- Email: [email protected]
The quest for the elusive factors that underpin neutrophil dysfunction in cirrhosis goes on
Letters to the Editor [4] Müller T, Buch S, Berg T, Hampe J, Stickel F. Distinct, alcohol-modulated effects of PNPLA3 genotype on progression of chronic hepatitis C. J Hepatol 2011;55:732–733. [5] Toyoda H, Kumada T. Favorable association between genetic polymorphisms near the IL28B gene and hepatic steatosis: direct or indirect? J Hepatol 2012;56:738–739. [6] Tillmann HL, Patel K, Muir AJ, Guy CD, Li JH, Lao XQ, et al. Beneficial IL28B genotype associated with lower frequency of hepatic steatosis in patients with chronic hepatitis C. J Hepatol 2011 [Epub ahead of print]. [7] Speliotes EK, Butler JL, Palmer CD, Voight BF, GIANT Consortium, MIGen Consortium, et al. PNPLA3 variants specifically confer increased risk for histologic nonalcoholic fatty liver disease but not metabolic disease. Hepatology 2010;52:904–912.
Pierre Pradat Department of Hepatogastroenterology, Hotel-Dieu, Lyon, France Jeanette J. McCarthy Institute for Genome Sciences and Policy, Duke University Medical Center, Durham, NC, USA ⇑
Hans L. Tillmann Duke Clinical Research Institute and Division of Gastroenterology, Duke University Medical Center, Durham, NC, USA ⇑ Tel.: +1 919 668 780; fax: +1 919 668 7164 E-mail address: [email protected]
Eric Trépo Department of Gastroenterology and Hepatopancreatology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium
The quest for the elusive factors that underpin neutrophil dysfunction in cirrhosis goes on To the Editor: We congratulate Tritto and colleagues on their recently published study [1], which aimed at extending our understanding of why patients with cirrhosis paradoxically exhibit features of impaired bacteriocidal capacity despite maintaining the ability to generate inflammation and bystander tissue damage. The neutrophil is a key component of the innate immune system and has been shown to play a vital role in the development of systemic inflammation and organ failure in sepsis [2]. The evidence base supporting neutrophil malfunction in acute and chronic liver disease continues to grow although factors that underpin this dysfunction are less clear and are likely to be related to a number of different factors [3–6]. In their study, Tritto and colleagues explore how the exposure of normal neutrophils to plasma from patients with varying severity and aetiology of liver disease in vitro may impact on their function. Furthermore, they aimed at investigating what role the presence of bacterial DNA, endotoxin and circulating cytokines within that plasma might play on neutrophil function and Tolllike receptor (TLR) expression. Lastly, they used a novel aseptic inflammation technique (blister study) to assess how neutrophils behave in vivo in 8 patients with Child Pugh A/B alcohol-related cirrhosis. The resulting data are both important and insightful but we would urge that these data need to be interpreted with caution at this stage due to several inherent flaws that exist in the study methodology. Firstly, further clarification on the control data is warranted. In the in vitro part of the study, the authors state that age- and sex-matched healthy volunteers were used as normal controls. No further details are given as to the control demographics or number of controls used, which appear to vary significantly in number between several figures in the manuscript. This is critical as the range of phagocytic capacity found in the healthy controls is vast (50,000–225,000 geometric mean of fluorescence intensity [GMFI]), suggesting the neutrophil function within the control group was highly variable and making valid statistical comparisons hard to achieve. A number of factors can influence neutrophil function in ‘healthy’ individuals including ethnicity, alcohol, tobacco habits and exercise within the previous 24 h which need to be controlled for. This is also fundamental when 1212
