From the AGA Journals

Blocking TLR9 may halt brain edema in acute liver failure

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Study shows interaction between immune function and brain disease

Acute liver failure is a devastating disease, which has a high mortality burden and often requires liver transplant. One of the major complications is cerebral edema that leads to encephalopathy and could be fatal. These brain changes are accompanied by inflammation, immune activation, and hyperammonemia, but further mechanistic approaches are needed.

Dr. Jasmohan S. Bajaj

The paper by Vijay et al. in this issue of Cellular and Molecular Gastroenterology and Hepatology studies the role of toll-like receptor 9 (TLR9) as a mediator of cerebral edema in a model of hyperammonemia. The authors use a novel combination of ammonium acetate and TLR9–/– mice to induce hyperammonemia while maintaining liver function, allowing direct evaluation of the receptor knockout’s effect on the subsequent development of brain edema. Further nuance is achieved by use of TLR9fl/fl mice crossed with mice expressing Cre recombinase under the control of the lysozyme promoter, generating macrophage and neutrophil conditional knockouts of TLR9. The results clearly demonstrate the absence of TLR9 prevents ammonia-induced increases in brain water, proinflammatory cytokine production, and hepatocyte swelling, which was reversed with the TLR9 antagonist ODN2088.

This data adds to the growing literature about the interaction between immune dysfunction and brain diseases such as schizophrenia, autism, depression, and multiple sclerosis. However, further studies in models of brain edema with concomitant liver failure, which are closer to the human disease process, are needed. This exciting investigation of neuroimmune regulation of brain edema could set the basis for new therapeutic options for the prevention and treatment of this feared complication of acute liver failure.

Jasmohan S. Bajaj, MD, AGAF, is professor in the division of gastroenterology, hepatology, and nutrition at Virginia Commonwealth University, Richmond. He reported no conflicts of interest.



A toll-like receptor 9 (TLR9) antagonist may eventually be used to combat brain edema in acute liver failure, according to investigators.

This prediction is based on results of a recent study involving mouse models, which showed that ODN2088, a TLR9 antagonist, could stop ammonia-induced colocalization of DNA with TLR9 in innate immune cells, thereby blocking cytokine production and ensuant brain edema, reported lead author Godhev Kumar Manakkat Vijay of King’s College London and colleagues.

“Ammonia plays a pivotal role in the development of hepatic encephalopathy and brain edema in acute liver failure,” the investigators explained in Cellular and Molecular Gastroenterology and Hepatology. “A robust systemic inflammatory response and susceptibility to developing infection are common in acute liver failure, exacerbate the development of ammonia-induced brain edema and are major prognosticators. Experimental models have unequivocally associated ammonia exposure with astrocyte swelling and brain edema, potentiated by proinflammatory cytokines.”

The investigators added that, “although the evidence base supporting the relationship between ammonia, inflammation, and brain edema is robust in acute liver failure, there is a paucity of data characterizing the specific pathogenic mechanisms entailed.” Previous research suggested that TLR9 plays a key role in acetaminophen-induced liver inflammation, they noted, and that ammonia, in combination with DNA, triggers TLR9 expression in neutrophils, which brought TLR9 into focus for the present study.

Along with wild-type mice, the investigators relied upon two knockout models: TLR9–/– mice, in which TLR9 is entirely absent, and LysM-Cre TLR9fl/fl mice, in which TLR9 is absent from lysozyme-expressing cells (predominantly neutrophils and macrophages). Comparing against controls, the investigators assessed cytokine production and brain edema in each type of mouse when intraperitoneally injected with ammonium acetate (4 mmol/kg). Specifically, 6 hours after injection, they measured intracellular cytokines in splenic macrophages, CD8+ T cells, and CD4+ T cells. In addition, they recorded total plasma DNA and brain water, a measure of brain edema.

Following ammonium acetate injection, wild-type mice developed brain edema and liver enlargement, while TLR9–/– mice and control-injected mice did not. After injection, total plasma DNA levels rose by comparable magnitudes in both wild-type mice and TLR9–/– mice, but did not change in control-injected mice, suggesting that ammonium-acetate injection was causing a release of DNA, which was binding with TLR9, resulting in activation of the innate immune system.

This hypothesis was supported by measurements of cytokines in T cells and splenic macrophages, which showed that wild-type mice had elevations of cytokines, whereas knockout mice did not. Further experiments showed that LysM-Cre TLR9fl/fl mice had similar outcomes as TLR9–/– mice, highlighting that macrophages and neutrophils are the key immune cells linking TLR9 activation with cytokine release, and therefore brain edema.

To ensure that brain edema was not directly caused by the acetate component of ammonium acetate, or acetate’s potential to increase pH, a different set of wild-type mice were injected with sodium acetate adjusted to the same pH as ammonium acetate. This had no impact on cytokine production, brain-water content, or liver-to-body weight ratio, confirming that acetate was not responsible for brain edema while providing further support for the role of TLR9.

Finally, the investigators treated wild-type mice immediately after ammonium acetate injection with the TLR9 antagonist ODN2088 (50 mcg/mouse). This treatment halted cytokine production, inflammation, and brain edema, strongly supporting the link between these ammonia-induced processes and TLR9 activation.

“These data are well supported by the findings of Imaeda et al. (J Clin Invest. 2009 Feb 2. doi: 10.1172/JCI35958), who in an acetaminophen-induced hepatotoxicity model established that inhibition of TLR9 using ODN2088 and IRS954, a TLR7/9 antagonist, down-regulated proinflammatory cytokine release and reduced mortality,” the investigators wrote. “The amelioration of brain edema and cytokine production by ODN2088 supports exploration of TLR9 antagonism as a therapeutic modality in early acute liver failure to prevent the development of brain edema and intracranial hypertension.”

The study was funded by the U.K. Institute of Liver Studies Charitable Fund and the National Institutes of Health. The investigators reported no conflicts of interest.

SOURCE: Vijay GKM et al. Cell Mol Gastroenterol Hepatol. 2019 Aug 8. doi: 10.1016/j.jcmgh.2019.08.002.

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