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Effect of sonication on B cell development and immunomodulatory functions of Bursa of Fabricius
Ultrasonics Sonochemistry 21 (2014) 1343–1348
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Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Effect of sonication on B cell development and immunomodulatory functions of Bursa of Fabricius Xiao-Dong Liu, Xin-Feng Li, Xiu-Li Feng, Bin Zhou, Rui-Bing Cao, Pu-Yan Chen ⇑ Division of Key Lab of Animal Disease Diagnosis and Immunology of China’s Department of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
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Article history: Received 16 January 2014 Received in revised form 14 February 2014 Accepted 14 February 2014 Available online 24 February 2014 Keywords: Bursa extract Sonication Immunomodulatory function Immunization experiment
a b s t r a c t A study was initiated with the objective of evaluating the effects of sonication treatment on important quality parameters of extract of Bursa of Fabricius. Sonication of extract was done (frequency 20 kHz and various amplitude levels) at 0 °C for 10 min, 30 min, 50 min, respectively. As results, the yield of bursa peptides significantly increased (p < 0.05). Then we found sonicated bursa extract promoted the content of bursin and the CFU pre-B formation, exerted immunomodulatory function on antigen-specific immune responses in C57/BL6 mice immunized with inactivated Japanese encephalitis b virus (JEV) vaccine, including enhancing JEV-specific antibody and cytokine production, T-cell immunophenotyping and lymphocyte proliferation. Findings of the present study suggested the sonication treatment of Bursa of Fabricius could improve the yield as well as the quality of bursa peptides, indicating that sonication is effective in processing of bursa extract and could be a potential process for future immuno-pharmacological use. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The Bursa of Fabricius (BF) is the primary humoral immune organ unique to birds [1], which is critical for early B-lymphocyte proliferation and differentiation [2–4]. B cell is named after BF for differentiation and development into immunocompetent lymphocyte in BF. However, in mammals, the B-cell-differentiating organ, equivalent to the T-cell-differentiating thymus, has not yet been identified [5], the BF therefore provides an invaluable model for studying basic immunology. The BF contains several biological active factors. For example, a high dose of chicken BF extract was shown to enhance blastogenic responses to T-cell mitogens [6]. Bursin is a candidate for the specific factor responsible for B-cell differentiation [7]. It selectively stimulates the differentiation of avian B cells, rather than that of T cells, from their precursors in vitro [5,8], and promotes immunoglobulin (Ig) switching from IgM to IgG [9]. Bursal anti-steroidogenic peptide is responsible for synchronizing B-cell division during embryogenesis and the neonatal period [10]. Bursal septpeptide (BSP)I and BSP-II can induce both humoral and cellular immune responses in vivo [11,12], and bursopentin (BP5) was shown to induce B-lymphocyte proliferation by various signaling pathways [13]. Bursal pentapeptide (BPP)-I exerts immune-inducing ⇑ Corresponding author. Tel.: +86 25 84396335. E-mail address: [email protected] (P.-Y. Chen). http://dx.doi.org/10.1016/j.ultsonch.2014.02.012 1350-4177/Ó 2014 Elsevier B.V. All rights reserved.
functions, including antitumor responses [14], while bursal peptide (BP)11 was found to regulate B-cell development and antigen-specific immune responses [15]. However, traditional techniques have many disadvantages, such as long time, low yield, and low activity of bio-factors. This is mainly caused by (1) decreased activity (2) aggregation and deposition of protein (3) low extraction efficacy. In order to overcome the problems, there is a dire need to introduce innovative, novel and effective technologies. Ultrasound treatment is an innovative emerging processing technique that could effectively improve the biological compounds [16]. The mechanism of ultrasound assisted extraction of bioactive compounds involves diffusion of the solvent through the cell matrix and washing out the cell contents. Ultrasound can also generate cavitation and microstreaming (microscopic fluid movement) effects that cause changes in a liquid medium [17]. Ultrasound is benefical in extraction because it increases the solvent uptake by the cell during sonication thereby increasing the extraction yield [18]. At present, many reports evaluating the effect of ultrasound on peptides. A recent study showed ultrasound can remarkably raise the enzymolysis efficiency and activity of ACE inhibitory peptides from wheat germ protein [19]. In this study, the objective was to investigate the effects of sonication treatment in detail on the yield and quality parameters including content of bursin, CFU pre-B, and antigen-specific immune responses of Bursa of Fabricius.