interpreting the blister studies, as 3 out of the 5 healthy controls had reduced phagocytic capacity of a similar magnitude to the cirrhotic patients and 2 patients stayed the same. This is somewhat at odds with the conclusion that the cirrhotic patients have impaired neutrophil phagocytic capacity in the blister fluid compared to controls. Moreover, there was a huge variation in GMFI in the cirrhotic patients (range 25,000–225,000). Four of the eight patients with cirrhosis also had evidence of impaired phagocytic capacity in peripheral blood at baseline, clouding the argument that stable cirrhotic patients did not have impairment of phagocytic capacity in peripheral blood but did in the inflammatory environment. Secondly, with regard to the TLR data, the baseline expression of TLR-2, 4 and 9 is not stated prior to exposure to cirrhotic plasma. This makes it difficult to interpret the impact of the cirrhotic plasma on the normal neutrophils. It might also have been better to have used healthy control plasma rather than phosphate-buffered saline which might have significantly influenced the neutrophil viability and the rate of apoptosis. It would also be interesting to determine whether the presence or absence of ascites had any relationship with baseline TLR-4 expression, which might be expected if this cohort has an increased rate of gut bacterial translocation. Lastly, we would be keen to learn more about the cohort (n = 34) with high oxidative burst. Did a large proportion of these patients have alcohol-related disease, ascites or encephalopathy? It is also fascinating to see that these patients had increased levels of IL-12, IFN-gamma and TNF-alpha. IL-12 and IFN-gamma are not cytokines that are typically associated with neutrophil activation and may have been released by other immune cell subtypes such as dendritic cells, macrophages and natural killer cells, which will undoubtedly have a role to play. In an ongoing prospective longitudinal cohort study of neutrophil morphology, phenotype and function at King’s College Hospital in over 125 patients with cirrhosis, we have shown that phagocytic dysfunction is universal in patients with cirrhosis (including those with Child Pugh A disease), which is reversible following liver transplantation [7]. Neutrophil dysfunction is likely to be determined by a large number of variables including plasma complement, immunoglobulin, sodium
Journal of Hepatology 2012 vol. 56 j 1207–1217
JOURNAL OF HEPATOLOGY and ammonia concentrations [8], the presence of hepatic encephalopathy [9], the presence of ascites and the pro- and anti-inflammatory cytokine milieu [10] which warrant further investigation. The quest for the elusive factor(s) that underpin neutrophil function therefore continues.
Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support Dr. Shawcross is funded by a 5 year Department of Health HEFCE Clinical Senior Lectureship. References [1] Tritto G, Bechlis Z, Stadlbauer V, Davies N, Frances R, Shah N, et al. Evidence of neutrophil functional defect despite inflammation in stable cirrhosis. J Hepatol 2011;55:574–581. [2] Brown KA, Brain SD, Pearson JD, Edgeworth JD, Lewis SM, Treacher DF. Neutrophils in development of multiple organ failure in sepsis. Lancet 2006;368:157–169. [3] Altin M, Rajkovic IA, Hughes RD, Williams R. Neutrophil adherence in chronic liver disease and fulminant hepatic failure. Gut 1983;24:746–750.
[4] Rajkovic IA, Williams R. Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis. Hepatology 1986;6:252–262. [5] Wyke RJ, Yousif-Kadaru AG, Rajkovic IA, Eddleston AL, Williams R. Serum stimulatory activity and polymorphonuclear leucocyte movement in patients with fulminant hepatic failure. Clin Exp Immunol 1982;50:442–449. [6] Mookerjee R, Stadlbauer V, Lidder S, Wright G, Hodges S, Davies N, et al. Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts outcome. Hepatology 2007;46:831–840. [7] Taylor N, Ma Y, Abeles R, Hussein M, Auzinger G, Vergani D, et al. Cirrhosis leads to impaired neutrophil phagocytosis and spontaneous oxidative burst which correlates with increasing IL-10 concentration and is reversed following liver transplantation. Hepatology 2009;50:121A, [Abstract]. [8] Shawcross D, Wright G, Stadlbauer V, Hodges S, Wheeler-Jones C, Pitsillides A, et al. Ammonia impairs neutrophil phagocytic function in liver disease. Hepatology 2008;48:1202–1212. [9] Shawcross DL, Shabbir SS, Taylor NJ, Hughes RD. Ammonia and the neutrophil in the pathogenesis of hepatic encephalopathy in cirrhosis. Hepatology 2010;51:1062–1069. [10] Taylor N, Abeles R, Ma Y, Wendon J, Shawcross D. A compensatory antiinflammatory response syndrome (CARS) triggered by neutrophil-induced oxidative stress is associated with chronic low grade hepatic encephalopathy in patients with advanced cirrhosis. J Hepatol 2010;52:S216, [Abstract].
