Modulation of the innate immune system by lipopolysaccharide in the proventriculus of chicks inoculated with or without Newcastle disease and infectious bronchitis vaccine

Abstract

This study aimed to determine whether the innate immune system in the proventriculus of broiler chicks responds to lipopolysaccharide (LPS) and whether this response is affected by Newcastle disease and infectious bronchitis (ND/IB) vaccination. Chicks were divided into 4 groups: nonvaccinated and injected with PBS or LPS (V-L- and V-L+), and vaccinated and injected with PBS or LPS (V+L- and V+L+). Vaccination was performed on d 1, and LPS was intraperitoneally injected on d 11 of age. The gene expression and protein levels of immune molecules, including toll-like receptors (TLRs), antimicrobial peptides, interleukin-1β (IL-1B), and immunoglobulin A (IgA) in the proventriculus and serum were analyzed. The results showed that the expression levels of TLR21 were higher in vaccinated (V+L-) group than in nonvaccinated (V-L-) group. Gene expression levels of avian β-defensin (AvBDs) and cathelicidin1 (Cath1) were not different among the 4 groups. However, the results of LC/MS analysis showed that the levels of AvBD2, 6, and 7 significantly increased after the LPS challenge in nonvaccinated and vaccinated chicks; the levels were higher in V-L+ and V+L+ than in V-L- and V+L-, respectively. Immunohistochemistry analysis revealed the localization of AvBD1 protein in the epithelial cells of the surface glands and AvBD2 and CATH1 in the heterophil-like cells in the lamina propria of surface glands. Although IL-1B gene expression and protein concentration in the proventriculus tissues were not different among the 4 groups, serum IL-1B levels were upregulated by LPS in both the nonvaccinated and vaccinated groups (V-L- vs. V-L+, V+L- vs. V+L+). Moreover, IgA levels in the proventriculus and serum were not affected by vaccination or LPS challenge. Taken together, we conclude that LPS derived from gram-negative bacteria upregulates the innate immune system, including antimicrobial peptide synthesis in the proventriculus. ND/IB vaccination may not significantly affect antimicrobial peptide synthesis in response to LPS; however, TLR21 expression is upregulated by that vaccination. The antimicrobial peptides synthesized in the proventriculus probably prevent pathogenic microbes from entering the intestine.

Keywords: chick proventriculus; innate immune system; lipopolysaccharide; vaccination.

Figure 1

Effects of Newcastle disease and infectious bronchitis (ND/IB) vaccination on the toll-like receptor (TLR) expression in the chick proventriculus. Day-old chicks were administered phosphate-buffered saline or ND/IB vaccine, and the proventriculus tissues were collected without lipopolysaccharide challenge (V-L- and V+L- groups, respectively) on d 11 post-hatching. The dots indicate the values for each individual. Bars represent mean ± SEM (n = 10). Asterisks indicate significant differences between the nonvaccinated and vaccinated groups (**P < 0.01, n = 10).

Figure 2

Effects of lipopolysaccharide (LPS) challenge on the gene expression of avian β-defensins (AvBDs) and cathelicidin 1 (Cath1) in the chick proventriculus with or without Newcastle disease and infectious bronchitis (ND/IB) vaccination. Day-old broiler chicks were inoculated with or without ND/IB vaccines, and then intraperitoneally injected with PBS or LPS 5 h before the examination at d 11 of age. Four experimental chick groups were designed: non-vaccinated and injected with PBS (V-L-), nonvaccinated and injected with LPS (V-L+), vaccinated and injected with PBS (V+L-), and vaccinated and injected with LPS (V+L+). Dots represent the values for each individual. Bars represent mean ± SEM (n = 10).

Figure 3

Effects of lipopolysaccharide (LPS) challenge on the levels of avian β-defensins (AvBDs) and cathelicidin 1 (CATH1) in the chick proventriculus with or without Newcastle disease and infectious bronchitis (ND/IB) vaccination. Day-old broiler chicks were inoculated with or without ND/IB vaccines, and then intraperitoneally injected with or without lipopolysaccharide 5 h before the examination at d 11 of age. The levels of AvBDs and CATH1 in the proventriculus were analyzed using liquid chromatography–mass spectrometry (LC/MS). See Figure 2 for the distribution of experimental chick groups V-L-, V-L+, V+L-, and V+L+. Individual values are indicated by dots. Bars represent mean ± SEM (n = 10). The asterisks indicate significant differences between the groups (*P < 0.05, **P < 0.05; Kruskal Wallis and Steel-Dwass tests).

Figure 4

Immunolocalization of avian β-defensins (AvBDs) and cathelicidin 1 (CATH1) in the proventriculus of a chick treated with Newcastle disease and infectious bronchitis (ND/IB) vaccine and lipopolysaccharide (LPS). (A and B) Sections of proventriculus mucosa under low and high magnifications, respectively. In the surface layer, mucosal folds (F) are formed, and surface glands (Sg) showing tubular structures are formed at the bottom of the folds. Formation of lymphocyte clusters can be seen. Note that the heterophils are localized in the whole lamina propria (arrows) and a part of them form small groups. (C) AvBD1 immunostaining. Note the AvBD1 immunopositive signals in the epithelial cells of surface glands (arrows). (D) AvBD2 immunostaining. Note that the AvBD2-positive cells are assembled to form groups (arrows). (E) CATH1 immunostaining. Note that the CATH1-positive cells are localized throughout the lamina propria (arrows). Abbreviations: Dg, deep glands; L, lumen; Lp, lamina propria; Ly, lymphocyte accumulation. Scale bars = 50 μm.

Figure 5

Effects of lipopolysaccharide (LPS) on the gene expression and concentration of interleukin 1β (IL-1B) in the proventriculus tissue and serum of chicks inoculated with or without Newcastle disease/infectious bronchitis (ND/IB) vaccine. Day-old broiler chicks were inoculated with or without ND/IB vaccine and were intraperitoneally injected with PBS or LPS 5 h before the examination at d 11 of age. IL-1B protein concentration was examined using enzyme-linked immunosorbent assay, and gene expression was analyzed using quantitative RT-PCR. See Figure 2 for the distribution of experimental chick groups V-L-, V-L+, V+L-, and V+L+. Individual values are indicated by dots. Bars represent mean ± SEM (n = 10). The asterisks indicate significant differences between the groups (*P < 0.05, **P < 0.01; Kruskal Wallis and Steel-Dwass tests).

Figure 6

Effects of Newcastle disease and infectious bronchitis (ND/IB) vaccination and lipopolysaccharide (LPS) on the IgA concentration in the proventriculus tissue and serum. Day-old broiler chicks were inoculated with or without ND/IB vaccines, followed by intraperitoneal injection with PBS or LPS 5 h before the examination at d 11 of age. IgA concentration was examined using enzyme-linked immunosorbent assay. See Figure 2 for the distribution of experimental chick groups V-L-, V-L+, V+L-, and V+L+. Individual values are indicated by dots. Bars represent mean ± SEM (n = 10).