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2. Materials and methods 2.1. Animal C57/BL6 mice (4–6 weeks old, 17–21 g) were obtained from Yang Zhou University (Yangzhou, China). The 21 old-day chickens were purchased from Qinglongshan farm (Nanjing, China). All the animal experimental procedures were performed in accordance with the institutional ethical guidelines for animal experiments. 2.2. Bursa extract and sonication treatment The BFs were collected and placed on ice. Then they were homogenized in 0.1 M phosphate buffer solution, pH 6.0 and centrifugated at 5000g for 10 min at 4 °C. The sonication of samples for 10, 30, 50, 70 was done at a temperature of 4 °C by using ultrasonic processor of 750 W (X0-650D, Xianou Technology Co., Nanjing, China). The frequency of 20 kHz and amplitude level of 10%, 20%, 40% were used. All the sample preparations and treatments were carried out in triplicate. Fresh untreated bursa extract was selected as control. 2.3. Determine of yield of bursa peptides, concentrations of Bursin and RP-HPLC analysis Total peptide concentration of sonicated bursa extracts was determined with Bicinchoninic Acid assay (BCA) (Beyotime biotechnology, China). The yield was calculated as micrograms of peptide produced per 10 g bursa consumed. 1 mg/ml of sonicated bursa extracts was collected to measure the bursin levels by the method of ELISA. Briefly, a sample or standard was added in ELISA plates (Nunc, UK) coated with capture antibody of bursin for 2 h at 37 °C, and then detected using a biotinylated anti-cytokine antibody, Vidin HRP, and tetramethylbenzidine. Color development was stopped with 2 M H2SO4, and optical densities were read at 450 nm. The concentrations of bursin were determined from the standard curve. 1 mg/ml of sonicated bursa extracts was analyzed by reversedphase (RP) high-performance liquid chromatography (HPLC) with a Zorbax C-18 (ODS) column (150 mm 4.6 mm, Agilent) using a linear gradient of acetonitrile (5–18%, EMD Chemicals) containing 0.1% TFA at a flow rate of 1 ml/min for 20 min at 30 °C. The elution was monitored at 280 nm with a UV–Visible diode-array detector (UV-DA).
with a 0.2 ml JEV inactivated antigen containing 1 mg/ml or without sonicated bursa extracts on days 0, and 14, respectively. As positive control, mice were intraperitoneally immunized twice at two weekly intervals with the same volume containing JEV inactivated antigen, and PBS was used as negative control. The sera were collected on the 14th day and 28th day to detect the antigen specific antibody responses (IgG) by ELISA method, respectively, as previously described [22]. 2.6. Lymphocyte proliferation, cytokine enzyme-linked immunosorbent assay (ELISA) and Flow cytometry On the fourteenth day after final immunization, mice were killed by cervical dislocation; their spleens were aseptically removed and cell suspensions made by passing them through a sterile cell strainer (Becton-Dickionson, USA). The cell suspensions were washed twice in RPMI-1640 medium supplemented with 10% FBS, and adjusted to 1 107 cells/ml, and inactivated JEV (105 PFU). After two days of incubation, the cell viability was measured by using the WST-1 Cell proliferation and cytotoxicity assay kit (Beyotime, Hangzhou, China) in accordance with the manufacturer’s instructions. Briefly, 10 ll of this reagent was added to each well containing 100 ll of cell suspension incubated for an additional 1 h. The absorbance at 450 nm was monitored and the reference wavelength was set at 630 nm. The percent viability of cells was calculated by comparison to that of untreated control cells. The amount of several cytokines (IL-4 and IFN-c) in the culture supernatants were measured by ELISA kits (RD, USA) according to the manufacturer’s instructions. Also, the culture cells were performed by three-color flow cytometry analysis (BD, LSR), by using different mixtures of specific mAbs CD3, CD4, and CD8 labeled with PE-Cy5, FITC or PE, respectively. Data analysis was done with the FACScan flow cytometer (BD, LSR) using the Cell Quest software. 2.7. Statistical analysis Results were expressed as means ± standard deviations (SD). The statistical significance of the observed differences was analyzed by 2-sided t test unless otherwise specified. p value <0.05 was considered to be significantly different. 3. Results and discussion 3.1. Effects of sonication on yield of bursa peptides of Bursa of Fabricius
2.4. CFU pre-B assay Single-cell suspensions were obtained from the bone marrow of c57 mice at 4–6 weeks of age. Cells were suspended in Iscove’s Modified Dulbecco’s medium containing 1% Methylcellulose, 2 mM L-glutamine, 10% fetal calf serum (FCS), 50 lM 2-mercaptoethanol, 0.1 g/L streptomycin, and 105 U/mL penicillin. Whole bone marrow cells were treated with the IL-7(10 ng/ml) and sonicated bursa extracts (both added immediately before plating), mixed with Methylcellulose, and plated in 35-mm culture dishes. The plates were incubated at 37 °C, 5% CO2. CFU-pre-B were counted on day 7 of culture as described [20]. Data are presented as the mean number of CFU/105 cells for triplicate samples ± SD. 2.5. Sensitization protocols and detection of antigen-specific Ab responses The immunomodulatory roles of the sonicated bursa extracts were examined in 6–8 weeks old BALB/c female mice, as previously reported [21], in which mice were intraperitoneally immunized
Results regarding the effects of sonication treatments on yield of bursa peptides of bursa extract are presented in Table 1. In the present study, we observed that the yield rise as the amplitude become higher. The yield rise leveled off between 40% and 60% (Table 1). High power ultrasound generated at high amplitude and low frequency (20–100 kHz), which is used in food processing, welding, and cleaning processes [23]. Ultrasonic extraction of various food components has been reported mainly for 20–40 kHz. In our study, we used 20 kHz and studied the changes of yield of Table 1 Effect of sonication treatments on Bursa peptide yield. Frequency (KHZ)
Amplitude (%)
US 10 (mg/g)
US 30 (mg/g)
20 20 20 20
10 20 40 60
3.74 ± 0.11c 4.08 ± 0.04b 4.25 ± 0.07a 4.31 ± 0.07a
4.11 ± 0.73d 4.46 ± 0.13c 4.97 ± 0.08b 5.15 ± 0.06a
Values with different letters in the same column (a–d) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min.
