Involvement of toll-like receptor 4 in concanavalin A-induced liver injury: Investigation on structure and permeability of intestinal wall
Mohd Hairulhisyam, Ngatiman
Mohd Hairulhisyam, Ngatiman
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Publication Date
2021-08-31
Type
Thesis
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Abstract
Concanavalin A (ConA) induces T cell-mediated liver injury in mice associated with altered microcirculation, cytokine signalling cascades, and increased intestinal permeability. Toll-like receptor 4 (TLR4) acts as a sensor for immune signals and plays a critical role in host defence. The binding of lipopolysaccharide (LPS) from the intestinal tract to TLR4 leads to liver injury. However, intestinal epithelium morphology and permeability with the liver injury remains unclear. In the current study, the involvement of TLR4 was investigated in ConA-induced liver injury, microcirculatory changes in the liver and intestinal tract and changes to gastrointestinal (GI) wall structure and function. Mice with a functional TLR4 (C3H/HeN), TLR4-defective (C3H/HeJ) mice, mice with TLR4 blockade using antibody and mice antibiotic-treated mice were administered ConA. C3H/HeN mice developed an acute liver injury with elevated plasma transaminases and necro-apoptotic hepatocytes changes. C3H/HeJ mice exhibited a delayed plasma transaminases peak and reduced necro-apoptotic hepatic changes. In C3H/HeN mice, plasma proinflammatory cytokines showed an early TNF-α peak, followed by IFN-γ and IL-6 elevation. However, C3H/HeJ mice developed early dominant IFN-γ peak, reduced TNF-α and IL-6 responses. The GI tract of C3H/HeN mice showed a compact, dense arrangement of villous projections, closely packed intervillous spacing without damaging mucosal architecture. In the C3H/HeJ mice, villous width was more prominent; however, intervillous space was smaller and closely packed. Stereological examination revealed increased early mucosal surface area per volume of ileal segments in CH3/HeJ mice. Colonic segments in CH3/HeN mice showed higher mucosal surface area at later time points. In addition, in the C3H/HeN mice, the microvilli appeared to be fused and cell membranes of adjacent cells in close apposition of tight junctions. The microvilli in the C3H/HeJ mice lacked the uniformity of height and diameter, with increased intervillous spaces at earlier time points. Tight junction surface density alteration in jejunal of C3H/HeJ was higher than in C3H/HeN mice, indicating tight junctional alteration. In the functional intestinal permeability study of C3H/HeN mice, we detected the significantly lower plasma concentration of oral fluorescein isothiocyanate-labelled dextran 4 kDa (FD4) with the only trace of mannitol and sucralose. However, we detected early jejunal sac clearance of FD4 followed by delayed ileal sac clearance in an ex vivo study in these mice. The C3H/HeJ mice showed a higher plasma concentration of oral FD4 with a non-significant trace of urine mannitol and sucralose. However, jejunal and ileal sacs exhibited delayed FD4 clearance. In the antibiotic-treated C3H/HeN mice, plasma transaminases, hepatic tissue necrotic volume, plasma TNF-α and IFN-γ, were significantly reduced following ConA. Plasma IL-6 reduction was delayed. In addition, plasma transaminases, hepatic tissue necrotic volume and plasma TNF-α were also significantly reduced in TLR4 antibody-treated mice compared to mice administered with ConA alone. An early fall in blood flow (laser Doppler) following ConA was found in the liver, jejunum, ileum, colon and spleen were observed in C3H/HeN mice. In C3H/HeJ mice, blood flow fell to a lesser extent in the liver, jejunum, ileum and colon but did not return to normal. Our results have demonstrated that LPS/TLR4 signalling is essential for necrotic liver injury secondary to intestinal wall layer disruption and increased proximal intestinal permeability following ConA administration. TLR4 is involved in the profoundly reduced GI microcirculation at the early phase of ConA administration, most likely via TNF-α in TLR4-functional mice and subsequently, IFN-γ delayed response TLR4-defective mice. LPS/TLR4 signalling plays a role in ConA-induced liver injury secondary to increased intestinal permeability.
Publisher
NUI Galway