Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9

Abstract

Background & aim: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model.

Methods: Ammonium acetate (NH₄-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9^-/- and Lysm-Cre Tlr9^fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH₄-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4⁺ and CD8⁺ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50μg/mouse) was evaluated.

Results: Following NH₄-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9^-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH₄-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9^fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9^-/- mice. Following NH₄-Ac injection, plasma tDNA levels increased in WT and Tlr9^-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells.

Conclusion: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.

Keywords: Ammonia; Brain edema; Immune dysfunction; Toll-like receptor 9.