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bursa peptides in different amplitude (from 10% to 60%), and found 40% and 60% are proper for bursa peptides yield. Then, we compared the effects of sonication treatments and the freeze treatment on yield of bursa peptides. We found that the yield of bursa peptides significant improved in sonicated bursa extract samples compared to control untreated bursa sample and freeze treatment. The observed increase in yield was from 2.822 to 4.171, 4.883, 5.589 and 5.564 mg/g in bursa extract sonicated for 10, 30, 50 and 70 min, respectively. There were no significant difference between 50 and 70 min of sonication, suggested that 50 min of sonication was enough for the treatment of sonication. Compared to sonication, the yields of bursa peptides in various time of freeze were significantly lower, from 3.356 to 3.873 mg/g for 1–7 times of freeze. Bursin is an essential peptide in the bursa peptides, which regulated B-cell differentiation and promotes immunoglobulin (Ig) switching from IgM to IgG [9]. We evaluated the effect of sonication and freeze in concentration of Bursin using ELISA method. The results showed that the concentration of Bursin significantly increased after sonication treatment, compared to control untreated bursa sample and various times of freeze (Fig. 1B). As bursa peptides from bursa extract have many useful biological compounds, we evaluated the effect of sonication on the yield of other bursa peptides beside Bursin using RP-HPLC (Fig. 2), the highest peaks were a–d. We found that each peaks of the sample treated for 50 min were the highest, the peaks of the sample treated for 30 min were also higher that the sample treated for 10 min. The changes in biological compounds might be attributed to the cavitations during sonication [23]. It has been reported that sonication treatment extracted more protein for extraction [24], we also found the similar results. The increase in yield might be due to high-pressure gradient by cavitations during sonication treatment that cause the breakdown of agglomerates to smaller ones and breakout of cells that increases of the solvent uptake and make the extract properly homogenized and more consistent. Various times of freeze can also breakout the cells and increase the solvent uptake, but may cause the aggregation and deposition of proteins and peptides that decreased the yield and activity. These results indicated that sonication is effective in processing of bursa extract which increased yield as well as the content of biological compounds. 3.2. Effects of sonication on B cell development of Bursa of Fabricius As Bursa of Fabricius is the central organ of B cell development [1], these sonicated bursa extract samples may affect the B cell development. To investigate this hypothesis, we compared the biological activity of sonicated bursa extract samples on B cell
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development by using CFU pre-B formation assays. As expected, the numbers of CFU pre-B which were treated with US 10 (ultrasounds treatment for 10 min), US 30 (ultrasounds treatment for 30 min) and US 50 (ultrasounds treatment for 50 min) were significantly increased at three different doses, compared to untreated bursa extract and the freeze treatment (P < 0.05). Furthermore, the numbers of CFU pre-B of US 50 were the highest among the three samples at 5 lg/ml, the numbers of CFU pre-B of US 30 and US 50 were higher than US 10 at 1 lg/ml and 25 lg/ml (Table 2). The B cell progenitors responding to IL-7 in the CFU-pre B cell assay are most likely cells at hte pre-pro,or C stage as described by Hardy et al. [25]. The changes in biological compounds might be attributed to the cavitations during sonication [23]. Various times of freeze may cause the aggregation and deposition of proteins and peptides that decreased the activity. These results indicated that sonication is effective in processing of bursa extract which increased the activity of bursa extract in vitro. 3.3. Effects of sonication on antibody response of Bursa of Fabricius The production of antibodies (humoral immunity) and action of sensitized T lymphocytes (cellular immunity) are vital components in the immune response. Cytokines are a super family of proteins and they induce multifarious biological effects by regulating the transcription of distinct sets of genes in different cell types in immune processes [26,27]. To determine whether sonication is effective in processing of bursa extract which modulate antibody responses in vivo, we injected mice with sonicated bursa extract (1 mg/kg) and JEV oil-adjuvant vaccine. We first assessed the antibody IgG profiles in sera day 14 from all groups (Fig. 3). Antibodies from mice immunized with JEV vaccine and US 10, US 30, US 50 were observed significantly increased (P < 0.05) than mouse immunized with JEV vaccine control. These results suggested that sonicated bursa extracts had fine inducing roles on antibody response. This increase may be attributed to the sonication treatments that increased the bursa peptides and macromolecules. 3.4. Effects of sonication on stimulation of primary cytokines responses of Bursa of Fabricius To evaluate whether the immunization with sonicated bursa extract and JEV vaccine in vivo enhance a specific cytokines response, the sera were collected from the immunized mice at 7th day after the second immunization to determine IL-4 and IFN-c cytokine productions by ELISA method. As shown in Table 3, IL-4 and IFN- c productions following the twice immunization procedure with US 10, US 30 and US 50 were significantly higher than in mouse received JEV vaccine control
Fig. 1. Effect of sonication on yield of bursa peptides and concentrations of Bursin. (A) Change in the yield of bursa peptides during sonication and various time of freeze of bursa extract were measured with BCA assay. Values with different letters in the same column (a–g) are significantly different (P < 0.05) from each other. (B) Change in the concentrations of Bursin during sonication and various time of freeze of bursa extract were measured with ELISA. Values with different letters in the same column (a–f) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min. US 70; ultrasounds treatment for 70 min. 1; 1 time of freeze. 3; 3 times of freeze. 5; 5 times of freeze. 7; 7 times of freeze.
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Fig. 2. The RP-HPLC assay of sonication of bursa extract. Letters (a–d) were the highest peaks. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min. US 70; ultrasounds treatment for 70 min.
