Effects of over-expression of TLR2 in transgenic goats on pathogen clearance and role of up-regulation of lysozyme secretion and infiltration of inflammatory cells

Abstract

Background: Toll-like receptor 2 (TLR2) is important to host recognition of invading gram-positive microbes. In goats, these microbes can cause serious mastitis, anthrax, tetanus, and other problems. Transgenic goats constitutively over-expressing TLR2 in many tissues serve as a suitable model for the study of the role of TLR2 over-expression in bacterial clearance.

Results: Capra hircus TLR2 over-expression vector (p3S-LoxP-TLR2) was used to generate transgenic goats by egg microinjection. The integration efficiency was 8.57%. Real-time PCR and immunohistochemical results confirmed that the goats over-expressing the TLR2 gene (Tg) expressed more TLR2 than wild-type goats (WT). Monocyte-macrophages from the bloodstreams of transgenic goats were stimulated with synthetic bacterial lipoprotein (Pam3CSK4) and by the promotion of interleukin-6 (IL-6) and IL-10 expression in vitro. The oxidative damage was significantly reduced, and lysozyme (LZM) secretion was found to be up-regulated. Ear tissue samples from transgenic goats that had been stimulated with Pam3CSK4 via hypodermic injection showed that transgenic individuals can undergo the inflammation response very quickly.

Conclusions: Over-expression of TLR2 was found to decrease radical damage to host cells through low-level production of NO and MDA and to promote the clearance of invasive bacteria by up-regulating lysozyme secretion and filtration of inflammatory cells to the infected site.

Figure 1

Transgenic goats over-expressing Capra hircus TLR2. (A) Structure of p3S-LoxP-TLR2 plasmids. (B) Southern blotting analysis, 1–3: positive samples; 4–7: negative samples; 8: control; 9, 10: p3S-Loxp-TLR2 plasmid. (C) Relative quantitative analysis of TLR2 mRNA expression under Pam3CSK4 stimulation. (D) Immunohistochemical study of TLR2 protein product: transgenic (Tg) group, wild-type (WT) group. Sections were double-stained with DAPI (blue) and TLR2-FITC (green).

Figure 2

Transgenic goats over-expressing Capra hircus TLR2 monocytes and macrophages under Pam3CSK4 stimulation. Promotion of IL-6 and IL-10 expression in transgenic goats, ELISA testing of changes in inflammatory cytokines: (A) IFN-γ, (B) IL-6, and (C) IL-10 expression.(D, E) Prevention oxidative damage in transgenic goats, levels of NO and MDA expression. (F) Up-regulation of lysozyme secretion in transgenic goats by ELISA. Data are means ± SE. * Values within the same concentration of Pam3CSK4 (1 μg/mL) were found to differ significantly between different groups at different times (P<0.05). Tg = transgenic group, WT = wild-type group.

Figure 3

Goats intradermally injected with 1 mg Pam3CSK4 in the ear. Rapid filtration of inflammatory cells in transgenic goats. Pathologic changes were examined microscopically (×400). Tg = transgenic group, WT = wild-type group.