Inhibition of Bcl-6 exp­ression ameliorates asthmatic characteristics in mice

doi:10.21203/rs.3.rs-2091729/v1

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Inhibition of Bcl-6 expression ameliorates asthmatic characteristics in mice

https://doi.org/10.21203/rs.3.rs-2091729/v1

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Background:

Bcl-6 is an essential factor control Tfh cells differentiation. T lymphocytes assistance B lymphocytes regulate IgE secretion is a crucial part of asthma. However, Bcl-6 direct effect on asthmatic characteristics, such as IgE secretion is unknown.

Methods:

We adopted OVA-induced BALB/c mice, and Bcl-6 small interfering RNA to inhibit Bcl-6 expression. There were five groups: Control, Blank carrier, Asthma, Bcl-6 siRNA interference, and DXM-treated. Then mice were euthanized, collected lungs tissues, peripheral blood, lymph nodes and spleen. Histopathological diagnosis, AR and LC of each group were detected. Expression levels of surface molecular markers on Tfh cells in pre-mentioned tissues were examined by FCM. Bcl-6 mRNA expression was detected by RT-PCR, and Bcl-6 protein grayscale was detected by Western-blot. Finally, the concentration of IgE and IgG₁ in peripheral blood and BALF were detected, and correlation analysis with the Tfh cells ratio in counterparts was applied.

Results:

Typical pathological changes were observed in lung tissue of asthmatic mice, that was alleviated by Bcl-6 antagonism and DXM. Asthmatic ones had increased AR and decreased LC, while Bcl-6 siRNA interference or DXM treatment could reduce AR and improve LC. FCM indicated that Tfh cells ratio in peripheral blood, lymph nodes, and spleen of asthmatic mice increased significantly comparing to control ones, and that also decreased significantly after Bcl-6 siRNA interferencing and DXM treatment. The trend occured in eosinophils ratio of BALF. Applying RT-PCR accessed the Bcl-6 mRNA expression in PBMCs, which was significantly higher of asthmatic mice than control ones. Whereas that was significantly decreased when Bcl-6 inhibited and DXM treated. Bcl-6 protein expression was similar to that of mRNA expression in Western-blot. As well as the IgE secretion in serum and BALF, B cells expression in PBMCs have the same trend. Besides, in asthmatic mice, the Tfh cells ratio in peripheral blood was strong positively correlated with the level of IgE in serum and BALF, but not that of IgG₁.

Conclusions:

Inhibition of Bcl-6 expression can ameliorate airway inflammation and airway hyper-responsiveness in asthmatic mice by blocking Tfh cell differentiation, which concomitantly reduces B cells regulation IgE production.