Table 2 Effect of sonication treatments on CFU-Pre-B values of Bursa of Fabricius. Treatment
1 lg/ml
5 lg/ml
25 lg/ml
Control Bursa extract Freeze US 10 US 30 US 50
18.33 ± 2.08e 22.67 ± 1.15d 25 ± 1c 31.33 ± 3.51b 51 ± 3.61a 52 ± 3a
18.33 ± 2.08f 34.67 ± 1.53e 40 ± 1d 46.67 ± 5.86c 60.67 ± 4.04b 71 ± 2.65a
18.33 ± 2.08d 40 ± 1.73c 48 ± 1b 56.33 ± 4.04b 73.33 ± 6.11a 80.33 ± 5.03a
Values with different letters in the same column (a–f) are significantly different (P < 0.05) from each other. Freeze; 5 times of freeze. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
Table 3 Effect of sonication treatments and inactivated JEV on Th1/Th2 cyotokin productions in splenocytes. Treatment
IL-4
IFN-c
PBS JEV vaccine JEV vaccine + US 10 JEV vaccine + US 30 JEV vaccine + US 50
32.78 ± 4.76d 68.62 ± 3.24c 122.70 ± 8.55b 132.16 ± 8.98b 167.26 ± 6.02a
36.69 ± 5.64e 66.17 ± 6.42d 88.98 ± 3.18c 104.43 ± 5.92b 126.39 ± 8.95a
Values with different letters in the same column (a–e) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
(P < 0.05). Furthermore, mice immunized with JEV vaccine and US 50 was able to produce the most significant increase of IL-4 and IFN-c cytokines (P < 0.05). IL-4 and IFN-c cytokine from mouse immunized with JEV vaccine and US 30 was not significantly increased compared to the mouse immunized with JEV vaccine and US 10. Th1 cells secrete IL-2, IFN-c. Th2 cells produce IL-4, IL-5 and IL-10. IFN-c is produced by stimulated T cells and has essential immunomodulatory effects [28]. IL-4 can promote B cell differentiation and enhance the production of antibodies by sensitized B cells [29]. These results suggested that the sonicated bursa extracts could induce Th1 and Th2 type cytokine immune responses. We conclude that sonicated bursa extracts are capable of enhancing T cells and T cell mediated immune responses, and balancing Th1 and Th2 responses. Fig. 3. Effects of sonication on antibody Response of Bursa of Fabricius. C57/BL6 mice were immunized with sonicated bursa extract and JEV vaccine, following the prime–boost vaccinations programs (days 0 and 14), respectively. Sera were collected on 14 day after twice immunizations for detecting the antibody (IgG) titers by ELISA. Values with different letters in the same column (a–c) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
3.5. Effects of sonication on Lymphocyte proliferation activity and T cell subpopulation of Bursa of Fabricius The level of lymphocyte proliferation is a significant method for measuring cell immunity [30]. To determine whether sonicated bursa extracts have an impact on the lymphocyte proliferation, the peripheral lymphocytes were prepared from the immunized
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Fig. 4. Effects of sonication on Lymphocyte proliferation activity and T cell subpopulation of Bursa of Fabricius. The lymphocytes were prepared from spleen of the immunized mice, and were stimulated with JEV antigen for 48 h. (A) Lymphocyte proliferation activity was detected by WST-1 incorporation. (B) T cell immunophenotyping was expressed as T-cell subtype percentages by flow cytometry. The data of samples immunized with JEV vaccine alone (Vaccine) was used as basic control. Values with different letters in the same column (a–d) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
mouse, and were stimulated with JEV antigen for 48 h to detect cell viability by WST-1 incorporation. The results showed that the proliferations of lymphocytes from mouse co injected with US 10, US 30 and US 50 were higher than that of cells from JEV vaccine injected control group (P < 0.05), respectively (Fig. 4A), and the proliferation of samples of US 50 was the highest. Also, the effect of sonicated bursa extracts on T cell subpopulation was assessed. The peripheral lymphocytes were prepared from the immunized mouse, and were stimulated with JEV antigen for 48 h. CD3+CD4+T cells and CD3+CD8+T cells were identified with anti-CD3 and anti-CD4+ or CD8+ markers by flow cytometry. As shown in (Fig. 4B), the enhancement of T cell immunophenotyping from mice immunized with JEV vaccine were observed. The results showed that US 10, US 30 and US 50 induced significantly increased percentage of CD3+CD4+T cell, and CD3+CD8+T cell from immunized mice (P < 0.05). 4. Conclusion In this study, we found that sonication treatment significantly increased the yield of bursa peptides. And sonicated bursa extract displays strong immuno-stimulating activity by promoting B cell development, and enhanced antibody production, inducing both of Th1 type (IFN-c) and Th2 type (IL-4) cytokines, increasing proliferations of splenic lymphocytes and subsets of CD4+T cells (CD3+CD4+), CD8 T cells (CD3+CD8+). This study suggested that sonication treatment of Bursa of Fabricius could be a potential process for future immuno-pharmacologcial use. Conflict of interest The authors declared no conflicts of interest. Acknowledgments This work was supported by the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the National Special Research Programs for Non-profit Trades, Ministry of Agriculture (No. 200803015). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to the Life Sciences Institute of Nanjing Agricultural University for providing RP-HPLC facilities and to the Institute of Biochemistry and Cell Biology, SIBS, CAS for providing mass spectrometric analysis.
