Introduction There is a marked propensity for patients with acute liver failure (ALF) and cirrhosis to develop sepsis and inflammation which may hasten the development of hepatic encephalopathy (HE) and cerebral oedema. Neutrophil dysfunction is an important biomarker of poor prognosis in liver failure and neutrophil TLR9 expression upregulates with ammonia exposure. There is a paucity of understanding regarding the relationship between neutrophil dysfunction and the development of HE, moreover there is a lack of predictive/prognostic biomarkers that differentiate ALF patients that will go on to develop HE. The aim of this study was to investigate the relationship between neutrophil TLR9 and development of HE.
Methods In healthy controls (n=12) and patients with ALF (n=12) and cirrhosis (n=50) we investigated neutrophil TLR9 expression using fluorochrome-conjugated monoclonal antibodies [CD16 (PE), CD11b (APC-Cy7) and TLR9 (APC) (after cytofixation/permeabilisation)] by flow cytometry in determining responses to endotoxin and bacterial challenge at baseline, and following 2 h stimulation with lipopolysaccharide (LPS) and ammonia. Pro- and anti-inflammatory cytokines were determined by CBA.
Results Baseline neutrophil TLR9 expression was significantly higher in patients with HE (ALF: Grade 3/4 vs controls: p<0.02, vs grade 0–2: p<0.03) (Cirrhotics: Grade 3/4 vs controls: p<0.03, vs grade 0–2: p<0.05). Moreover their baseline TLR9 expression was associated with severity of HE and higher IL6 and IL8 levels. CD16 expression was downregulated by a median of 45% (range 25%–85%) in ALF patients with grade 3/4 HE compared to controls and in cirrhotics by a median of 88% (range 5%–90%) (Grade 3/4 vs controls: p<0.05). Baseline CD11b expression did not differ between controls and patients. Exposure to LPS and ammonia upregulated TLR9 and CD11b and downregulated CD16.
Conclusion Neutrophil TLR9 expression in patients with ALF and cirrhosis serves as a useful biomarker that differentiates those who develop high grade HE from those who do not. High baseline TLR9 expression and low CD16 expression promote a pro-inflammatory cytokine milieu that may help to explain the propensity to develop infection and why inflammation hastens the development of HE. TLR9 antagonists may be of therapeutic value in restoring neutrophil activity.
Competing interests None declared.
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