Godhev K. Manakkat Vijay Nicholas J. Taylor ⇑ Debbie Lindsay Shawcross Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London SE5 9RS, UK ⇑ Tel.: +44 020 3299 3216; fax: +44 020 3299 3167 E-mail address: [email protected] (D.L. Shawcross)
Reply to: ‘‘The quest for the elusive factors that underpin neutrophil dysfunction in cirrhosis goes on’’ To the Editor: We thank Vijay and colleagues for their appreciation and interest in our paper evaluating some features of neutrophil dysfunction in cirrhosis of different severity [1]. They raise three points which are addressed below: 1. The first issue raised was over concerns for the high variability of phagocytic capacity in the group of healthy controls and the numerous factors influencing neutrophil function, which may hinder the statistical evaluation of differences. We used plasma from 21 healthy controls who were non-smokers and had not drunk alcohol in the previous 48 h. For cytokine assays we used 10 healthy controls. With regards to variability, we would suggest that it was even higher in the cirrhosis group, with some patients showing higher phagocytic capacity than controls. In general, variability is a normal component of all biologic phenomena and an ever-present bystander in all experimental models. Statistical methods are specifically aimed at evaluating whether the differences between two groups are secondary to casual variability or not. In this specific experiment, despite some overlap of phagocytic capacity between patients and control, the probability of the null-hypotesis was calculated as less than 1 in 10,000, a quite reassuring value. On the other hand, one may argue that also some negative results (i.e. accepting the null hypothesis or non-sta-
tistically significant differences) can actually be the consequence of high variability. In fact, in our discussion we focused only on the results supported by strong statistical significance. In addition, the observations of Vijay and colleagues give us the opportunity to stress once again that all patients with liver cirrhosis are not the same and better understanding of the reason for such wide variations is needed. It is therefore remarkable the constant and invariable near to complete loss of phagocytic capacity of neutrophils migrating to the skin blisters of cirrhotic patients, indicating that patients with cirrhosis have evidence of marked neutrophil dysfunction. 2. The ‘‘control’’ TLR expression in Fig. 4 refers to neutrophils incubated with healthy control’s plasma as stated in the ‘‘Subjects’’ paragraph and similar to all other experiments as stated in the Materials and methods section. 3. We found a trend towards higher oxidative burst in patients with alcoholic aetiology, ascites or encephalopathy (not statistically significant). We preferred to focus and comment only on results supported by strong statistical significance. The preliminary data Vijay et al. refer to are interesting and we understand consistent with our results. Further understanding of the underlying mechanisms will allow development of new strategies that can reduce the risk of infection in liver disease.
Journal of Hepatology 2012 vol. 56 j 1207–1217
1213
Pierre Pradat Department of Hepatogastroenterology, Hotel-Dieu, Lyon, France Jeanette J. McCarthy Institute for Genome Sciences and Policy, Duke University Medical Center, Durham, NC, USA ⇑
Hans L. Tillmann Duke Clinical Research Institute and Division of Gastroenterology, Duke University Medical Center, Durham, NC, USA ⇑ Tel.: +1 919 668 780; fax: +1 919 668 7164 E-mail address: [email protected]
Eric Trépo Department of Gastroenterology and Hepatopancreatology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium
The quest for the elusive factors that underpin neutrophil dysfunction in cirrhosis goes on To the Editor: We congratulate Tritto and colleagues on their recently published study [1], which aimed at extending our understanding of why patients with cirrhosis paradoxically exhibit features of impaired bacteriocidal capacity despite maintaining the ability to generate inflammation and bystander tissue damage. The neutrophil is a key component of the innate immune system and has been shown to play a vital role in the development of systemic inflammation and organ failure in sepsis [2]. The evidence base supporting neutrophil malfunction in acute and chronic liver disease continues to grow although factors that underpin this dysfunction are less clear and are likely to be related to a number of different factors [3–6]. In their study, Tritto and colleagues explore how the exposure of normal neutrophils to plasma from patients with varying severity and aetiology of liver disease in vitro may impact on their function. Furthermore, they aimed at investigating what role the presence of bacterial