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Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Effect of sonication on B cell development and immunomodulatory functions of Bursa of Fabricius Xiao-Dong Liu, Xin-Feng Li, Xiu-Li Feng, Bin Zhou, Rui-Bing Cao, Pu-Yan Chen ⇑ Division of Key Lab of Animal Disease Diagnosis and Immunology of China’s Department of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
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Article history: Received 16 January 2014 Received in revised form 14 February 2014 Accepted 14 February 2014 Available online 24 February 2014 Keywords: Bursa extract Sonication Immunomodulatory function Immunization experiment
a b s t r a c t A study was initiated with the objective of evaluating the effects of sonication treatment on important quality parameters of extract of Bursa of Fabricius. Sonication of extract was done (frequency 20 kHz and various amplitude levels) at 0 °C for 10 min, 30 min, 50 min, respectively. As results, the yield of bursa peptides significantly increased (p < 0.05). Then we found sonicated bursa extract promoted the content of bursin and the CFU pre-B formation, exerted immunomodulatory function on antigen-specific immune responses in C57/BL6 mice immunized with inactivated Japanese encephalitis b virus (JEV) vaccine, including enhancing JEV-specific antibody and cytokine production, T-cell immunophenotyping and lymphocyte proliferation. Findings of the present study suggested the sonication treatment of Bursa of Fabricius could improve the yield as well as the quality of bursa peptides, indicating that sonication is effective in processing of bursa extract and could be a potential process for future immuno-pharmacological use. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The Bursa of Fabricius (BF) is the primary humoral immune organ unique to birds [1], which is critical for early B-lymphocyte proliferation and differentiation [2–4]. B cell is named after BF for differentiation and development into immunocompetent lymphocyte in BF. However, in mammals, the B-cell-differentiating organ, equivalent to the T-cell-differentiating thymus, has not yet been identified [5], the BF therefore provides an invaluable model for studying basic immunology. The BF contains several biological active factors. For example, a high dose of chicken BF extract was shown to enhance blastogenic responses to T-cell mitogens [6]. Bursin is a candidate for the specific factor responsible for B-cell differentiation [7]. It selectively stimulates the differentiation of avian B cells, rather than that of T cells, from their precursors in vitro [5,8], and promotes immunoglobulin (Ig) switching from IgM to IgG [9]. Bursal anti-steroidogenic peptide is responsible for synchronizing B-cell division during embryogenesis and the neonatal period [10]. Bursal septpeptide (BSP)I and BSP-II can induce both humoral and cellular immune responses in vivo [11,12], and bursopentin (BP5) was shown to induce B-lymphocyte proliferation by various signaling pathways [13]. Bursal pentapeptide (BPP)-I exerts immune-inducing ⇑ Corresponding author. Tel.: +86 25 84396335. E-mail address: [email protected] (P.-Y. Chen). http://dx.doi.org/10.1016/j.ultsonch.2014.02.012 1350-4177/Ó 2014 Elsevier B.V. All rights reserved.
functions, including antitumor responses [14], while bursal peptide (BP)11 was found to regulate B-cell development and antigen-specific immune responses [15]. However, traditional techniques have many disadvantages, such as long time, low yield, and low activity of bio-factors. This is mainly caused by (1) decreased activity (2) aggregation and deposition of protein (3) low extraction efficacy. In order to overcome the problems, there is a dire need to introduce innovative, novel and effective technologies. Ultrasound treatment is an innovative emerging processing technique that could effectively improve the biological compounds [16]. The mechanism of ultrasound assisted extraction of bioactive compounds involves diffusion of the solvent through the cell matrix and washing out the cell contents. Ultrasound can also generate cavitation and microstreaming (microscopic fluid movement) effects that cause changes in a liquid medium [17]. Ultrasound is benefical in extraction because it increases the solvent uptake by the cell during sonication thereby increasing the extraction yield [18]. At present, many reports evaluating the effect of ultrasound on peptides. A recent study showed ultrasound can remarkably raise the enzymolysis efficiency and activity of ACE inhibitory peptides from wheat germ protein [19]. In this study, the objective was to investigate the effects of sonication treatment in detail on the yield and quality parameters including content of bursin, CFU pre-B, and antigen-specific immune responses of Bursa of Fabricius.
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2. Materials and methods 2.1. Animal C57/BL6 mice (4–6 weeks old, 17–21 g) were obtained from Yang Zhou University (Yangzhou, China). The 21 old-day chickens were purchased from Qinglongshan farm (Nanjing, China). All the animal experimental procedures were performed in accordance with the institutional ethical guidelines for animal experiments. 2.2. Bursa extract and sonication treatment The BFs were collected and placed on ice. Then they were homogenized in 0.1 M phosphate buffer solution, pH 6.0 and centrifugated at 5000g for 10 min at 4 °C. The sonication of samples for 10, 30, 50, 70 was done at a temperature of 4 °C by using ultrasonic processor of 750 W (X0-650D, Xianou Technology Co., Nanjing, China). The frequency of 20 kHz and amplitude level of 10%, 20%, 40% were used. All the sample preparations and treatments were carried out in triplicate. Fresh untreated bursa extract was selected as control. 2.3. Determine of yield of bursa peptides, concentrations of Bursin and RP-HPLC analysis Total peptide concentration of sonicated bursa extracts was determined with Bicinchoninic Acid assay (BCA) (Beyotime biotechnology, China). The yield was calculated as micrograms of peptide produced per 10 g bursa consumed. 1 mg/ml of sonicated bursa extracts was collected to measure the bursin levels by the method of ELISA. Briefly, a sample or standard was added in ELISA plates (Nunc, UK) coated with capture antibody of bursin for 2 h at 37 °C, and then detected using a biotinylated anti-cytokine antibody, Vidin HRP, and tetramethylbenzidine. Color development was stopped with 2 M H2SO4, and optical densities were read at 450 nm. The concentrations of bursin were determined from the standard curve. 1 mg/ml of sonicated bursa extracts was analyzed by reversedphase (RP) high-performance liquid chromatography (HPLC) with a Zorbax C-18 (ODS) column (150 mm 4.6 mm, Agilent) using a linear gradient of acetonitrile (5–18%, EMD Chemicals) containing 0.1% TFA at a flow rate of 1 ml/min for 20 min at 30 °C. The elution was monitored at 280 nm with a UV–Visible diode-array detector (UV-DA).
