Investigation on the regulatory role of a20 gene and molecular mechanisms involved in controlling dendritic cell physiology

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.

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-inactive DCs group and control group. 3 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. 4 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 5 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 DC- 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 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 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. 6 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. 7 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). 8 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 9 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 10 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 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 11 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. 12 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 A20 inhibits migration in pDCs when exposed with TgPRP 13 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 TgPRP- matured pDCs was inhibited by the presence of A20. 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. 14 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 7- AAD 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 15 cytokines such as TNF-α, INF-γ, and IL-2 do 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. 16 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 TgPRP- challenged pDCs, whereas activations of these molecules in A20- silenced 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 17 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 18 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 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. 19 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. 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 20 Besides, the contents of the pro-inflammatory cytokines IL-6, IL- 12p40, 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, IL- 12p40, TNF-α and IFN-γ were observed, while fludarabine significantly reduced level of TNF-α only in both control and A20- silenced 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 A20- silenced mature pDCs remained unaltered (Fig 3.13). 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 21 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 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 22 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. 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 23 function is reported for the first time. Upon LPS treatment, A20 participates in attenuating DCs

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