DNA, endotoxin and circulating cytokines within that plasma might play on neutrophil function and Tolllike receptor (TLR) expression. Lastly, they used a novel aseptic inflammation technique (blister study) to assess how neutrophils behave in vivo in 8 patients with Child Pugh A/B alcohol-related cirrhosis. The resulting data are both important and insightful but we would urge that these data need to be interpreted with caution at this stage due to several inherent flaws that exist in the study methodology. Firstly, further clarification on the control data is warranted. In the in vitro part of the study, the authors state that age- and sex-matched healthy volunteers were used as normal controls. No further details are given as to the control demographics or number of controls used, which appear to vary significantly in number between several figures in the manuscript. This is critical as the range of phagocytic capacity found in the healthy controls is vast (50,000–225,000 geometric mean of fluorescence intensity [GMFI]), suggesting the neutrophil function within the control group was highly variable and making valid statistical comparisons hard to achieve. A number of factors can influence neutrophil function in ‘healthy’ individuals including ethnicity, alcohol, tobacco habits and exercise within the previous 24 h which need to be controlled for. This is also fundamental when 1212
interpreting the blister studies, as 3 out of the 5 healthy controls had reduced phagocytic capacity of a similar magnitude to the cirrhotic patients and 2 patients stayed the same. This is somewhat at odds with the conclusion that the cirrhotic patients have impaired neutrophil phagocytic capacity in the blister fluid compared to controls. Moreover, there was a huge variation in GMFI in the cirrhotic patients (range 25,000–225,000). Four of the eight patients with cirrhosis also had evidence of impaired phagocytic capacity in peripheral blood at baseline, clouding the argument that stable cirrhotic patients did not have impairment of phagocytic capacity in peripheral blood but did in the inflammatory environment. Secondly, with regard to the TLR data, the baseline expression of TLR-2, 4 and 9 is not stated prior to exposure to cirrhotic plasma. This makes it difficult to interpret the impact of the cirrhotic plasma on the normal neutrophils. It might also have been better to have used healthy control plasma rather than phosphate-buffered saline which might have significantly influenced the neutrophil viability and the rate of apoptosis. It would also be interesting to determine whether the presence or absence of ascites had any relationship with baseline TLR-4 expression, which might be expected if this cohort has an increased rate of gut bacterial translocation. Lastly, we would be keen to learn more about the cohort (n = 34) with high oxidative burst. Did a large proportion of these patients have alcohol-related disease, ascites or encephalopathy? It is also fascinating to see that these patients had increased levels of IL-12, IFN-gamma and TNF-alpha. IL-12 and IFN-gamma are not cytokines that are typically associated with neutrophil activation and may have been released by other immune cell subtypes such as dendritic cells, macrophages and natural killer cells, which will undoubtedly have a role to play. In an ongoing prospective longitudinal cohort study of neutrophil morphology, phenotype and function at King’s College Hospital in over 125 patients with cirrhosis, we have shown that phagocytic dysfunction is universal in patients with cirrhosis (including those with Child Pugh A disease), which is reversible following liver transplantation [7]. Neutrophil dysfunction is likely to be determined by a large number of variables including plasma complement, immunoglobulin, sodium
Journal of Hepatology 2012 vol. 56 j 1207–1217
JOURNAL OF HEPATOLOGY and ammonia concentrations [8], the presence of hepatic encephalopathy [9], the presence of ascites and the pro- and anti-inflammatory cytokine milieu [10] which warrant further investigation. The quest for the elusive factor(s) that underpin neutrophil function therefore continues.
Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support Dr. Shawcross is funded by a 5 year Department of Health HEFCE Clinical Senior Lectureship. References [1] Tritto G, Bechlis Z, Stadlbauer V, Davies N, Frances R, Shah N, et al. Evidence of neutrophil functional defect despite inflammation in stable cirrhosis. J Hepatol 2011;55:574–581. [2] Brown KA, Brain SD, Pearson JD, Edgeworth JD, Lewis SM, Treacher DF. Neutrophils in development of multiple organ failure in sepsis. Lancet 2006;368:157–169. [3] Altin M, Rajkovic IA, Hughes RD, Williams R. Neutrophil adherence in chronic liver disease and fulminant hepatic failure. Gut 1983;24:746–750.