with a 0.2 ml JEV inactivated antigen containing 1 mg/ml or without sonicated bursa extracts on days 0, and 14, respectively. As positive control, mice were intraperitoneally immunized twice at two weekly intervals with the same volume containing JEV inactivated antigen, and PBS was used as negative control. The sera were collected on the 14th day and 28th day to detect the antigen specific antibody responses (IgG) by ELISA method, respectively, as previously described [22]. 2.6. Lymphocyte proliferation, cytokine enzyme-linked immunosorbent assay (ELISA) and Flow cytometry On the fourteenth day after final immunization, mice were killed by cervical dislocation; their spleens were aseptically removed and cell suspensions made by passing them through a sterile cell strainer (Becton-Dickionson, USA). The cell suspensions were washed twice in RPMI-1640 medium supplemented with 10% FBS, and adjusted to 1 107 cells/ml, and inactivated JEV (105 PFU). After two days of incubation, the cell viability was measured by using the WST-1 Cell proliferation and cytotoxicity assay kit (Beyotime, Hangzhou, China) in accordance with the manufacturer’s instructions. Briefly, 10 ll of this reagent was added to each well containing 100 ll of cell suspension incubated for an additional 1 h. The absorbance at 450 nm was monitored and the reference wavelength was set at 630 nm. The percent viability of cells was calculated by comparison to that of untreated control cells. The amount of several cytokines (IL-4 and IFN-c) in the culture supernatants were measured by ELISA kits (RD, USA) according to the manufacturer’s instructions. Also, the culture cells were performed by three-color flow cytometry analysis (BD, LSR), by using different mixtures of specific mAbs CD3, CD4, and CD8 labeled with PE-Cy5, FITC or PE, respectively. Data analysis was done with the FACScan flow cytometer (BD, LSR) using the Cell Quest software. 2.7. Statistical analysis Results were expressed as means ± standard deviations (SD). The statistical significance of the observed differences was analyzed by 2-sided t test unless otherwise specified. p value <0.05 was considered to be significantly different. 3. Results and discussion 3.1. Effects of sonication on yield of bursa peptides of Bursa of Fabricius
2.4. CFU pre-B assay Single-cell suspensions were obtained from the bone marrow of c57 mice at 4–6 weeks of age. Cells were suspended in Iscove’s Modified Dulbecco’s medium containing 1% Methylcellulose, 2 mM L-glutamine, 10% fetal calf serum (FCS), 50 lM 2-mercaptoethanol, 0.1 g/L streptomycin, and 105 U/mL penicillin. Whole bone marrow cells were treated with the IL-7(10 ng/ml) and sonicated bursa extracts (both added immediately before plating), mixed with Methylcellulose, and plated in 35-mm culture dishes. The plates were incubated at 37 °C, 5% CO2. CFU-pre-B were counted on day 7 of culture as described [20]. Data are presented as the mean number of CFU/105 cells for triplicate samples ± SD. 2.5. Sensitization protocols and detection of antigen-specific Ab responses The immunomodulatory roles of the sonicated bursa extracts were examined in 6–8 weeks old BALB/c female mice, as previously reported [21], in which mice were intraperitoneally immunized
Results regarding the effects of sonication treatments on yield of bursa peptides of bursa extract are presented in Table 1. In the present study, we observed that the yield rise as the amplitude become higher. The yield rise leveled off between 40% and 60% (Table 1). High power ultrasound generated at high amplitude and low frequency (20–100 kHz), which is used in food processing, welding, and cleaning processes [23]. Ultrasonic extraction of various food components has been reported mainly for 20–40 kHz. In our study, we used 20 kHz and studied the changes of yield of Table 1 Effect of sonication treatments on Bursa peptide yield. Frequency (KHZ)
Amplitude (%)
US 10 (mg/g)
US 30 (mg/g)
20 20 20 20
10 20 40 60
3.74 ± 0.11c 4.08 ± 0.04b 4.25 ± 0.07a 4.31 ± 0.07a
4.11 ± 0.73d 4.46 ± 0.13c 4.97 ± 0.08b 5.15 ± 0.06a
Values with different letters in the same column (a–d) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min.
