Lipopolysaccharide induces acute bursal atrophy in broiler chicks by activating TLR4-MAPK-NF-κB/AP-1 signaling

Abstract

We investigated the mechanisms that induce atrophy of the chicken bursa of Fabricius (BF) upon lipopolysaccharide (LPS) treatment in young chicks. LPS treatment resulted in ∼36% decrease in bursal weight within 36 h (P < 0.01). Histological analysis showed infiltration of eosinophilic heterophils and nucleated oval shaped RBCs in or near blood vessels of the BF from LPS-treated chicks. Scanning electron micrographs showed severe erosion and breaks in the mucosal membrane at 12 h and complete exuviation of bursal mucosal epithelial cells at 36 h. We observed decreased cell proliferation (low PCNA positivity) and increased apoptosis (high TUNEL and ssDNA positivity) in the BF 12-72 h after LPS treatment. RNA-seq analysis of the BF transcriptome showed 736 differentially expressed genes with most expression changes (637/736) 12 h after LPS treatment. KEGG pathway analysis identified TLR4-MAPK-NF-κB/AP-1 as the key signaling pathway affected in response to LPS stimulation. These findings indicate LPS activates the TLR4-MAPK-NF-κB/AP-1 signaling pathway that mediates acute atrophy of the chicken bursa of Fabricius by inducing inflammation and apoptosis.

Keywords: Immunology; acute atrophy; bursa of Fabricius; chicken; lipopolysaccharide; transcriptional analysis.

Figure 1. Bursal weight and index in chicks treated with LPS

A. Comparison of weights of bursa of Fabricius (BF) in chicks at different time points (0-120 h) after treatment with 50 mg/kg LPS or saline. B. Comparison of bursal index in chicks at different time points (0-120 h) after treatment with 50 mg/kg LPS or saline. C. Comparison of weights of bursa of Fabricius (BF) in chicks at 36 h after treatment with different doses of LPS or saline. D. Comparison of bursal index in chicks at 36 h after treatment with different doses of LPS or saline. As shown, LPS induces acute bursal atrophy in chicks. Note: * denotes P < 0.05; ** denotes P < 0.01.

Figure 2. Analysis of bursal morphology after LPS treatment

A. Representative images of H&E stained BF tissue sections at 12, 36 and 72 h after saline (control) or LPS i.p. injection. As shown, BF specimens from LPS-treated chicks show histopathological changes compared to the control. Arrows indicate blood vessel and arrow heads represent accumulation of RBC or inflammatory cells. Note: Scale bars = 50 μm. B. Representative scanning electron micrographs of BF sections at 12, 36 and 72 h after saline (control) or LPS i.p. injection showing the ultra structure of BF mucosal surface. Bursa tissue sections in LPS stimulated group show severe erosion and breaks in mucosal surface at 12 h, complete exuviation of bursal mucosal epithelial cells at 36 h and slight restoration of the disrupted mucosal surface at 72 h. Magnification = 500x. Arrowheads represent normal, smooth and intact bursal follicles on mucosal surface in saline group while arrows indicate broken, eroded and sloughed mucosal surface in LPS stimulated group. Data represent at least five tissue sections per chick per group (n = 3 at each time point).

Figure 3. LPS treatment upregulates apoptosis and inhibits proliferation in chick BF

A. Representative images of immunostaining with anti-ssDNA monoclonal antibody of BF tissue sections at 12, 36 and 72 h after saline (control) or LPS i.p. injection. Arrows show single stranded DNA (ssDNA) staining as a dark brown product (arrows). Integral optical density (IOD) analysis showed that in comparison to the saline group, ssDNA expression was upregulated in LPS treated group, especially at 12 (IOD ratio saline vs LPS; 8801:14453.67) and 36 h (IOD ratio saline vs LPS; 10135:16781.5). B. Representative images of TUNEL assay of BF tissue sections at 12, 36 and 72 h after saline (control) or LPS i.p. injection. Scale bars = 100 μm. IOD analysis shows increased TUNEL- positive (apoptotic) cells with brown stained nuclei in BF from LPS-treated chicks, especially at 36h time point (IOD ratio saline vs LPS; 2054.67:3439.67). C. Scale bars = 50μm. Serial tissue sections of chicken bursa of Fabricius were immuno-stained with PCNA antibody (proliferation marker) at 0h, 12 h, 36 h and 72 h post saline (control) or LPS stimulation C. Representative images of immunohistochemical staining of PCNA (proliferation) in BF tissue sections at 12, 36 and 72h after saline (control) or LPS i.p. injection. Arrows show PCNA positive signals as light brown dots. PCNA expression was downregulated in LPS treatment group compared to control. Integral optical density (IOD) of PCNA expression significantly decreased at 12 (IOD ratio saline vs LPS; 6266.67:4310) and 36 h (IOD ratio saline vs LPS; 6390:3822) after LPS treatment. Scale bars = 100μm; Note: * denotes P < 0.05; ** denotes P < 0.01. Data represent microscopic examination of at least five tissue sections per chick per group (n = 3 at each time point).

