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

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.


INTRODUCTION
The bursa of Fabricius (BF) is an avian specific immune organ.It has a specialized microenvironment that supports the differentiation of B cells and production of antibodies [1,2].Acute atrophy of BF is observed during various infectious diseases including bursal disease virus [3,4], Marek's disease virus [5,6], Fowl adenoviruses [7], Escherichia coli, fowl typhoid and Salmonella [8][9][10].Salmonella enterica serovar Typhimurium (STm) is a gram-negative facultative anaerobic bacterium that causes serious clinical disease in newly hatched chicks [11], which is characterized by severe diarrhea, dehydration, and increased mortality [12].STm infection results in immune suppression, cytokine imbalance and disruption of lymphoid tissue architecture [13].The JNK signaling pathway plays a critical role during STm-induced thymic injury in mice [14,15].Although BF plays a significant role in STm infection in poultry [16], only few scientific reports have described the transcriptional changes in BF following STm infection in avian species.
Since the acquired immune system in the neonatal chicken is not completely functional until the first week, the innate immune system plays defensive crucial role for the chicks [17].Bursa is important in the innate immunity of neonatal chicken and < 0.1% BF to body weight indicates infection in broilers [18,19].TLRs are key mediators of innate immune responses to various infections [20].For gram negative bacteria, the endotoxin LPS is recognized by TLR2 and TLR4 [21,22].LPS Research Paper: Immunology www.impactjournals.com/oncotargetstimulation disrupts the histo-morphological organization of different organs including BF of chicken [23,24].Reduced body and bursa weight are also characteristic of salmonella LPS stimulated chickens [25].NF-κB and AP-1 are two key down-stream transcriptional factors of the TLR4 signaling pathways [26], which induce various inflammatory factors including TNF-α, IL-6, IL-8, IL-1b [26,27].Furthermore, chronic inflammation results in apoptosis of immune cells [28].Inflammation in response to LPS results in induction of pro-apoptotic factors including nitric oxide, TNF-α and glucocorticoids [29,30].Although studies have described the immune response and atrophy of immune organs mediated by infectious diseases in model organisms like mice, rats as well as humans, the molecular mechanism of bursal atrophy is still largely unknown.Therefore, in the current study, we investigated the molecular mechanisms involved in acute bursal atrophy in young chickens induced by Salmonella LPS.

Increased apoptosis and decreased proliferation in bursa after LPS treatment
In-situ cell apoptosis in the bursa was analyzed by the formamide-MAb assay which recognizes damaged single-strand DNA (ssDNA) in early apoptotic cells.We observed increased ssDNA in bursa from LPS treatment group compared to the control (Figure 3A).Integral optical density (IOD) of ssDNA expression in LPS treatment group was increased at 12 (P < 0.05) and 36 h (P < 0.01) (Figure 3A).TUNEL assay showed that bursal sections from LPS treatment group had increased number of TUNEL-positive cells compared to control.High IOD value at 36h confirmed increased apoptotic cells in bursa from LPS-treated chicks compared to control (Figure 3B).Further, immunohistochemical analysis showed low PCNA expression in LPS group compared to the control group at 12 and 36 h (P < 0.05) suggesting decreased cell proliferation upon LPS treatment (Figure 3C).

Identification of differential expressed genes (DEGs) in bursa after LPS stimulation
The RNA-seq statistics pertaining to the average total reads, mapping reads and unique matching for each of the 18 samples are shown in Figure 4A.The differentially expressed genes (DEGs) between LPS and control (saline) group were analyzed by Random-Sampling Empirical Myerson (RSEM) software.Overall, there were 736 DEGs with 417 downregulated and 319 upregulated genes at all the three time points (12, 36 and 72 h; Figure 4B).Among these, we observed 637 DEGs (387 downregulated and 250 upregulated) at 12 h (Figure 4C), 24 DEGs (12 downregulated and 12 upregulated) at 36h and 75 DEGs (18 downregulated and 57 upregulated) at 72 h (Supplementary Table 1).Maximum DEGs were observed at 12 h time point.The top ten upregulated and downregulated DEGs at 12h are shown in Figure 4D.To validate the RNA-seq data, eight genes (AvBD2, ZNF217, BPI, MAPK9/JNK, AVD, IFNGR, PIK3CG, DOCK10) were randomly selected at 12h for qRT-PCR analysis, which confirmed the RNA-seq findings.Moreover, there was high correlation between qRT-PCR and RNA-seq data (r = 0.954; Figure 4E).