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bursa peptides in different amplitude (from 10% to 60%), and found 40% and 60% are proper for bursa peptides yield. Then, we compared the effects of sonication treatments and the freeze treatment on yield of bursa peptides. We found that the yield of bursa peptides significant improved in sonicated bursa extract samples compared to control untreated bursa sample and freeze treatment. The observed increase in yield was from 2.822 to 4.171, 4.883, 5.589 and 5.564 mg/g in bursa extract sonicated for 10, 30, 50 and 70 min, respectively. There were no significant difference between 50 and 70 min of sonication, suggested that 50 min of sonication was enough for the treatment of sonication. Compared to sonication, the yields of bursa peptides in various time of freeze were significantly lower, from 3.356 to 3.873 mg/g for 1–7 times of freeze. Bursin is an essential peptide in the bursa peptides, which regulated B-cell differentiation and promotes immunoglobulin (Ig) switching from IgM to IgG [9]. We evaluated the effect of sonication and freeze in concentration of Bursin using ELISA method. The results showed that the concentration of Bursin significantly increased after sonication treatment, compared to control untreated bursa sample and various times of freeze (Fig. 1B). As bursa peptides from bursa extract have many useful biological compounds, we evaluated the effect of sonication on the yield of other bursa peptides beside Bursin using RP-HPLC (Fig. 2), the highest peaks were a–d. We found that each peaks of the sample treated for 50 min were the highest, the peaks of the sample treated for 30 min were also higher that the sample treated for 10 min. The changes in biological compounds might be attributed to the cavitations during sonication [23]. It has been reported that sonication treatment extracted more protein for extraction [24], we also found the similar results. The increase in yield might be due to high-pressure gradient by cavitations during sonication treatment that cause the breakdown of agglomerates to smaller ones and breakout of cells that increases of the solvent uptake and make the extract properly homogenized and more consistent. Various times of freeze can also breakout the cells and increase the solvent uptake, but may cause the aggregation and deposition of proteins and peptides that decreased the yield and activity. These results indicated that sonication is effective in processing of bursa extract which increased yield as well as the content of biological compounds. 3.2. Effects of sonication on B cell development of Bursa of Fabricius As Bursa of Fabricius is the central organ of B cell development [1], these sonicated bursa extract samples may affect the B cell development. To investigate this hypothesis, we compared the biological activity of sonicated bursa extract samples on B cell
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development by using CFU pre-B formation assays. As expected, the numbers of CFU pre-B which were treated with US 10 (ultrasounds treatment for 10 min), US 30 (ultrasounds treatment for 30 min) and US 50 (ultrasounds treatment for 50 min) were significantly increased at three different doses, compared to untreated bursa extract and the freeze treatment (P < 0.05). Furthermore, the numbers of CFU pre-B of US 50 were the highest among the three samples at 5 lg/ml, the numbers of CFU pre-B of US 30 and US 50 were higher than US 10 at 1 lg/ml and 25 lg/ml (Table 2). The B cell progenitors responding to IL-7 in the CFU-pre B cell assay are most likely cells at hte pre-pro,or C stage as described by Hardy et al. [25]. The changes in biological compounds might be attributed to the cavitations during sonication [23]. Various times of freeze may cause the aggregation and deposition of proteins and peptides that decreased the activity. These results indicated that sonication is effective in processing of bursa extract which increased the activity of bursa extract in vitro. 3.3. Effects of sonication on antibody response of Bursa of Fabricius The production of antibodies (humoral immunity) and action of sensitized T lymphocytes (cellular immunity) are vital components in the immune response. Cytokines are a super family of proteins and they induce multifarious biological effects by regulating the transcription of distinct sets of genes in different cell types in immune processes [26,27]. To determine whether sonication is effective in processing of bursa extract which modulate antibody responses in vivo, we injected mice with sonicated bursa extract (1 mg/kg) and JEV oil-adjuvant vaccine. We first assessed the antibody IgG profiles in sera day 14 from all groups (Fig. 3). Antibodies from mice immunized with JEV vaccine and US 10, US 30, US 50 were observed significantly increased (P < 0.05) than mouse immunized with JEV vaccine control. These results suggested that sonicated bursa extracts had fine inducing roles on antibody response. This increase may be attributed to the sonication treatments that increased the bursa peptides and macromolecules. 3.4. Effects of sonication on stimulation of primary cytokines responses of Bursa of Fabricius To evaluate whether the immunization with sonicated bursa extract and JEV vaccine in vivo enhance a specific cytokines response, the sera were collected from the immunized mice at 7th day after the second immunization to determine IL-4 and IFN-c cytokine productions by ELISA method. As shown in Table 3, IL-4 and IFN- c productions following the twice immunization procedure with US 10, US 30 and US 50 were significantly higher than in mouse received JEV vaccine control
Fig. 1. Effect of sonication on yield of bursa peptides and concentrations of Bursin. (A) Change in the yield of bursa peptides during sonication and various time of freeze of bursa extract were measured with BCA assay. Values with different letters in the same column (a–g) are significantly different (P < 0.05) from each other. (B) Change in the concentrations of Bursin during sonication and various time of freeze of bursa extract were measured with ELISA. Values with different letters in the same column (a–f) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min. US 70; ultrasounds treatment for 70 min. 1; 1 time of freeze. 3; 3 times of freeze. 5; 5 times of freeze. 7; 7 times of freeze.
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Fig. 2. The RP-HPLC assay of sonication of bursa extract. Letters (a–d) were the highest peaks. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min. US 70; ultrasounds treatment for 70 min.