Bcl-6

Tfh

Asthma

Airway Hyper-responsiveness

Mouse Model

Bronchial asthma is a chronic inflammatory airway disease caused by immune system hyper-sensitivity to innocuous environmental antigens that result in activation a range of immune cells such as eosinophils, macrophages, neutrophils and Th2 cells, as well as production of inflammatory cytokines and chemokines by these cells[1, 2]. The secretion of IgE by B lymphocytes with the assistance of T lymphocytes is one of the central parts of anaphylaxis in asthma. The exact mechanism of asthma pathogenesis is not entirely clear, but previous research suggests that an imbalance of Th1/Th2 cells is associated with asthma response[3, 4]. More recent studies indicated that, in contrast to the previous line of thinking that Th2 cells assistant the function of B cell, Tfh cells are the major factor in induction of B cell differentiation, proliferation and activation as well as class switching of the immunoglobulin antibodies that could play essential roles in formation of asthma characteristics[5, 6]. Tfh cells in mice and humans are characterized by high expression of CXC chemokine receptor 5 (CXCR5), Inducible T-cell co-stimulator (ICOS), programmed cell death protein 1 (PD-1) [7–9]. IL-21 is the most important cytokine secreted by Tfh cells and can promote the adaptive proliferation of germinal center B cells. Bcl-6 is the most important transcription factor that controls the differentiation of initial T cells into Tfh cells. Deficiency in Bcl-6 will inhibit differentiation of Tfh cells into T cells, which can block formation of germinal centers (GCs), impaired immunoglobulin maturation, and cause overall dysfunction of humoral immunity[7, 10]. Bcl-6 inhibits differentiation of other CD4 + T cell subsets by inhibiting the transcription factor Blimp-1[11]. In our previous study[12], we found that in PBMCs of asthma patients, the expression of Bcl-6 mRNA was increased and the expression of molecular markers on Tfh cell surface was enhanced. After treatment with DXM, the expression of Bcl-6 mRNA and the number of Tfh cells were down-regulated. Therefore, we believe that Tfh cells have enhanced differentiation in asthmatic patients, and this may be involved in the proliferation and activation of B cells, resulting in increased levels of IgE and IgG₁, thus participating in the inflammatory response of asthma. Therefore, We will observe the effects of inhibition of Tfh cell differentiation on B cell differentiation and Ig secretion level, airway inflammation and airway reactivity in asthmatic mice after injection of Bcl-6 siRNA, which may provide a new idea for elucidating the pathogenesis of asthma.

1. The Mouse models

A mouse model of allergic asthma induced by ovalbumin (OVA) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was established. A total of 75 female BALB/c mice (6–8 weeks, 20-22g) were purchased from the Animal Experiment Center, and divided into 5 groups of 15 mice each. All experimental protocols were approved by the Animal Ethics Committee. Mice in the asthmatic group were immunized intraperitoneally with 0.2ml aluminum hydroxide containing 100µg of OVA twice at an interval of 2 weeks, with nasal inhalation of OVA given at the same time as the second intraperitoneal immunization. The same method was used for nasal immunization with 100 µg OVA at day 25, 26, and 27 after the first immunization. Experimental analyses were performed within 24 hours after completion of the last immunization. Mice in the control group were immunized with an equal volume of normal saline. Mice in the blank carrier group received with 100 µg blank siRNA (5′- TTCTCCGAACGTGTCACGT-3′, GenePharma, China) via tail vein injection on day 1, 8, 15, and 22 after the immunized with an equal volume of normal saline but not OVA. Mice in the Bcl-6 siRNA interference asthmatic group received 100 µg Bcl-6 siRNA (5′- GCACGCTAGTGATGTTCTTCT-3′, GenePharma, China) via tail vein injection on day 1, 8, 15, and 22 after the first OVA immunization, and the OVA immunization process was consistent with that of the asthma group. Both siRNA used HIV-1, which deleted all the coding genes, as the carrier, and no HIV-1 protein was expressed when mediating the expression of the target gene. Mice in the DXM treatment group were executed with 0.1mg/kg DXM in the vein tail after the last OVA immunization.

2. Hematoxylin-Eosin staining of lung tissue

Lung tissues was fixed with 10% neutral formaldehyde for 12–24 hours, dehydrated, and then embedded in paraffin, Hematoxylin-Eosin staining was carried out on 5 µm-thick section. 2 pathologists blinded to the experimental groups evaluated histopathological changes in lung tissues using an optical microscope.

3. Detection of airway resistance and lung compliance in mice

Airway hyper-responsiveness was assessed by measuring airway compliance and lung resistance before and after exposure to ascending concentrations of methacholine (Mch, 4.0–32.0 mg/mL, Sigma-Aldrich) dissolved in phosphate buffered saline (PBS). Here, mice were anesthetized within 24 h after the last OVA stimulation and disposed with a whole-body volumetric meter to record the baseline resistance (RI, cmH₂O*sec/L) and compliance (Cdyn, ml/cmH₂O). Ascending concentrations of methacholine (Mch; 4, 8, 16, 32 mg/ml) were given intranasally and the mean resistance and compliance were calculated from measurements collected 20 mins after administration.