Bioinformatics analysis of DEGs
GO annotation of DEGs was performed at all the time points (12 h, 36 h and 72 h).All the DEGs were enriched in all three major GO classes, namely, biological process, cellular component and molecular function.
Moreover, most DEGs were enriched in GO terms at 12 h.At 12 h, 992 genes were annotated in biological processes, 631 genes in cellular components and 257 genes in molecular function (Figure 5A).The top three enriched biological processes were cellular (139 genes), metabolic (120 genes) and single-organism (119 genes) processes (Figure 5B).The top three enriched cellular components were cell (141 genes), cell part (141 genes) and organelle part (108 genes) (Figure 5B).The top three enriched molecular function categories were binding (130 genes), catalytic activity (76 genes) and structural molecule activity (13 genes) (Figure 5B).KEGG enrichment analysis of DEGs at 12 h showed enrichment of 135 DEGs in cellular processes, 118 in environmental information system, 84 in genetic information processing, 324 human diseases, 158 metabolism and 213 organismal system related to the signaling pathways (Figure 5C).The details of these DEGs in each signaling pathway are listed in Supplementary Tables 1 and 2.

DISCUSSION
Acute atrophy of bursa is observed in various infectious diseases including Salmonella infection.In the present study, we found that LPS stimulation markedly reduced bursal weight and index.Also, the structural integrity of bursa was disrupted due to inflammation.We also identified that TLR4-MAPK-NF-κB/AP-1 signaling pathway played an important role in acute atrophy of bursa induced by LPS.
In this study, we observed morphological manifestation of atrophy at 36 h after LPS treatment.However, in regards to gene expression changes, the most significant time point was 12 h.Therefore, the molecular response to the LPS stimulation was earlier than the appearance of the morphological atrophy phenotype.In our previous study, we found thymus atrophy at 36 h and gene expression changes at 12 h after LPS treatment [14,15].Therefore, both bursa and thymus show similar phenotype changes in response to LPS treatment.In addition, there were no significant changes in bursal weight between LPS treatment and control group at 72 h and 120 h suggesting compensatory growth after atrophy in the LPS treatment group.SEM results showed that the structure of bursa was disrupted at 12 h and damaged considerably at 36 h after LPS treatment.However, it recovered at 72 h, but was not completely restored at 120 h.In a previous study, reduction in feeding efficiency and growth performance along with compensatory growth mechanism was observed in broiler chicks following LPS challenge [31].Therefore, although the weight of bursa was similar to the control group at 72 h, its function was not fully restored.
We also observed increased apoptosis and decreased proliferation at 12 and 36 h after LPS treatment, which resulted in the bursal atrophy.Many studies have confirmed that inflammation induces cellular apoptosis [32][33][34].In our previous study in chicken, inflammation induced apoptosis in thymus at 12, 36 and 72 h after LPS treatment [14,15].In the present study, we observed infiltration of eosinophilic heterophils and nucleated oval shaped RBCs in or near blood vessels in chicken bursa at 12, 36 and 72 h time points.Enhanced heterophilic granules accumulated in the medulla of bursal follicle at 36 h post LPS stimulation.Moreover, RNA-seq results showed upregulation of a number of inflammatory factors after LPS treatment.All these results indicated that inflammation due to LPS resulted in cellular apoptosis lead to bursal atrophy.
The RNA-seq data showed that many DEGs were significantly enriched in immune response pathways.Also, integrated pathway analysis revealed that TLR4-MAPK-NF-κB/AP-1 was the major pathway responsible for atrophy of bursa induced by LPS.The expression of major genes in this signaling pathway, including TLR4, MAPK9, NF-κB and Fos/Jun (two subunits of AP-1) were significantly up-regulated after LPS treatment.Previous studies have confirmed that TLR4 is the receptor of LPS [35][36][37].TLR4 is required for inflammatory responses after intravenous LPS injection [38].MAPK9 is induced and activated by TLR4 with mediation by PI3K/AKT phosphorylated kinases [39,40].
In conclusion, our study demonstrates that LPS treatment induces TLR4-MAPK-NF-κB/AP-1 signaling pathway resulting in increased inflammation and apoptosis in the bursa, thereby leading to its atrophy.

Ethics statement
The present study was approved by the Animal Care and Use Committee of Huazhong Agricultural University (HZAU), Wuhan, China.

