نوع مقاله : مقاله پژوهشی
عنوان مقاله English
نویسندگان English
Introduction: The growing prevalence of antibiotic resistance in the poultry industry has created a critical demand for safe, effective, and natural alternatives to conventional antimicrobials. Antimicrobial peptides (AMPs) are key components of the innate immune system, functioning as the first line of defense against invading pathogens. These molecules possess broad-spectrum activity against bacteria, viruses, and fungi, while also modulating immune responses. Consequently, they are considered promising alternatives to antibiotics in poultry production. Understanding the transcriptional regulation of AMPs in response to immune challenges is essential for evaluating their potential application. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, is widely used as an immune stimulant because it activates inflammatory pathways via pattern recognition receptors (PRRs). Studying LPS-induced immune responses provides valuable insights into host defense mechanisms even in the absence of active infection. This study aimed to investigate the transcriptomic response of the liver of broiler chickens (Gallus gallus) to LPS stimulation, with a particular focus on the expression of AMP-related genes. By identifying differentially expressed genes (DEGs), enriched pathways, and regulatory factors, we sought to provide a comprehensive overview of how AMPs are transcriptionally and post-transcriptionally modulated under inflammatory stress.
Materials and Methods: RNA-seq data were obtained from an experiment in which broiler chickens were injected intraperitoneally with 1 mg/kg of LPS derived from Escherichia coli. Control animals received no treatment. Liver samples were collected 24 h post-injection to capture acute immune responses. Total RNA was extracted and sequenced using Illumina sequencing platform, producing high-quality paired-end reads. The raw reads underwent quality control (QC) steps to remove low-quality bases and adapters. Clean reads were then aligned to the Gallus gallus reference genome (version Galgal7) to ensure accurate transcript alignment. Gene expression levels were quantified, and differential expression analysis was performed between LPS-treated and control groups. DEGs were identified using adjusted p-values and |log2 fold change| thresholds. Functional enrichment analysis was conducted using Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Additionally, transcription factors (TFs) and microRNAs (miRNAs) predicted to regulate DEGs were analyzed to reveal upstream and post-transcriptional regulatory mechanisms.
Results and Discussion: Transcriptomic profiling revealed a total of 2,949 DEGs in broiler chicken liver following LPS stimulation, including 1,552 upregulated and 1,397 downregulated genes. This extensive transcriptional reprogramming highlights the strong impact of LPS on the hepatic immune landscape. Among the most notable findings was the upregulation of AMP‑related genes such as AvBD13, AvBD9, AvBD8, and CATH3. These peptides, belonging to the avian β-defensin and cathelicidin families, are crucial for direct antimicrobial defense and immune modulation. Their induction demonstrates that LPS alone, without the presence of live pathogens, is sufficient to trigger innate host defense responses. Enrichment analyses identified several innate immune signaling pathways as significantly enriched, including the Toll-like receptor (TLR), NOD-like receptor (NLR), and MAPK signaling cascades. These pathways are central to pathogen recognition and cytokine-mediated immune responses. Activation of the TLR4 pathway reflects canonical LPS sensing by hepatocytes and immune cells in the liver. Further investigation revealed that transcription factors such as IRF1, NF-κB, and ICSBP play central roles in orchestrating this immune response. These TFs regulate downstream AMP expression and cytokine production, thereby bridging pathogen recognition with effector functions. In parallel, miRNAs, including gga-miR-130a-3p and gga-miR-16-5p, were predicted to modulate post-transcriptional regulation of immune-related genes. This highlights the layered regulatory mechanisms by which chickens fine-tune their immune responses. The observed transcriptomic signature of LPS-stimulated livers closely resembles the immune activation patterns associated with necrotic enteritis, a major poultry disease caused by Clostridium perfringens. This suggests that the LPS model is a valuable experimental system to study inflammatory responses relevant to disease conditions, even in the absence of infection. Importantly, the activation of AMPs under these conditions reinforces their potential as natural alternatives to antibiotics for maintaining poultry health and reducing reliance on conventional antimicrobials.
Conclusion: This study demonstrates that intraperitoneal LPS challenge induces extensive transcriptional reprogramming in the liver of broiler chickens. AMP‑related genes, including AvBD13, AvBD9, AvBD8, and CATH3, were significantly upregulated, reflecting their role in innate immune defense. Key signaling pathways such as TLR, NLR, and MAPK were activated, with transcription factors IRF1, NF-κB, and ICSBP identified as master regulators. Moreover, miRNAs such as gga-miR-130a-3p and gga-miR-16-5p were implicated in fine-tuning these immune responses. Together, these results underscore the importance of AMPs in poultry immunity and highlight LPS stimulation as an effective experimental model for studying host defense mechanisms. The findings provide valuable insights for the development of AMP‑based strategies as sustainable alternatives to antibiotics in the poultry industry.
کلیدواژهها English
Authors retain the copyright. This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0)