4. Collection of the cells in PBMCs, spleens, and bronchial lymph node cells

After anesthetizing mice with 1% pentobarbital sodium solution, a 1ml syringe was used to collect blood from the heart. The blood was centrifuged at 3,000 rpm/min for 10 min and the serum was stored at -20°C for use in an Enzyme Linked Immunosorbent Assay (Elisa). The heparinized blood was 1:1 diluted with PBS and then layered over Lymphoprep (Nycomed, Norway). PBMCs were separated by centrifugation at 2,500 rpm for 20 min at room temperature. Spleens were collected after euthanasia then placed in Petri dishes, where we cut it into small pieces and gently crushed with a grinder. Then, 4 ml erythrocyte lysate was added and incubated for 5 minutes before centrifugation for 5 minutes at 1,000 g. The supernatant was discarded and the pellet was resuspended in 5 ml RPMI1640 medium (Gibco, USA) containing 10% FBS and then filtered through a nylon filter membrane. The extracted spleen cells were again centrifuged and diluted. Bronchial lymph node cells were collected using the same method as that used for the spleen.

5. Detection of surface marker of Tfh cells by FCM in PBMCs, spleens, and bronchial lymph node tissues

After the collection of the cells in PBMCs, spleens, and bronchial lymph node cells. The cells were resuspended and incubated with FITC-anti-mouse-CD4, APC-anti-mouse-CXCR5, PE-anti-mouse-ICOS, PE-Cy5-anti-mouse-CD19 (all from eBioscience, USA) at 4°C for 40 min in the dark. Then labeled cells were then washed with PBS and resuspended in 500 µl PBS immediately before FCM (Epics Altra; Beckman, USA). The levels of surface antigens were expressed as the percentage of positive cells.

6. Collection of the TfH cells and quantification of eosinophils in BALF

After anesthesia and collection of blood, the neck was dissected to expose the trachea. Tracheal intubation was performed with an intravenous indwelling needle. BALF was collected with 0.5 mL of PBS solution containing 1 mM EDTA for 3 times, and the collected fluid was centrifuged at 4 ℃ (1,500 rpm/min, 5 min). The cells were resuspended in 1 ml of PBS containing 1% BSA and a 10 µl aliquot was used to determine the cells count. The residual liquid was centrifuged at 4℃ again, and the previously mentioned steps were repeated. After centrifugation, the supernatant was removed, and the concentration of cells was adjusted to 1×10⁶ cells/ml. A 100 µl smear of cells suspension was taken from each slide (TXD3 cell smear centrifuge, 500 rpm/min, 1 min). The slides were air-dried before staining with Jimsa kit according to the manufacturer's instructions. Eosinophils were counted by cell classification after drying Under the microscope.

7. Quantitative PCR analysis of mRNA expression in PMBCs

Total RNA was extracted from PBMCs with Trizol (Invitrogen, USA) according to the manufacturer's protocols. Total RNA (1 µg) was transcribed into cDNA using a ReverTra Ace RT kit (TOYOBO, Japan). The specific primers for human Bcl-6 were: 5′-CCGGCACGCTAGTGATGTT-3′ (F), 5′-TGTCTTATGGGCTCTAAACTGCT − 3′ (R). β-actin was used as internal control and the primers were: 5′-AGCCATGTACGTAGCCATCC-3′ (F), 5′-ACCCTCATAGATGGGCACAG-3′ (R). To half-quantify Bcl-6 expression in Tfh cells, real-time PCR (RT-PCR) was performed using a ROTOR Gene 3000 system with 5ng cDNA in a total reaction volume of 20 µl. SYBR Green Supermix (TaKaRa, Japan) was used to indicate the PCR products. Amplification was conducted using the following conditions: initial denaturation at 95°C for 2min, then 40 cycles at 95°C for 15s, 57°C for 20s and 72°C for 20s. All measurements were performed in duplicate. The relative expression of the target gene was calculated using the comparative 2-ΔΔCt method[13], wherein Ct indicates the cycle number threshold at which amplification is exponential.