Experimental design and lipopolysaccharide (LPS) stimulation
Healthy one-day-old Cobb strain broiler chicks were obtained from a local breeding company (Zhengda, Wuhan, China).The chicks were reared under a conventional housing environment similar to commercial broiler husbandry conditions.Water and feed were provided ad libitum without any medication and vaccination.In a separate study, the effect of different doses of LPS (i.e., 12.5, 25 and 50 mg/kg b.w.) on bursa weight was determined.In the present study, the chicks were intraperitoneally (i.p.) injected with 50mg/kg salmonella LPS (L7261; Sigma-Aldrich, St. Louis, MO, USA) in 0.5ml saline (0.75 % NaCl solution); control chicks received only 0.5ml saline i.p. www.impactjournals.com/oncotarget

Western blot analysis
Western blotting was performed following previously described methods [51].Briefly, the frozen specimens were powdered in liquid nitrogen and homogenized in lysis buffer with a protease inhibitor enzyme.The supernatants were vortexed, incubated on ice and centrifuged at 12,000 × g for 5 min.The protein concentrations was measured using BCA protein quantification kit (Vazyme™ biotyech.co., ltd.).Equal amounts of total proteins (40 μg) were subjected to 10% SDS-PAGE (30 min at 80 volts and after that 60 min at 100 volts).Then, the separated proteins were transferred onto a PVDF membrane (Merck Millipore, USA).The membranes were incubated against rabbit anti-TLR4 (A00017-Boster, 1:400) and anti-human β-actin (sc-47778-Santa Cruz, 1:1000) antibodies for 12 h and after washing in 1X TBST buffer thrice, were incubated with peroxidase conjugated secondary antibody (1:3000) for 30 min (Boster, China).The blots were developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and visualized using ChemiDoc-It™ Imaging System.

Quantitative real-time PCR (qRT-PCR)
Total RNA from bursal tissue samples was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer`s instructions.The genomic DNA (gDNA) was removed by treating RNA with RNase-free DNase I (Fermentas, Opelstrasse, Germany).RevertAid First Strand cDNA Synthesis Kit (Fermentas, Opelstrasse, Germany) was used to synthesize of first strand cDNA.For qPCR, 10μl total reaction mixture was comprised of 5μl SYBR Select Master Mix for CFX (Applied Biosystems), 2μl of each forward and reverse primer and 1μl of template cDNA.The qPCR reactions were performed on a Bio-Rad CFX Connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA).The qPCR conditions were as follows: pre-denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 20 s.All samples were run in triplicate and relative gene expression levels were quantified using the ΔΔCt method using β-actin as control.Primers sequences for qPCR are given in Table 3.

RNA-seq and data analysis
TRIzol (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from BF samples and the quality of RNA was assessed using Nanodrop spectrophotometer (Thermo, Waltham, MA, USA) and by gel electrophoresis.For high-throughput sequencing (RNA-seq), 18 BF RNA samples (three time points at 12, 36 and 72 h; three biological replicates at each time point and two treatments,namely, saline and LPS) were selected in equal quantities.Briefly, after construction of strand-specific cDNA libraries, sequencing was performed on Illumina HiSeq 4000 platform by BGI Co., Ltd.(Shenzhen, China).The original raw data was saved in FASTQ/FQ file format.Bowtie2 was used for mapping of clean reads to reference gene and HISAT to reference chicken genome (release: Gallus gallus 5.0).The gene expression was quantified by FPKM (fragments per kilobase per million) method using RSEM (Random-Sampling Empirical Myerson) quantification tool [52].NOISeq method was used to screen differentially expression genes (DEGs) between two groups (saline vs. LPS) at 12, 36 and 72 h by using absolute fold change ≥ 2 and divergent probability ≥ 0.8 [53].
For the purpose of functional classification of genes, Gene Ontology (GO) database that offers an up to date database for terminology and comprehensive illustration of the characteristics of genes and gene products in a given organism species was used [54].DEGs were annotated on the basis of three ontologies, namely, biological process, molecular function and cellular component.Similarly, Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg) was used to discover the link between differential genes and various KEGG pathways and to determine functional pathway analysis [55].

Statistical analysis
GraphPad Prism version 5.0 was used for statistical analyses.The data is shown as mean ± standard deviation (SD).Independent-samples t test was applied to calculate significant differences between groups in the same tissue regions.Bonferroni's multiple comparisons test after one-way ANOVA test was applied to calculate statistical significance among multiple sample sets versus control samples.Differences were considered significant if P < 0.05 i.e., *P < 0.05, **P < 0.01 and ***P < 0.001.

Availability of supporting data
All the relevant datasets supporting the findings of the current study have been provided in the article and its supporting information.The raw sequence reads (RNAseq data) associated with this study have been submitted to the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA) under accession code SRP093225.

Figure 1 :
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 :
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 :
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 :
Figure 4: Analysis and validation of differentially expressed genes in LPS-treated chicken bursa.A. Statistics of highthroughput sequencing reads aligned against reference genome.B. Scatter plot diagram showing log value of gene expression of LPStreated 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 log 2 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 :
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 LPStreated 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 :
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.