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. 2017 Jan 3;12(1):e0169154.
doi: 10.1371/journal.pone.0169154. eCollection 2017.

Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens

Tamiru N Alkie  1 , Khaled Taha-Abdelaziz  1   2 , Neda Barjesteh  1 , Jegarubee Bavananthasivam  1 , Douglas C Hodgins  1 , Shayan Sharif  1
Affiliations

Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens

Tamiru N Alkie et al. PLoS One. .

Abstract

Natural or synthetic Toll-like receptor (TLR) ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide) polymer nanoparticles (PLGA NPs) was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line) were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4) induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN)-γ and IFN-β until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens) by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1β until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Nitric oxide (NO) production from chicken macrophages (six replicates/group) stimulated with encapsulated and soluble forms of three TLR ligands, measured by Griess reagent system.
(A). LPS; (B). Pam3CSK4, and (C). CpG ODN. Significance (P <0.05) between delivery systems (encapsulated and soluble TLR ligands) within a treatment dose was determined. P-LPSHi—encapsulated high dose LPS; P-LPSlo—encapsulated low dose LPS; LPSHi—soluble high dose LPS; LPSlo—soluble low dose LPS. P-PamHi—encapsulated high dose Pam3CSK4; P-Pamlo—encapsulated low dose Pam3CSK4; PamHi—soluble high dose Pam3CSK4; Pamlo—soluble low dose Pam3CSK4. Similar designations were made for CpG ODN. * indicates significant difference.
Fig 2
Fig 2. Expression profiles of IFN-γ for (A). LPS; (B). Pam3CSK4, and (C). CpG ODN.
Chicken macrophages (six replicates/group) were treated with encapsulated and soluble forms of the three TLR ligands and mean fold expression of IFN-γ in treated groups were compared to the cell culture medium treated group. Significance (P <0.05) was tested between delivery systems (encapsulated and soluble TLR ligands) within a dose and at a given time point. * indicates significant difference.
Fig 3
Fig 3. Expression level of IFN-β by stimulation with CpG ODN.
Chicken macrophages (six replicates/group) were treated with encapsulated and soluble CpG ODN. Mean fold expression of IFN-β in treated groups were compared to the cell culture medium treated group. Significance (P <0.05) was tested between delivery systems within a dose and at a given time point. * indicates significant difference.
Fig 4
Fig 4. IL-1β mRNA expression in chicken macrophages stimulated with (A). LPS; (B). Pam3CSK4; and (C) CpG ODN.
Chicken macrophages (six replicates/group) were treated with encapsulated and soluble forms of the three TLR ligands and mean fold expression of IL-1β in treated groups were compared to the cell culture medium treated group. Significance (P <0.05) was tested between delivery systems within a dose and at a given time point. * indicates significant difference.
Fig 5
Fig 5. Quantification of IFN-γ (A, B), IL-1β (C, D) and IL-8 (E, F) expression in spleen of chickens intramuscularly injected with encapsulated and soluble LPS.
At 3, 18, 48 and 96 hr post-injection, spleens were collected and the relative expression of cytokine genes was determined. Bars represent mean fold expression of cytokines in treated chickens compared to the control chickens. Cytokine gene expressions were compared between delivery systems (encapsulated and soluble) within a dose and at a given time point. * indicates significant difference at P <0.05.
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