8. Protein expression assay by Western-blot analysis in PMBCs

Protein expression levels of Bcl-6 were determined by Western-blot analysis. Proteins isolated from PBMCs were separated by electrophoresis on 10% SDS-polyacrylamide gels in RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 0.2 mM EDTA; pH 8.0). Proteins were then electrically transferred onto a nitrocellulose membrane by electroblotting at 4°C. The membrane was blocked with 5% nonfat dried milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TTBS) at room temperature for 2 h and then probed overnight at 4°C with specific primary antibodies in 2% nonfat dried milk containing TTBS. Rabbit polyclonal anti-Bcl-6 antibody (1:1000, Abcam, Cambridge, MA) was used to detect the target proteins. Monoclonal anti-actin antibody was used as an internal control. The membrane was incubated for 45 min at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000) in 2% nonfat dried milk containing TTBS, and the target proteins were visualized with enhanced chemiluminescence reagent (ECL; Pierce, USA).Blots were imaged using a Fusion Fx7 image acquisition system (Vilber, France) and bands were quantified by densitometry using Image J software.

9. Detection of the levels of IgE and IgG1 in serum and BALF by ELISA

Blood and BALF were collected as described above and stored at -20°C for ELISA. The levels of IgE and IgG₁ in serum of blood and BALF were measured using ELISA kits following the manufacturer's instructions (eBioscience, USA). The concentration range of assay was 0.137-100 ng/mL for IgE and 3.13–200 ng/mL for IgG1. All samples were measured in duplicate. Undetectable values were assigned an arbitrary value of half the sensitivity limit.

Statistical analysis

Data are presented as the Mean ± Standard Deviation (SD), median and 25th-75th quartile or percentages. Comparison between groups was made by analysis of variance followed by Dunnett's test, Mann-Whitney or χ² test. Pearson and Spearman correlation analysis was also applied to explore the relationships between the percentage rate of Tfh cells and IgE, IgG₁ in both serum and BALF. All the statistical analysis was conducted using SPSS 24.0 (Chicago, Illinois, USA) with P < 0.05 indicating statistically significant differences. All figures were performed using Graph-Pad Prism (Version 7.0, GraphPad Software, San Diego, CA, USA).

1. Effects of Bcl-6 siRNA interference on lung histopathology in asthmatic mice

The asthmatic mouse model was achieved successfully. Histopathology of HE stained sections revealed that, compared with the normal mice (Fig. 1.A), the asthmatic mice (Fig. 1.C) had thicker bronchial mucosa, lumen stenosis, and proliferation of inflammation cells around blood vessels and airway. Eosinophils and lymphocytes were the main type of infiltrating cells and could damage alveolar epithelial in Fig. 1.C. The histopathology of the blank carrier group (Fig. 1.B) hasn’t much change comparing with the control group, this means that the lentiviral vector itself has no significant effect on inflammation and airway structure in mice. Tail vein injection of lentivirus carrying Bcl-6 siRNA (Fig. 1.D) or DXM (Fig. 1.E) on mice could alleviate airway stenosis and decrease inflammatory cells infiltration.

2. Effects of Bcl-6 siRNA interference on airway resistance and lung compliance in asthmatic mice

Compared with the control group (AR, Mean 2.60 ± 0.10 cmH₂O*sec/L, 32mg/ml Mch; LC, Mean 0.08 ± 0.01, ml/cmH₂O), there was no significant change in airway resistance (AR, Mean 3.80 ± 0.90 cmH₂O*sec/L, 32mg/ml Mch; P > 0.05) and lung compliance (LC, Mean 0.065 ± 0.010, ml/cmH₂O; P > 0.05) in the siRNA blank carrier group. Suggesting that the lentivirus vector itself had no significant effect on AR and LC compare to control mice. However, AR (Mean 9.50 ± 1.50 cmH2O*sec/L, 32mg/ml Mch; P < 0.05) was significantly increased and LC (Mean 0.02 ± 0.005, ml/cmH₂O; P < 0.01) was significantly decreased in asthmatic mice, when compare to the contral ones. It was obvious that Bcl-6 siRNA interference and DXM treatment reduced AR and improved LC in asthmatic mice. (Fig. 2)

