in the regulation of T gondii infection is unknown Recently in several cell types, A20 is also known to have an inhibitory effect on STAT-1 activation, which prevents the death from T gondii infection in mice 3.2.5 Roles of A20 in functions of CD11bDCs through STAT1 signalling 3.2.5.1 A20 inhibits cytokine productions of CD11bDC through partial STAT1 signalling Fig 3.15 Effects of A20 on the cytokine productions of CD11bDC via STAT1 signalling 21 Concentrations of inflammatory cytokines TNF-α and IL-10 secreted in cell culture in the presence or absence of fludarabine were checked As shown in Fig 3.15, the inhibitory effects of A20 on TNF-α and IL-10 were significantly blunted when fludarabine was added to the cell culture The evidence indicated that the effect of A20 in suppressing the release of cytokines was sensitive to STAT1 signalling pathway 3.2.5.2 A20 inhibits migration of CD11bDC through partial STAT1 signalling Fig 3.16 Effects of A20 on CD11bDC migration via STAT1 signalling Cell migration is a hallmark of DC maturation induce T cell activation and proliferation in secondary lymphoid organs Mature DCs express the high level of CCR7, thus they are facilitated to migrate toward CCR7 ligands, such as CCL21, which is expressed in LNs Upon LPS treatment, DC migration was enhanced and the effect was further increased following A20 silencing However, inhibition of STAT1 signaling by using fludarabine did not affect the migration of A20-silenced DCs, indicating that migration of DCs was dependent on the presence of A20 and independent from STAT1 signaling A20 has been reported to inhibit DC activation and maturation, however STAT1-dependent suppressing effect of A20 on DC 22 function is reported for the first time Upon LPS treatment, A20 participates in attenuating DCs functions through NF-κB signalling pathways Differently, we observed that A20 downregulated the phosphorylation of STAT1 (Fig 3.11) that induces transcriptional gene expression such as inflammatory cytokines Activation of STAT-1 is also followed by chemotactic activity in DCs The effective migration of DCs to LNs is a prerequisite for their role as regulators of T lymphocyte function CCL21, a CCR7 ligand, is constitutively expressed by peripheral lymphatic endothelial cells and LN stroma cells, which elicit their effects to catch mature DCs homing The present study indicated the role of A20 as a negative regulator of migratory capacity towards CCL-21 independently from STAT1 pathway in Cd11bDCs (Figure 3.16), suggesting that A20 sensitive DC migration might be dependent on other signaling pathways rather than STAT1 In summary, the regulatory role of A20 for DCs classes is different For the CD11bDC, when exposed to LPS, the A20 protein did not affect apoptosis but inhibited cytokine production related to the transcription factor STAT1, while A20 protein inhibited DC migration is controlled by CCR7 but is independent of this STAT1 signal stream These results are expected to be related to the inflammatory response in STAT1-associated disease For pDC, after exposure to profilin, the A20 protein participates in hindering maturation and secretion of cytokines and cell migration via NF-κB and STAT1 signaling pathways 23 Chapter CONCLUSIONS To conclude Roles of A20 in regulating the physiology of dendritic cells ⁃ When challenging to TgPRF, A20 inhibits some cellular physiological processes including maturation (through the expression of MHC II and CD40), cytokine secretion (such as IL-6, IL-12p40, TNF-α and IFN-γ) and movement of pDCs - Protein A20 partially inhibits cytokine secretion of CD11bDC (IL-6 and TNF-α) upon exposure to TgPRF - However, when exposed to LPS and some cytokines (such as IL-2, IL-10, TNF-α and INF-γ), A20 does not affect apoptosis of DCs Effects of A20 on some physiological functions of DCs through molecular signal NF-κB and STAT1 - Protein A20 interferes with phosphorylation of the signaling molecules IκB-α and STAT1 but does not affect the STAT3 signaling pathway - A20 suppresses the maturation of pDC through NF-κB and inhibits inflammatory response through STAT1 but the role of A20 in inhibiting migration is independent of STAT1 Recommendation The expression level of A20 is related to the biological activity of immune cells including DCs through several molecular signals such as NF-κB and STAT1 Abnormal expression of the A20 was also found in many cancer patients, including Leukemia Therefore, the next research will focus on: Identifying the relationship between A20 and the pathogenesis of some type of Leukemia, such as T-ALL or B-ALL to propose a treatment for Leukemia combination with A20 target gene therapy or cell target therapy involving the A20 24 NEW FINDINGS OF THE THESIS  A20 participates in hindering maturation, cytokine secretion and migration of pDCs when exposed to TgPRF antigen via NF-κB and STAT1 signaling pathways The impact of the A20 on the biological activity of pDC may be related to the immune response in T gondii infection in humans and animals  Role of IL-10 inducing apoptosis in DCs may be related to TNF-α release via STAT1 signal and independent of A20 Other cytokines such as IL-2, TNF-α and INF-γ not affect apoptosis LIST OF WORKS HAS BEEN PUBLISHED Pham Ngoc Duy, Nguyen Thu Thuy, Bui Kieu Trang, Nguyen Hoang Giang, Nguyen Thi Hong Van, Nguyen Thi Xuan Regulation of NF-κB- and STAT1-mediated plasmacytoid dendritic cell functions by A20 PLoS ONE, 2019, 14(9): e0222697 Nguyen Thi Xuan, Nguyen Thu Thuy Regulation of inflammatory response by A20 through STAT-1 signalling in dendritic cells Journal of Biotechnology, 2019 17(2) 241-247 Nguyen Thu Thuy, Nguyen Thi Xuan Regulation of apoptosis by IL-10 and the association with STAT-1 signaling molecule in dendritic cells Journal of Biology, 2019 41(1) 109-116 25 ... the role of A20 regulating physiological processes through molecular signaling in DCs, including the phosphorylation of IκB-α, STAT1 and STAT3 Chapter OVERVIEW 1.1 Protein A20 Protein A20 is a tumor-stimulating... 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. .. between A20 and the pathogenesis of some type of Leukemia, such as T-ALL or B-ALL to propose a treatment for Leukemia combination with A20 target gene therapy or cell target therapy involving the A20