[4] Rajkovic IA, Williams R. Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis. Hepatology 1986;6:252–262. [5] Wyke RJ, Yousif-Kadaru AG, Rajkovic IA, Eddleston AL, Williams R. Serum stimulatory activity and polymorphonuclear leucocyte movement in patients with fulminant hepatic failure. Clin Exp Immunol 1982;50:442–449. [6] Mookerjee R, Stadlbauer V, Lidder S, Wright G, Hodges S, Davies N, et al. Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts outcome. Hepatology 2007;46:831–840. [7] Taylor N, Ma Y, Abeles R, Hussein M, Auzinger G, Vergani D, et al. Cirrhosis leads to impaired neutrophil phagocytosis and spontaneous oxidative burst which correlates with increasing IL-10 concentration and is reversed following liver transplantation. Hepatology 2009;50:121A, [Abstract]. [8] Shawcross D, Wright G, Stadlbauer V, Hodges S, Wheeler-Jones C, Pitsillides A, et al. Ammonia impairs neutrophil phagocytic function in liver disease. Hepatology 2008;48:1202–1212. [9] Shawcross DL, Shabbir SS, Taylor NJ, Hughes RD. Ammonia and the neutrophil in the pathogenesis of hepatic encephalopathy in cirrhosis. Hepatology 2010;51:1062–1069. [10] Taylor N, Abeles R, Ma Y, Wendon J, Shawcross D. A compensatory antiinflammatory response syndrome (CARS) triggered by neutrophil-induced oxidative stress is associated with chronic low grade hepatic encephalopathy in patients with advanced cirrhosis. J Hepatol 2010;52:S216, [Abstract].
Godhev K. Manakkat Vijay Nicholas J. Taylor ⇑ Debbie Lindsay Shawcross Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London SE5 9RS, UK ⇑ Tel.: +44 020 3299 3216; fax: +44 020 3299 3167 E-mail address: [email protected] (D.L. Shawcross)
Reply to: ‘‘The quest for the elusive factors that underpin neutrophil dysfunction in cirrhosis goes on’’ To the Editor: We thank Vijay and colleagues for their appreciation and interest in our paper evaluating some features of neutrophil dysfunction in cirrhosis of different severity [1]. They raise three points which are addressed below: 1. The first issue raised was over concerns for the high variability of phagocytic capacity in the group of healthy controls and the numerous factors influencing neutrophil function, which may hinder the statistical evaluation of differences. We used plasma from 21 healthy controls who were non-smokers and had not drunk alcohol in the previous 48 h. For cytokine assays we used 10 healthy controls. With regards to variability, we would suggest that it was even higher in the cirrhosis group, with some patients showing higher phagocytic capacity than controls. In general, variability is a normal component of all biologic phenomena and an ever-present bystander in all experimental models. Statistical methods are specifically aimed at evaluating whether the differences between two groups are secondary to casual variability or not. In this specific experiment, despite some overlap of phagocytic capacity between patients and control, the probability of the null-hypotesis was calculated as less than 1 in 10,000, a quite reassuring value. On the other hand, one may argue that also some negative results (i.e. accepting the null hypothesis or non-sta-
tistically significant differences) can actually be the consequence of high variability. In fact, in our discussion we focused only on the results supported by strong statistical significance. In addition, the observations of Vijay and colleagues give us the opportunity to stress once again that all patients with liver cirrhosis are not the same and better understanding of the reason for such wide variations is needed. It is therefore remarkable the constant and invariable near to complete loss of phagocytic capacity of neutrophils migrating to the skin blisters of cirrhotic patients, indicating that patients with cirrhosis have evidence of marked neutrophil dysfunction. 2. The ‘‘control’’ TLR expression in Fig. 4 refers to neutrophils incubated with healthy control’s plasma as stated in the ‘‘Subjects’’ paragraph and similar to all other experiments as stated in the Materials and methods section. 3. We found a trend towards higher oxidative burst in patients with alcoholic aetiology, ascites or encephalopathy (not statistically significant). We preferred to focus and comment only on results supported by strong statistical significance. The preliminary data Vijay et al. refer to are interesting and we understand consistent with our results. Further understanding of the underlying mechanisms will allow development of new strategies that can reduce the risk of infection in liver disease.
Journal of Hepatology 2012 vol. 56 j 1207–1217
1213