3. Effects of Bcl-6 siRNA interference on Tfh cells expression in asthmatic mice

After collecting cells for flow fluorescence labeling, CD4 + lymphocytes were loop-gated and then the expression ratio of CXCR5 + ICOS + lymphocytes (Tfh cells) in the spleen (Fig. 3.A), lymph nodes (Fig. 3.B) and peripheral blood (Fig. 3.C) was measured. The number of cells having expression patterns consistent with Tfh cells was significantly higher in the asthma group compared to the control group, Bcl-6 interference group, and DXM-treated group. (P < 0.001, respectively). There was no significant difference between the control group and the blank carrier (P > 0.05). Figure 4 showed representative FCM results of the surface markers expression. The specific data indicated that in the peripheral blood, lymph nodes and spleen of different groups of mice, asthmatic mice have more TFH cells, the mice treated with Bcl-6 siRNA also have the same obvious Tfh cells expression decreased as the DXM-treated ones.

4. Effects of Bcl-6 siRNA interference on eosinophil expression in BALF of asthmatic mice

Eosinophils are monitoring indicator of allergic diseases. Here, we used the eosinophils ratio in BALF from mice as a monitoring parameter to compare asthmatic characteristics among the experimental groups. Both the Bcl-6 interference group ( Mean 32.82 ± 7.8%; %) and DXM treated group (Measn 22.57 ± 5.6; %) had a significant downward trend in the eosinophil ratio compared to the asthma group (Measn 46.16 ± 11.51; %) ( P < 0.001; P < 0.001) (Fig. 5). Besides, no significant difference between control mice and blank carrier ones.

5. Effects of Bcl-6 siRNA interference on Bcl-6 gene expression and protein levels in asthmatic mice

We used the comparative 2-ΔΔCt method with RT-PCR result to calculate the relative expression of Bcl-6 in the experimental groups. Compared to the control group (Mean 1 ± 0; folds), Bcl-6 expression was upregulated in asthmatic mice (Mean 4.5 ± 1.8; folds). (P < 0.001). In mice treated with Bcl-6 siRNA (Mean 2.2 ± 0.5; folds), Bcl-6 expression was down-regulated contrast to the asthmatic group. (P < 0.01) In mice treated with DXM (Mean 1.8 ± 0.5; folds), Bcl-6 expression was further down-regulated compared to the asthmatic group. (P < 0.01) (Fig. 6. A). Western-blot indicated similar change in protein levels (Fig. 6. B). And the relative expression level of Bcl-6/β-actin protein band gray scale was measured by Image J, as showed in Fig. 6. C.

6. Effects of Bcl-6 siRNA interference on B cells expression, IgE, and IgG ₁ secretion in asthmatic mice

The IgE and IgG₁ concentration of in serum and BALF from mice in the different experimental groups were detected. There was a significant statistical difference between each of the experimental group for serum IgE, especially between the Bcl-6 interference group (Mean 1980 ± 410; ng/ml) and DXM treated group (Mean 2500 ± 600; ng/ml) compared with the asthma group (Mean 3200 ± 600; ng/ml) (P < 0.001; P < 0.001) (Fig. 7A). Meanwhile, BALF from the asthma group (Mean 61 ± 10.5; ng/ml) showed a significant difference in IgE compare to the DXM treatment group (Mean 47 ± 11; ng/ml), and showed a significant difference to asthma group (p < 0.01), whereas no significant difference was seen when compared to the Bcl-6 siRNA interference group (Mean 50 ± 9.3; ng/ml) (P > 0.05). At the same time, when comparison had been implemented between the Tfh in blood and IgG₁ both in serum and BALF, no significant difference was achieved. (Fig. 7B and D). This result suggests that Bcl-6 intervention similar to DXM treatment, could have a significant inhibitory effect on IgE formation, but not that of IgG₁. In addition, the number of CD19 + B cells in PBMCs from asthmatic mice (Mean 6.8 ± 2.4; %) was higher than that of control mice (Mean 2.5 ± 0.8; %) (P < 0.01), but there was no significant difference between the blank carrier mice (Mean 3.4 ± 1.1; %) and the control mice. In mice interfered with Bcl-6 siRNA (Mean 4.2 ± 1.1; %) or DXM (Mean 3.5 ± 0.9; %), the ratio of CD19 + B cells/PBMCs was lower than that in the asthma mice (P < 0.01) (Fig. 7E). There was also a strong positive correlation (r = 0.650; P < 0.01) between CD19+/PBMCs (%) and Tfh/PBMCs (%). (Fig. 7F)