Table 2 Effect of sonication treatments on CFU-Pre-B values of Bursa of Fabricius. Treatment
1 lg/ml
5 lg/ml
25 lg/ml
Control Bursa extract Freeze US 10 US 30 US 50
18.33 ± 2.08e 22.67 ± 1.15d 25 ± 1c 31.33 ± 3.51b 51 ± 3.61a 52 ± 3a
18.33 ± 2.08f 34.67 ± 1.53e 40 ± 1d 46.67 ± 5.86c 60.67 ± 4.04b 71 ± 2.65a
18.33 ± 2.08d 40 ± 1.73c 48 ± 1b 56.33 ± 4.04b 73.33 ± 6.11a 80.33 ± 5.03a
Values with different letters in the same column (a–f) are significantly different (P < 0.05) from each other. Freeze; 5 times of freeze. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
Table 3 Effect of sonication treatments and inactivated JEV on Th1/Th2 cyotokin productions in splenocytes. Treatment
IL-4
IFN-c
PBS JEV vaccine JEV vaccine + US 10 JEV vaccine + US 30 JEV vaccine + US 50
32.78 ± 4.76d 68.62 ± 3.24c 122.70 ± 8.55b 132.16 ± 8.98b 167.26 ± 6.02a
36.69 ± 5.64e 66.17 ± 6.42d 88.98 ± 3.18c 104.43 ± 5.92b 126.39 ± 8.95a
Values with different letters in the same column (a–e) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
(P < 0.05). Furthermore, mice immunized with JEV vaccine and US 50 was able to produce the most significant increase of IL-4 and IFN-c cytokines (P < 0.05). IL-4 and IFN-c cytokine from mouse immunized with JEV vaccine and US 30 was not significantly increased compared to the mouse immunized with JEV vaccine and US 10. Th1 cells secrete IL-2, IFN-c. Th2 cells produce IL-4, IL-5 and IL-10. IFN-c is produced by stimulated T cells and has essential immunomodulatory effects [28]. IL-4 can promote B cell differentiation and enhance the production of antibodies by sensitized B cells [29]. These results suggested that the sonicated bursa extracts could induce Th1 and Th2 type cytokine immune responses. We conclude that sonicated bursa extracts are capable of enhancing T cells and T cell mediated immune responses, and balancing Th1 and Th2 responses. Fig. 3. Effects of sonication on antibody Response of Bursa of Fabricius. C57/BL6 mice were immunized with sonicated bursa extract and JEV vaccine, following the prime–boost vaccinations programs (days 0 and 14), respectively. Sera were collected on 14 day after twice immunizations for detecting the antibody (IgG) titers by ELISA. Values with different letters in the same column (a–c) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
3.5. Effects of sonication on Lymphocyte proliferation activity and T cell subpopulation of Bursa of Fabricius The level of lymphocyte proliferation is a significant method for measuring cell immunity [30]. To determine whether sonicated bursa extracts have an impact on the lymphocyte proliferation, the peripheral lymphocytes were prepared from the immunized
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Fig. 4. Effects of sonication on Lymphocyte proliferation activity and T cell subpopulation of Bursa of Fabricius. The lymphocytes were prepared from spleen of the immunized mice, and were stimulated with JEV antigen for 48 h. (A) Lymphocyte proliferation activity was detected by WST-1 incorporation. (B) T cell immunophenotyping was expressed as T-cell subtype percentages by flow cytometry. The data of samples immunized with JEV vaccine alone (Vaccine) was used as basic control. Values with different letters in the same column (a–d) are significantly different (P < 0.05) from each other. US 10; ultrasounds treatment for 10 min. US 30; ultrasounds treatment for 30 min. US 50; ultrasounds treatment for 50 min.
mouse, and were stimulated with JEV antigen for 48 h to detect cell viability by WST-1 incorporation. The results showed that the proliferations of lymphocytes from mouse co injected with US 10, US 30 and US 50 were higher than that of cells from JEV vaccine injected control group (P < 0.05), respectively (Fig. 4A), and the proliferation of samples of US 50 was the highest. Also, the effect of sonicated bursa extracts on T cell subpopulation was assessed. The peripheral lymphocytes were prepared from the immunized mouse, and were stimulated with JEV antigen for 48 h. CD3+CD4+T cells and CD3+CD8+T cells were identified with anti-CD3 and anti-CD4+ or CD8+ markers by flow cytometry. As shown in (Fig. 4B), the enhancement of T cell immunophenotyping from mice immunized with JEV vaccine were observed. The results showed that US 10, US 30 and US 50 induced significantly increased percentage of CD3+CD4+T cell, and CD3+CD8+T cell from immunized mice (P < 0.05). 4. Conclusion In this study, we found that sonication treatment significantly increased the yield of bursa peptides. And sonicated bursa extract displays strong immuno-stimulating activity by promoting B cell development, and enhanced antibody production, inducing both of Th1 type (IFN-c) and Th2 type (IL-4) cytokines, increasing proliferations of splenic lymphocytes and subsets of CD4+T cells (CD3+CD4+), CD8 T cells (CD3+CD8+). This study suggested that sonication treatment of Bursa of Fabricius could be a potential process for future immuno-pharmacologcial use. Conflict of interest The authors declared no conflicts of interest. Acknowledgments This work was supported by the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the National Special Research Programs for Non-profit Trades, Ministry of Agriculture (No. 200803015). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to the Life Sciences Institute of Nanjing Agricultural University for providing RP-HPLC facilities and to the Institute of Biochemistry and Cell Biology, SIBS, CAS for providing mass spectrometric analysis.
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