The Long Pentraxin PTX3 Is of Major Importance Among Acute Phase Proteins in Chickens

Abstract

The expression level of acute phase proteins (APPs) mirrors the health status of an individual. In human medicine, C-reactive protein (CRP), and other members of the pentraxin family are of significant relevance for assessing disease severity and prognosis. In chickens, however, which represent the most common livestock species around the world, no such marker has yet gained general acceptance. The aim of this study was therefore, to characterize chicken pentraxin 3 (chPTX3) and to evaluate its applicability as a general marker for inflammatory conditions. The mammalian and chicken PTX3 proteins were predicted to be similar in sequence, domain organization and polymeric structure. Nevertheless, some characteristics like certain sequence sections, which have varied during the evolution of mammals, and species-specific glycosylation patterns, suggest distinct biological functions. ChPTX3 is constitutively expressed in various tissues but, interestingly, could not be found in splenic tissue samples without stimulation. However, upon treatment with lipopolysaccharide (LPS), PTX3 expression in chicken spleens increased to 95-fold within hours. A search for PTX3 reads in various publicly available RNA-seq data sets of chicken spleen and bursa of Fabricius also showed that PTX3 expression increases within days after experimental infection with viral and bacterial pathogens. An experimental infection with avian pathogenic E.coli and qPCR analysis of spleen samples further established a challenge dose-dependent significant up-regulation of chPTX3 in subclinically infected birds of up to over 150-fold as compared to untreated controls. Our results indicate the potential of chPTX3 as an APP marker to monitor inflammatory conditions in poultry flocks.

Keywords: LPS; acute phase proteins; avian pathogenic E. coli; chicken; inflammation; next generation sequencing; pentraxin.

Figure 1

Homology of PTX3 in various species. (A) Assessment of protein sequence similarity and identity of PTX3 amino acid sequences of various species, obtained by pairwise alignment. The highest degree of similarity is observed among PTX3 sequences of mammals. The Gallus gallus PTX3 sequence (bold) is highly similar to PTX3 of all examined species. (B) Phylogenetic analysis by Maximum Likelihood method. The tree with the highest log likelihood (−3,880.21) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Figure 2

Western blot analysis of PTX3-Flag resolved by SDS-PAGE indicates that chPTX3 has a polymeric N-glycosylated structure. (A) Purified chPTX3-Flag from cell culture supernatant under reducing conditions; shows successful expression of the recombinant protein. (B) PTX3-Flag under non-reducing (lane B1) and reducing (lane B2) conditions. ChPTX3 is constituted as a polymer. (C) Reduced chPTX3-Flag before (lane C1) and after (lane C2) N-deglycosylation. ChPTX3 contains N-glycosylated residues.

Figure 3

Mean constitutive PTX3 expression in chicken tissue samples from RNA-seq dataset PRJEB12891. RNA-seq data was downloaded from the Short Read Archieve (SRA) database for a total of 168 samples (21 tissues per bird) and blasted for PTX3 reads. Relative expression of PTX3 was assessed for each sample. Data shown represent the mean value of the respective tissue; error bars indicate standard deviation from the mean. ChPTX3 expression pattern varies prominently between tissues. The highest expression is observed in skin and kidney, the least in muscle, ovary, and spleen. Lymphoid tissue samples, except for spleen, express moderate amounts of PTX3 in chickens. N = 8.

Figure 4

Constitutive PTX3 expression in chicken lymphoid tissue samples, analyzed by RT-PCR. RT-PCR analysis was used to amplify PTX3 transcripts in various tissues. UV-visualization of a subsequent agarose gel electrophoresis is shown. The (top) represents PTX3, the (bottom) shows GAPDH expression in the same samples. RT-PCR underscores RNA-seq data analysis of chicken lymphoid tissues. NTC, Non-template control.

Figure 5

Analysis of RNA-seq data from study PRJNA340891 for PTX3 expression in spleens of LPS-treated chickens. RNA-seq data was downloaded from the Short Read Archieve (SRA) database for a total of 32 samples and blasted for PTX3 reads. Relative expression of PTX3 was assessed for each sample. Data shown represent the mean value of the respective group; error bars indicate standard deviation from the mean. PTX3 expression changes in response to a 3.5 h mock (PBS) or LPS treatment in Broiler (A) and Fayoumi chickens (B). LPS enhances PTX3 expression in spleens of Broiler chickens to 95-fold and to 3.5-fold in Fayoumi chickens. ^*p ≤ 0.05, ^**p ≤ 0.01. N = 8.

Figure 6

PTX3 expression in chicken spleen and liver after LPS treatment, as determined by qRT-PCR. Spleens and livers of a total of 12 chickens, of which 6 had received LPS intravenously 3 h before scarification, were subjected to RNA extraction. cDNA samples were analyzed using qRT-PCR. Data shown represent the mean value of the respective group; error bars indicate standard deviation from the mean. Mean 40-dCT in control (CTR) spleens is 26.6, and 35.6 in spleens of LPS-treated chickens (LPS). CTR livers display a 40-dCT of 30.2, LPS livers of 34.9. These numbers convert to an expression fold change of 508 for spleens and 26 for livers. The induction of chPTX3 in the LPS group is highly significant (^***p ≤ 0.005) for both tissues. N = 6.

Figure 7

Expression changes of PTX3 in spleens of line 7₂ chickens at 14 hpi, 3 dpi, and 7 dpi with low, intermediate or high doses of APEC O1, or mock injection, determined by qRT-PCR. Mean 40-dCT values for controls (white bars) and low (7.3 × 10⁴ CFU; light gray bars), intermediate (1.1 × 10⁶ CFU; medium gray bars), and high (8.8 × 10⁶ CFU; dark gray bars) doses were 21.6, 24.7, 27.1, and 28.9, respectively, at 14 hpi. These numbers depict a non-significant expression fold change of 9 between control and low, a very significant fold change of 46 for intermediate (^**p ≤ 0.01), and a highly significant fold change of 154 for high dose APEC O1 (^***p ≤ 0.005). Expression changes for APEC-infected birds relative to controls at 3 dpi and 7 dpi were not significant. ChPTX3 expression changes in a dose-dependent manner at 14 h post infection. Data shown represent the mean value of the respective group; error bars indicate standard deviation from the mean. N = 3 PBS controls, N = 6 APEC O1 low and intermediate dose inoculated birds, N = 5 APEC O1 high dose inoculated birds.

Figure 8

Correlation of PTX3 expression and number of viable bacteria in chicken spleens at 14 hpi after low, intermediate and high dose APEC O1 infection. PTX3 expression correlates significantly (p = 0.0313) with the amount of viable bacteria inoculated in the tissue. N = 17. CFU/g, colony forming units per gram of splenic tissue.