7. Correlation analysis of Tfh cells with IgE and IgG ₁ secretion in blood and BALF.

A correlation analysis indicated a strong positive correlation between Tfh and IgE in both blood (r = 0.840; P < 0.001) (Fig. 8.A) and BALF (r = 0.795; P < 0.001) (Fig. 8.C). However, correlation analysis between Tfh and IgG₁ in blood and BALF, both fail to show a significant difference.(Fig. 8.B and D)

Tfh cells function in many areas of the body and in multiple pathogenic process in allergies, tumors, and autoimmune diseases.[14, 15] We were acknowledged that Tfh cells form a subgroup of CD4 + T cells that assist in antibody production by B cells in response to antigenic challenges and are also essential for the germinal centers (GC) formation.[16–18] When T cells are activated by dendritic cells (DC) in T cell regions, they differentiate into precursor Tfh (Pre-Tfh) cells regular B cell follicles.[19, 20] With continuous ICOS stimulation of B cells and the down-regulation expression of other molecules, Tfh cells further develop into B follicular cells and remain in the germinal center as Tfh (GC-Tfh) cells.[7, 21, 22] Bcl-6 is an essential component of this complex cellular process because it promotes expression of CXCR5 and inhibits expression of the transcription factors T-bet, GATA-3 and RoR-γt, which are essential for inducing other effector T cell subsets, such as Th1, Th2 and Th17 cells.[23, 24]. However, the precise mechanism for the genetic regulation of Tfh differentiation is not fully characterized.

Based on the above previous research facts, we speculate that inhibition of Bcl-6 may have a related effect on asthma, so we completed the above study and obtained data to support our hypothesis. In this study, we successfully established the asthma model by using the generally accepted modeling method, and successfully explored the effect on the mouse model of asthma by inhibiting the expression of Bcl-6. The effects of inhibition of Bcl-6 expression in asthma were comprehensively demonstrated through the levels of cell, protein, and airway mechanics, that is, from micro to macro-level. The Bcl-6 siRNA interference technology was a mature and successfully applied method to study Tfh in mice. [25] Regarding the microstructure display, our study clearly showed the characteristics of the alveoli, peripheral blood vessels and inflammatory cell infiltration of mice in each study group, which is consistent with our expectation. The asthmatic mice had obvious airway edema and inflammatory cell infiltration surrounding, while in the blank carrier mice. There were still a lot of inflammatory cells infiltration in the airway and without edema. In Bcl-6 siRNA interference mice, there was a lot of inflammatory cell infiltration surrounding the airway. Finally, the DXM treatment mice, there was no airway edema and inflammatory cell infiltration, but the surrounding alveoli were significantly destroyed and fused because of OVA's effect.

Airway resistance and compliance in mice are reliable indicators for assessing airway hyper-responsiveness in asthma. Figure 2 is the highlight of our article. After slowly increasing the stimulation dose of methacholine to mice, the airway resistance of mice gradually increased. Compared with other groups, the airway resistance of asthmatic mice showed a significant increase in stimulation of 8 mg/ml methacholine. The increasing significant difference was even more dramatic after reaching a methacholine concentration of 32 mg/ml. The data from the figure indicate that Bcl-6 siRNA interference has almost the same effect of ameliorating airway resistance as DXM treated.

