Dysfunction of Circulating Polymorphonuclear Leukocytes and Monocytes in Ambulatory Cirrhotics

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Supplementary file

Dysfunction of circulating polymorphonuclear leukocytes and monocytes in ambulatory cirrhotics predicts patient outcome

Supplementary Patients and methods

Data collection

Alcohol consumption among patients with alcoholic liver disease (ALD) was quantified by means of measurement of phosphatidyl-ethanol (PEth) in whole blood, a direct ethanol metabolite which has been established as a sensitive and specific marker of prolonged alcohol overconsumption and a measure of prolonged (last 2 weeks) alcohol intake [1, 2]. Alcohol consumption in the month prior to enrollment was also assessed using a timeline follow-back method [3, 4], which is a well-established method to gather detailed drinking data in alcohol dependent individuals [5]. In short, participants completed a retrospective log based on a monthly calendar to indicate daily alcohol drinking to provide the total number of alcoholic drinks consumed in the previous 30 days prior to enrollment. This quantity was then divided by the total number of drinking days (within the previous 30 days) to calculate drinks per drinking day [3, 4]. Patients with ALD cirrhosis were further classified into current drinkers if they reported alcohol consumption at timeline follow-back and/or confirmed by a raised blood PEth. In cases that quantifiable blood PEth in self-reported abstainers was found, patients were classified as current drinkers.

Supplementary Results

Correlation between percentage of bursting cells and burst intensity (MFI)

As expected, the percentage of bursting cells correlated positively with burst intensity (MFI) in our study. This correlation was observed for both PMNs and monocytes after stimulation with E.coli (r=0.370, p=0.004 and r=1.000, p=0.002, respectively) and stimulation with PMA (r=0.679, p<0.001 and r=1.000, p<0.001, respectively).

Correlation between oxidative burst and phagocytosis

In our study, we observed a correlation between resting oxidative burst (MFI) and phagocytic capacity (MFI) for both PMNs (r=0.392, p=0.008) and monocytes (r=0.351, p=0.019). Apart from this, we found no significant correlation between oxidative burst and phagocytosis for PMNs or monocytes (p>0.05 for all, data not shown).

Relation of monocyte function with pro-inflammatory cytokine levels

The percentage of resting monocytes producing ROS were correlated with IL-6 (r=0.236, p=0.085) and TNFα levels (r=0.277, p=0.041). E.coli stimulated monocyte burst tended to be related to IL-6 (r=0.229, p=0.089) and TNFα levels (r=0.233, p=0.081). Moreover, impaired phagocytic capacity of monocytes tended to correlate with increased levels of TNFα (r=-0.261, p=0.080).

Correlation between CRP levels and PMN/monocyte function

Plasma CRP levels were available in 59/60 (98%) patients at inclusion in the study. Plasma CRP levels at inclusion were low (median CRP: 2mg/L, interquartile range: 4.4, range: 0.6-45). When correlation analysis was performed, no significant correlations between CRP levels and PMN or monocyte function were observed (r= 0.202-0.961, p>0.05 for all; data not shown).

Relation of PMN and monocyte function with patient outcome

Sites of first bacterial infection experienced by the study patients during follow-up were: pneumonia (n=2), urinary tract infection (n=3), spontaneous bacterial peritonitis (n=3), skin/soft-tissue infection (n=3), cholangitis (n=2) and secondary peritonitis (n=2).

Supplementary Table.Polymorphonuclear leukocyte and monocyte function in patients with ALD vs. non-ALD cirrhosis

Non-ALD cirrhosis (n=29) / ALD cirrhosis (n=31)
Male gender / 20 (69) / 22 (71)
Age (years) / 64 (56-70) / 63 (57-67)
MELD score / 6 (6-8) / 6 (6-8)
Prior or current decompensation / 6 (21)a / 17 (55)
Abstainers
Current drinkers / 8 (26)
23 (74)
PMN function
Resting oxidative burst, %* / 0.4 (0.2-0.8) / 0.5 (0.3-0.9)
Resting oxidative burst, MFI / 3.6 (3.1-3.9) / 3.1 (2.9-3.6)
E.coli oxidative burst, MFI / 20.5 (15.2-26.3) / 18.2 (13.5-22.8)
PMA oxidative burst, MFI / 19.9 (10.5-34.2) / 13.3 (7.7-24.1)
Phagocytic activity, %** / 96.3 (87.6-98.1) / 94.7 (87.9-97.8)
Phagocytic capacity, MFI / 52.2 (44.4-74.7) / 51.7 (35.1-69.6)
Monocyte function
Resting oxidative burst, %* / 0.7 (0.3-2.4) / 0.7 (0.3-1.8)
Resting oxidative burst, MFI / 2.8 (2.2-3.3) / 3.0 (2.7-3.2)
E.coli oxidative burst, MFI / 5.3 (4.0-5.5) / 5.2 (4.2-5.9)
PMA oxidative burst, MFI / 4.6 (3.5-6.7) / 4.1 (3.3-6.1)
Phagocytic activity, %** / 90.2 (75.4-94.2) / 87.1 (74.1-95.4)
Phagocytic capacity, MFI / 33.3 (20.5-40.2) / 29.1 (15.2-39.1)
Cytokines
S-IL-6, ng/L / 4 (3-8)a / 9 (6-12)
P-IL-8, ng/L / 5 (3-9) / 9 (5-22)
S-TNFα, ng/L / 14 (5-14) / 13 (11-16)

Data are expressed as n (%) or median (interquartile range) as appropriate

Data on phagocytosis were available in 23/29 (79%) patients with non-ALD and in 23/31 (74%) patients with ALD cirrhosis(in 6/8 (75%) abstainers and 17/23(74%) current drinkers)

The median alcohol intake per drinking day during the last 30 days prior to study inclusion reported in timeline follow-back method by ALD patients was 12 gr ethanol/day (range 0-96). 7 (30%) ALD patients had ≥1 points scored on AUDIT questions 4-6 (range 1-9) (a score 0 in these questions of AUDIT imply the potential presence or incipience of alcohol dependence)

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ALD, alcoholic liver disease; MELD, model for end-stage liver disease; PMN, polymorphonuclear leukocyte; MFI, median fluorescence intensity; PMA, phorbol-12-myristate-13-acetate; IL, interleukin; TNF, tumor necrosis factor; AUDIT, Alcohol Use Disorders Identification Test

*Percent of bursting cells

**Percent of cells undergoing phagocytosis

Supplementary figure legends

Figure 1. Receiver operating characteristics curves for the utility of E.coli stimulated PMN oxidative burst in predicting the occurrence of A) acute-on-chronic liver failure, and B) severe sepsis within the first year following inclusion in the study

PMN, polymorphonuclear leukocytes; MFI, median fluorescence intensity

Figure 2. Receiver operating characteristics curves for the utility of E.coli stimulated monocyte oxidative burst in predicting the occurrence of A) severe sepsis, B) acute-on-chronic liver, and C) death or transplant within the first year following inclusion in the study

MFI, median fluorescence intensity

Supplementary References

[1]Aradottir S, Asanovska G, Gjerss S, Hansson P, Alling C. PHosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients. Alcohol Alcohol 2006;41:431-437.

[2]Wurst FM, Thon N, Aradottir S, et al. Phosphatidylethanol: normalization during detoxification, gender aspects and correlation with other biomarkers and self-reports. Addict Biol 2010;15:88-95.

[3]Epstein EE, Labouvie E, McCrady BS, