AbstractAmmonia plays a central role in the pathogenesis of cerebral oedema in paracetamol-induced acute liver failure (PALF). Infection and inflammation play an important synergistic role in its development. Toll-like receptors (TLRs) sense pathogens and induce inflammation but whether this contributes to the development of cerebral oedema in PALF remains unknown. I postulated that ammonia-induced cerebral oedema and immune dysfunction are mediated by TLR9 and aimed to determine whether this could be prevented in a hyperammonemic TLR9 knockout mouse model.
TLR9 expression on circulating neutrophils and their function in PALF was assessed. To examine the influence of PALF plasma and endogenous DNA on TLR9 expression, healthy neutrophils were incubated with PALF plasma with/without DNase. Ammonium acetate (NH4-Ac) was injected intraperitoneally in wild type Black6 (WT-B6), TLR9-/- B6 mice and TLR9fl/fl LysCre B6 mice with TLR9 deleted from neutrophils and macrophages. The TLR9 antagonist ODN2088 was also evaluated.
Neutrophil TLR9 correlated with plasma IL-8 and ammonia concentration and increased with severity of hepatic encephalopathy and systemic inflammation. Healthy neutrophil TLR9 expression increased upon stimulation with PALF plasma which was abrogated by pre-incubation with DNase. Following NH4-Ac stimulation, intracellular cytokine (IFN-γ, TNF-α and IL-6) production of lymphocytes and macrophages were increased in WT-B6 mice compared to controls. This was accompanied by increased brain water however in TLR9-/-, cytokine production and brain water content were decreased. This was seen similarly in WT-B6 administered the TLR9 antagonist ODN2088 in conjunction with NH4-Ac. TLR9fl/fl LysCre mice had decreased cytokine production and brain water compared to the TLR9fl/fl group following NH4-Ac injection. Total DNA levels were increased in the circulation after NH4-Ac injection.
In summary, ammonia-induced cerebral oedema and immune dysfunction are mediated through TLR9 and DNA dependent. The amelioration of brain oedema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early PALF to prevent progression to cerebral oedema.
|Date of Award||2017|
|Supervisor||Solon Pissis (Supervisor) & Costas Iliopoulos (Supervisor)|