By measuring the expression of Tfh cells, we found that the lymph nodes, peripheral blood and spleen of asthmatic mice all showed significant higher Tfh expression, and through Bcl-6 siRNA intervention, the Tfh expression in the interference mice was significantly lower than that in the asthmatic ones. The exact data indicated the Tfh expression ratio in immune organs of asthmatic mice was also significantly increased (Fig. 4), and Bcl-6 siRNA intervention mice had a similar effect to the DXM treatment ones. A significant decrease in Tfh expression was achieved. However, the possible reasons for the decline not reaching the effect of DXM treatment are, firstly, the limited number of statistical samples; and secondly, the inhibition of Tfh expression during DXM treatment may have multiple effects, which means more than Bcl-6 siRNA effect. At the same time, we are familiar with many side effects which DXM applied in clinical practice may trigger. That is why we seek further breakthroughs in asthma treatment, striving to relieve symptoms while reducing the impact of side effects.

In our study, we found that the expression of IgE and Tfh showed a significant correlation, but the correlation between Tfh and IgG₁ was not significant. Previous studies have also made it clear that Tfh accelerates the transformation of IgG₁ into IgE, and another way is that IgM can directly transform into IgE. [26–29] So in our study, we also verified that there is no meaningful correlation between IgG₁ expression and Tfh. At the same time, we can also observe that there is a large difference in the orders of magnitude of IgE between BALF and serum, which is one of the reasons why there is no obvious difference by now. However, whether there are other mechanisms that will affect the difference in IgE secretion in serum and BALF that require further study.

At the same time, we also understand that the mechanism of Bcl-6 regulating Tfh cells is very complex, the regulation of Bcl-6 in CD4 + T cells is controlled by multiple signal pathways, some of which are shared with non-Tfh cells or participate in a strong negative feedback loop.[16] And another study showed Bcl-6 represses its own expression in CD4 + T cells, mediated in conjunction with corepressor Ncor1, in a negative autoregulatory feedback loop at the Bcl-6 promoter, dampening Tfh and GC-Tfh cell accumulation.[30] In fact, in previous study, it has been proved that Tfh cells were positively correlated with B cells, plasma IgE and IL-21 levels in patients with acute asthma, suggesting that Tfh cells and IL-21 may promote airway IgE transition to participate in the pathogenesis of asthma by stimulating B cell proliferation.[12] The current study is the advance of previous research to verify the effect of Tfh cell differentiation on B cell differentiation and function in asthmatic mouse model. During this period, related articles have also explained that Tfh cells and their signature cytokines IL-21 are closely related to the occurrence of asthma, especially the specialized subsets of Tfh cells, such as Tfh-2 cells, Tfh-13 cells, and TFR cells, which closely regulate the production of asthma IgE.[31] And other cytokines, such as the production of IL-4 by Tfh cells may be necessary for any IgE induction, but it is not sufficient to produce high affinity IgE.[32] Also others believe that an overall structure of Tfh differentiation and gene regulation in a parsimonious model in which Bcl-6 serves as the apex of a repressor-of-repressors network.[30]

However, there limitations also occurred in our study. First, although OVA is commonly used to induce asthma in mice, it is rarely associated with human asthma. As such, more relevant antigens such as house dust mite (HDM) or cockroach extracts could be used to induce asthma in other study designs.[33] Secondly, the regulatory mechanism of Bcl-6 is comprehensive and involves immunity, autoimmunity, and other factors that have yet not be identified.[34] Besides, a variety of transcription factors in addition to Bcl-6 have been shown to regulate Tfh differentiation.[35–37] Third, although we observed correlation between effector protein components and secretion of IgE and IgG₁, larger sample sizes and further research at the genetic levels are needed. Last, the subtle profound influence mechanism involves research on gene coding related to Bcl-6 is not very clear. Those are worthy of further exploration. Furthermore, What we have confirmed also needs to be profoundly explored at a more subtle level.

