Lipopolysaccharide mediates immuno-pathological alterations in young chicken liver through TLR4 signaling

Abstract

Background: Lipopolysaccharide (LPS) induces acute liver injury and the complex mechanisms include the activation of toll like receptor 4 (TLR4) signaling pathway in many species. However, immuno-pathological changes during TLR4 signaling under LPS stress in acute liver injury is poorly understood in avian species. The present investigation was therefore carried out to evaluate these alterations in TLR4 signaling pathway during acute liver injury in young chickens.

Results: After intraperitoneal injection of LPS or saline, liver samples were harvested at 0, 2, 6, 12, 24, 36, 72 and 120 h (n = 6 at each time point) and the microstructures were analyzed by hematoxylin and eosin (H&E) staining. Alanine aminotransferase (ALT) and caspase-3 enzyme activity was assessed by enzyme-linked immunosorbent assay (ELISA). Proliferative cell nuclear antigen (PCNA), single stranded DNA (ssDNA) and TLR4 protein expressions were determined by immunohistochemistry. Gene expressions of PCNA, caspase-3, caspase-8, TLR4 and its downstream molecules were analyzed by quantitative polymerase chain reaction (qPCR). LPS injection induced significantly higher ALT activity, severe fatty degeneration, necrotic symptoms, ballooning degeneration, congestion, enhanced inflammatory cell infiltration in liver sinusoids, decreased proliferation, increased apoptosis and significant up-regulation in TLR4 and its downstream molecules (MyD88, NF-κB, TNF-α, IL-1β and TGF-β) expression at different time points.

Conclusions: This study indicated that TLR4 signaling and its downstream molecules along with certain cytokines play a key role in acute liver injury in young chickens. Hence, our findings provided novel information about the histopathological, proliferative and apoptotic alterations along with changes in ALT and caspase-3 activities associated with acute liver injury induced by Salmonella LPS in avian species.

Keywords: Acute injury; Chicken; Lipopolysaccharide; Liver; Toll-like receptor 4.

Fig. 1

Effect of lipopolysaccharide on histomorphology and ALT activity and in chicken liver. Following intraperitoneal LPS treatment in chickens at different time points, H&E staining was performed on liver serial tissue sections. Stellate macrophages (Kupffer cells) in perisinusoidal areas ①, diffuse infiltration of fat vacuoles indicating fatty infiltration ②, dilated central vein ③ and sinusoidal capillaries ④, reduction in size of a few hepatocytes ⑤, dissociated liver cells from each other in hepatic cords ⑥, dilated hepatic sinusoids along with fibrocytes proliferation in perisinusoidal areas ⑦, intracytoplasmic infiltration of variable size and shape fat vacuoles ⑧, dilated hepatic sinusoids ⑨, infiltration of oval shaped nucleated RBCs ⑩, cytoplasmic fat vacuoles have pushed hepatocyte nuclei at periphery ⑪, reduction in size of a few hepatocytes ⑫ and intense inflammatory cells infiltration around the portal area ⑬ (a). After LPS stimulation, alanine aminotransferase (ALT) activity was measured from liver tissues at 0 h, 2 h, 6 h, 12 h, 24 h, 36 h, 72 h and 120 h by ELISA technique (b). The letter C represents saline (control) group and L represents LPS group. The numbers represent the hours after stimulation. **P < 0.01

Fig. 2

Effect of LPS stimulation on hepatic cell proliferation in chicken liver. After intraperitoneal LPS injection in chicks at different time points, PCNA protein expression was assessed in liver tissue by immunohistochemistry using anti-PCNA antibody, PCNA positive product was mainly distributed around the portal and biliary epithelial cells and more concentrated expression was present on epithelial cell near portal area in saline group at 6 h, 12 h, 24 h, and 72 h as compared to LPS group (a). Quantification of PCNA expression from liver tissue images was accomplished by image-pro plus (IPP) computer software where IOD represents integrated optical density (b). The analysis of PCNA gene expression was performed by real-time quantitative RT-PCR and normalized by the expression of actin beta (ACTB) (c). The letter C represents saline (control) group and L represents LPS group. The numbers represent the hours after stimulation. *P < 0.05, **P < 0.01

Fig. 3

Effect of LPS stimulation on hepatocyte apoptosis in chicken liver. Following intraperitoneal LPS injection in chicks at different time points, single stranded DNA (ssDNA) protein expression was assessed in liver tissues by immunohistochemistry using anti-ssDNA antibody, ssDNA positive product was extensively distributed in biliary epithelial cells and hepatic sinosoidal endothelial cells in LPS group at 6 h, 12 h, 24 h, and 72 h as compared to PBS (saline) group (a). Quantification of ssDNA expression from liver tissue images was accomplished by image-pro plus (IPP) computer software where IOD represents integrated optical density (b). The activity of caspase-3 enzyme was measured by ELIZA technique and the expressions of caspase-3 and caspase-8 genes were also determined by quantitative RT-PCR and normalized by the expression of actin beta (ACTB) (c). The letter C represents saline (control) group and L represents LPS group. The numbers represent the hours after stimulation. *P < 0.05, **P < 0.01

Fig. 4

Effect of LPS stimulation on TLR4 expression in chicken liver. After intraperitoneal LPS injection in chicks at different time points, TLR4 protein expression was assessed in liver tissue by immunohistochemistry using anti-TLR4 antibody, TLR4 positive product was mainly distributed on hepatocytes. In LPS group, strong TLR4 expression was present at 6 h, 12 h, 24 h, and 72 h as compared to saline group (a). Quantification of TLR4 expression from liver tissue images was accomplished by image-pro plus (IPP) computer software where IOD represents integrated optical density (b). The analysis of TLR4 gene expression was performed by quantitative RT-PCR and normalized by the expression of actin beta (ACTB) (c). The letter C represents saline (control) group and L represents LPS group. The numbers represent the hours after stimulation. *P < 0.05, **P < 0.01

Fig. 5

Effect of LPS stimulation on downstream molecules of TLR4 signaling and cytokines in chicken liver. Following intraperitoneal LPS stimulation in chicks at 0 h, 2 h, 6 h, 12 h, 24 h, 36 h, 72 h and 120 h, the expressions of MyD88, NF-κB, TNF-α, TGF-β and IL-1β genes were determined by real-time quantitative PCR (qRT-PCR) and normalized by the expression of actin beta (ACTB). The letter C represents saline (control) group and L represents LPS group. The numbers represent the hours after stimulation. *P < 0.05, **P < 0.01