Immunoregulatory and Antimicrobial Activity of Bovine Neutrophil β-Defensin-5-Loaded PLGA Nanoparticles against Mycobacterium bovis

Abstract

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex imposing a high zoonotic threat to human health. The limited efficacy of BCG (Bacillus Calmette-Guérin) and upsurges of drug-resistant tuberculosis require new effective vaccination approaches and anti-TB drugs. Poly (lactic-co-glycolic acid) (PLGA) is a preferential drug delivery system candidate. In this study, we formulated PLGA nanoparticles (NPs) encapsulating the recombinant protein bovine neutrophil β-defensin-5 (B5), and investigated its role in immunomodulation and antimicrobial activity against M. bovis challenge. Using the classical water-oil-water solvent-evaporation method, B5-NPs were prepared, with encapsulation efficiency of 85.5% ± 2.5%. These spherical NPs were 206.6 ± 26.6 nm in diameter, with a negatively charged surface (ζ-potential -27.1 ± 1.5 mV). The encapsulated B5 protein from B5-NPs was released slowly under physiological conditions. B5 or B5-NPs efficiently enhanced the secretion of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-10 in J774A.1 macrophages. B5-NPs-immunized mice showed significant increases in the production of TNF-α and immunoglobulin A (IgA) in serum, and the proportion of CD4⁺ T cells in spleen compared with B5 alone. In immunoprotection studies, B5-NPs-immunized mice displayed significant reductions in pulmonary inflammatory area, bacterial burden in the lungs and spleen at 4-week after M. bovis challenge. In treatment studies, B5, but not B5-NPs, assisted rifampicin (RIF) with inhibition of bacterial replication in the lungs and spleen. Moreover, B5 alone also significantly reduced the bacterial load in the lungs and spleen. Altogether, our findings highlight the significance of the B5-PLGA NPs in terms of promoting the immune effect of BCG and the B5 in enhancing the therapeutic effect of RIF against M. bovis.

Keywords: Mycobacterium bovis; PLGA; antimicrobial activity; bovine neutrophil β-defensin-5; immunoregulation; nanoparticles.

Figure 1

Experimental design for evaluation of immunoregulatory and antimicrobial activities of B5 protein nanoparticles (B5-NPs). (A) Flow chart depicting the pre-exposure strategy. Groups of BALB/c mice (n = 10 per group) were immunized subcutaneously with 10⁶ CFU of Bacillus Calmette–Guérin (BCG) in 100 µL of PBS. At four weeks before infection, mice were immunized intranasally with B5 (2.5 mg/kg) or B5-NPs (50 mg/kg) three times every three weeks. (B) Flow chart depicting the post-exposure strategy. Mice were challenged intranasally with 110 CFU of M. bovis. After 4 weeks, the mice were treated subcutaneously with B5 (5 mg/kg), B5-NPs (100 mg/kg), or respectively combined with rifampicin (RIF) (10 mg/kg) until week 6.

Figure 2

Physicochemical and morphological characterization of B5-NPs. (A) The purified recombinant B5 from B5-expressing pichia pastoris cells that was used for the preparation of B5-NPs (left lane). The protein recovered from B5-NPs after 7 days of in vitro release (middle lane) and protein molecular weight ladder (right lane) were subjected to Tricine-SDS-PAGE followed by Coomassie blue staining. (B) Western blotting identification of the purified B5 by using anti-His·tag antibody. (C) The release profile of B5 protein from B5-NPs in vitro. B5-NPs suspended in PBS (50 mg/mL) were incubated at 37 °C for indicated time periods, and the release of B5 protein was estimated by micro-BCA assay. The initial burst release caused >45% of B5 protein to be released within 24 h (inner graph), followed by slower-release kinetics that reached ∼68% of B5 release by 7 days (outer graph). (D,E) Scanning electron microscopy observed the morphological characterization of B5-NPs. (F,G) The physicochemical characterization of B5-NPs was determined by dynamic light scattering (F) and ζ-potential analysis (G).

Figure 3

B5 and B5-NPs induce pro-inflammatory and anti-inflammatory cytokine production in macrophages. (A–C) ELISA analysis of TNF-α (A), IL-1β (B), and IL-10 (C) production in J774A.1 macrophages stimulated with B5, B5-NPs, or PBS-NPs for 24 h. B5: BNBD5, B5-NP: BNBD5- PLGA NPs, PBS-NP: PBS- PLGA NPs. All data are shown in vitro representing the mean ± SD of three independent experiments (** p < 0.01; *** p < 0.001).

Figure 4

B5-NPs induce cytokines and antibodies production in BCG-immunized mice. (A–E) Concentrations of TNF-α (A), IL-1β (B) and IL-10 (C) in serum, IgG (D) in serum and IgA in serum (E) or bronchoalveolar lavage fluid (BALF) (F) were examined by ELISA. Serum and BALF were collected three weeks after the last immunization. All data shown represent the mean ± SD of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

The percentage of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ lymphocytes within splenocytes. (A–C) Flow cytometry analysis percentage of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ lymphocytes within splenocytes. At week 13, isolated spleen cells from mice were stained with PerCP-Cy5.5-conjugated anti-CD3, FITC-conjugated anti-CD4, APC-conjugated anti-CD8. CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T-cell subgroup were detected via flow cytometry. Data collected are expressed as the mean ± SD of three independent experiments (* p < 0.05; ** p < 0.01).

Figure 6

B5-NPs reduce pathological damage of lung. (A,B) Histopathological evaluation of lung tissues was performed with hematoxylin and eosin (H&E) staining (A) and acid-fast staining (B) four weeks after challenge with M. bovis strain. The top panel (A) shows images at ×100 magnification (scale bars, 100 μm); the bottom panel (B) shows images at ×1000 magnification (scale bars, 10 μm). (C–F) The organ coefficient of lung (C) and spleen (D), the number of viable bacteria in the lungs (E) and spleen (F) were determined 4 weeks after challenge with M. bovis strain. (G) Percentage of lung area involved in inflammation lesions relative to total lung area used for morphometric analysis. Data shown are representative of six mice per group. Data collected are expressed as the mean ± SD. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 7

The therapeutic effect of B5 combined with RIF on M. bovis-infected mice. (A,B) Histopathological evaluation of lung tissues of M. bovis-infected mice was performed with H&E staining (A) and acid-fast staining (B) two weeks after treatment with B5, B5-NPs, or combined with rifampicin (RIF), respectively. The top panel (A) shows images at ×100 magnification (scale bars, 100 μm); the bottom panel (B) shows images at ×1000 magnification (scale bars, 10 μm). (C–F) The organ coefficient of lung (C) and spleen (D), and the number of viable bacteria in the lungs (E) and spleen (F) were determined 2 weeks after treatment. (G) Percentage of lung area involved in inflammation lesions relative to total lung area used for morphometric analysis. Data shown are representative of six mice per group. Data collected are expressed as the mean ± SD. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 8

Effects of B5 combined with RIF on TNF-α secretion in serum and BALF. (A,B) Concentrations of TNF-α in serum (A) and BALF (B) were examined by ELISA. Serum and BALF were collected two weeks after the last treatment. All data showing represent the mean ± SD of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001).