J Vet Sci.  2015 Dec;16(4):501-507. 10.4142/jvs.2015.16.4.501.

Isolation and immunomodulatory activity of bursal peptide, a novel bursal peptide from the chicken bursa of Fabricius

• Liu XD
• Qian Y
• Jung YS
• Chen PY

• KMID: 2133633
• DOI: http://doi.org/10.4142/jvs.2015.16.4.501

Abstract

The bursa of Fabricius (BF), which is unique to birds, serves as the central humoral immune organ and plays a significant role in B lymphocyte differentiation. In this study, a new bursal peptide (BP-IV) was isolated from BF, which promoted colony-forming unit pre-B formation and regulated B cell differentiation. BP-IV also exerted immunomodulatory effects on antigen-specific immune responses via both humoral and cellular immunity in chicken and mice that had been immunized with inactivated avian influenza virus (AIV; H9N2 subtype), including enhancing AIV-specific antibody and cytokine production. The results of this study provided novel insights into the use of a potential candidate reagent for B cell development and future immuno-pharmacological use.

Keyword

B cell development; bursal peptide; immunomodulatory experiment; immunomodulatory function

MeSH Terms

Animals
Birds
Bursa of Fabricius*
Cell Differentiation
Chickens*
Immunity, Cellular
Influenza in Birds
Lymphocytes
Mice
Stem Cells

Figure

• 

  Fig. 1 Purification of bursal peptide BP-IV. (A) The bursal extracts from healthy young chickens were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a 4.6 × 250 mm SinoChrom ODS-BP (Eliteplc, China) RP-HPLC affinity column equilibrated in aqueous 0.1% TFA and developed with a linear acetonitrile gradient (from 0.5% to 50%). BP-IV was eluted in the arrow-marked peak. (B) BP-IV is composed of KNEVEEEAKTP, and was analyzed by DNA Star.

• 

  Fig. 2 BP-IV modulates B cell development. (A) Bone marrow cells were plated in 35-mm dishes at 2.5 × 104/mL in methycellulose medium. Various concentrations of BP-IV and BP-IV-scrambled (from 1 to 25 µg/mL) were added to the culture. Control cells (0 µg/mL) were treated with BSA (1 µg/mL). The cells were cultivated for 7 days, after which the produced colonies (CFU pre-B) were scored. All experiments were repeated three times independently. Values with different letters in the same column (a-d) are significantly different (p < 0.05). Data were expressed as the means ± SD (n = 3). (B) BM cells were cultured in the presence of IL-7 (10 ng/mL) with BP-IV or BP-IV-scrambled (25 µg/mL) for 7 days. The cell population was then analyzed by staining with antibodies against B220, IgM, CD43, and IgD, followed by FACS analysis. The proportion of each population was defined as the percentage of the entire cell population. All experiments were repeated independently three times. Values with different letters in the same column (a-d) are significantly different (p < 0.05) from each other. Data were expressed as the means ± SD. (C) Relative PU.1 gene expression. BM cells were grown in IL-7. On day 3, BP-IV or BP-IV-scrambled (25 µg/mL) was added to half of the culture for 24 h. Cells were sorted on day 4 into pro-B (B220+CD43+IgM-), pre-B (B220+CD43+IgM-), and immature/mature (iMB/MB) cells (B220+CD43-IgM+). (D) Total RNA was extracted. Real-time PCR analysis was performed on cDNA using PU.1-specific primers as described in the materials and methods. Figures show normalized values from a single experiment and fold-change over untreated from three independent experiments. scr, scrambled.

• 

  Fig. 3 Immunomodulatory roles of BP-IV on chicken immunization regimen. Chickens were twice immunized with BP-IV or BP-IV-scrambled and avian influenza virus (AIV) vaccine. (A) The induction of AIV-specific antibodies was measured by hemagglutination inhibition assay. (B) The sera were collected from the immunized chickens to determine IL-4 and IFN-γ cytokine production by enzyme-linked immunosorbent assay (ELISA). (C) Lymphocytes were prepared from the blood of immunized chickens, then stimulated with AIV antigen for 48 h to detect cell viability by MTT incorporation. All experiments were repeated independently three times. Values with different letters in the same column (a-d) are significantly different (p < 0.05) from each other. Data were expressed as the means ± SD (n = 10) from two independent experiments. PBS, phosphate buffered saline.

• 

  Fig. 4 Immunomodulatory roles of BP-IV on mouse immunization regimen. Mice were immunized with BP-IV or BP-IV-scrambled and AIV vaccine following the prime-boost vaccinations programs (days 0 and 14), respectively. (A) Sera were collected on day 14 after the two immunizations for use in antibody (IgG) titers by ELISA. (B) On day 7 after the second immunization, sera were collected to detect cytokine IL-4 and IFN-γ. (C) T cell immunophenotyping was expressed as the T-cell subtype percentages by flow cytometry. All experiments were repeated independently three times. Values with different letters in the same column (a-d) are significantly different (p < 0.05) from each other. Data were expressed as the mean ± SD (n = 5) from two independent experiments.

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