Taken together, our results in asthmatic mouse model indicated that inhibition of Bcl-6 expression can negatively affect formation of Tfh and turn decrease the production of B cells, sequentially decreasing IgE secretion in serum and BALF, and ameliorate airway inflammation as well as negatively impact airway hyper-responsiveness. Additional research is needed to define the exact mechanism of formation of Tfh cells and cell subsets in asthma. And the research result does benefit to human beings still has a long road to go.

AHR, airway hyper-resistance; AR, airway resistance; BALF, bronchoalveolar lavage fluid; Bcl-6, B cell lymphoma-6; BSA, Bovine Serum Albumin; cDNA, complementary DNA; DXM, Dexamethasone; EDTA, Ethylene Diamine Tetraacetie Acid; FBS, Fetal bovine serum; FCM, flow cytometry; GC, germinal center; HDM, house dust mite; HE, hematoxylin-eosin; ICOS, Inducible T-cell costimulator; LC, lung compliance; MFI, mean fluorescent intensity; OVA, ovalbumin; PAS, Periodic Acid-Schiff; PBMCs, Peripheral blood mononuclear cells; PBS, phosphate buffered saline; RT-PCR, real time polymerase chain reaction; SD, standard deviation; TBS, Tris-buffered saline; Tfh, T follicular helper cells; mRNA, messaenger Ribonucleic Acid.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Central Hospital of Wuhan.

Consent for publication

All authors agree to publish.

Availability of data and materials

The datasets analyzed during the current study were made available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research supported by National Natural Science Foundation of China (No. 81400020) and Scientific Research Project of Wuhan Municipal Health and Family Planning Commission (No. WX19C30).

Authors’ contributions 

ZCZ and XX had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. ZCZ and XX performed the statistical analysis and drafted the manuscript. GS conceptualized the study, and participated in its design and coordination and helped to draft the manuscript. ZCZ, XX, TWJ, WYF, YZ, LXY, YXW, LXF and YSF completed all laboratory operation content and data sorting. All authors read and approved the final manuscript.

Acknowledgments

We thank Dr Li Liu and Dr Ling Liu's assistance during the preparation of this manuscript.

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Inhibition of Bcl-6 expression ameliorates asthmatic characteristics in mice

Status:

Version 1

Abstract

Background:

Methods:

Results:

Conclusions:

Figures

Introduction

Materials And Methods

1. The Mouse models

Statistical analysis

Results

1. Effects of Bcl-6 siRNA interference on lung histopathology in asthmatic mice

2. Effects of Bcl-6 siRNA interference on airway resistance and lung compliance in asthmatic mice

3. Effects of Bcl-6 siRNA interference on Tfh cells expression in asthmatic mice

4. Effects of Bcl-6 siRNA interference on eosinophil expression in BALF of asthmatic mice

Dissussion

Conclusions

Abbreviations

Declarations

References

Status:

Version 1

Inhibition of Bcl-6 exp­ression ameliorates asthmatic characteristics in mice

Status:

Version 1

Abstract

Background:

Methods:

Results:

Conclusions:

Figures

Introduction

Materials And Methods

1. The Mouse models

Statistical analysis

Results

1. Effects of Bcl-6 siRNA interference on lung histopathology in asthmatic mice

2. Effects of Bcl-6 siRNA interference on airway resistance and lung compliance in asthmatic mice

3. Effects of Bcl-6 siRNA interference on Tfh cells expression in asthmatic mice

4. Effects of Bcl-6 siRNA interference on eosinophil expression in BALF of asthmatic mice

Dissussion

Conclusions

Abbreviations

Declarations

References

Status:

Version 1

Inhibition of Bcl-6 expression ameliorates asthmatic characteristics in mice