Figure 4. Analysis and validation of differentially expressed genes in LPS-treated chicken bursa

A. Statistics of high-throughput sequencing reads aligned against reference genome. B. Scatter plot diagram showing log value of gene expression of LPS-treated BF samples (Y-axis) versus and log value of gene expression of saline treated BF samples (X-axis). The blue color indicates downregulated genes, orange color represents upregulated genes and brown color designates unchanged genes. Top legends on each figure show statistics of screening threshold values. C. The diagram shows total numbers of differential expressed genes (DEGs) at 12, 36 and 72h after saline or LPS treatments. D. Top 10 up-regulated and down-regulated DEGs at 12 h post saline or LPS treatments. The plus numbers designate upregulated log values while negative numbers represent downregulated log values of DEGs. E. Comparison and correlation analysis of log₂ values of differential gene expression of eight genes (AvBD2, ZNF217, BPI, MAPK9, AVD, IFNGR1, PIK3CG and DOCK10) assessed by RNA-seq and qRT-PCR experiments.

Figure 5. Gene Ontology (GO) and KEGG functional pathway analysis of DEGs in LPS-treated chicken bursa

A. Gene Ontology (GO) statistical analysis in chicken bursa of Fabricius at 12, 36 and 72 h time points of RNA seq data from saline and LPS-treated chicken bursal samples. B. Diagram showing GO terms and their designated number of DEGs at 12 h post saline or LPS treatment chicken BF samples. X-axis shows number of DEG while Y-axis represents GO terms. Three different colors designate three ontologies of GO terms; blue color represents biological process, green color indicates cellular components and the red color shows molecular functions. C. Diagram showing number of DEGs involved in KEGG enrichment analysis at 12 h post saline or LPS treatment in chicken BF samples.

Figure 6. Functional pathway analysis and graphical illustration of molecular mechanism of LPS induced bursal atrophy

A. Diagram showing TLR4-MAPK-NF-κB/AP-1 pathway analysis of differentially regulated genes in LPS-treated BF. The red colored text represents upregulated genes; blue designates downregulated genes and black designates neighboring genes in TLR4-MAPK-NF-κB/AP-1 pathway in chicken bursa of Fabricius at early time point. The figure was drawn in Pathway Builder Tool. 2 software. B. Representative images of immunostained BF sections from LPS and saline control group chicks using anti-TLR4 antibody at 12, 36 and 72 h time points. As seen, TLR4 expression is enhanced in the LPS treated BF compared to control. Integral optical density (IOD) shows increased TLR4 expression at 12 and 36 h after LPS treatment. Scale bars = 50μm. Note: * denotes P < 0.05; ** denotes P < 0.01. Data represent microscopic examination of at least five tissue sections per chick per group (n = 3 at each time point). Also shown are immunoblots showing increased TLR4 expression in LPS-treated BF compared to control. C. Representative images of immunostained BF sections from LPS and saline control group chicks using anti-NFkB-p50 antibody at 12, 36 and 72 h time points. IOD analysis shows increased NFkB expression at all 3 time points in LPS-treated BF (arrows) compared to control. Scale bars = 50μm. * denotes P < 0.05; ** denotes P < 0.01. Data represent microscopic examination of at least five tissue sections per chick per group (n = 3 at each time point). D. QRT-PCR analysis of TLR4, MyD88, NFκB, HRas, FOS and JUN-D mRNA expression at 12, 36 and 72 h post LPS treatment as validation of key components of TLR4-MAPK-NF-κB/AP-1 pathway.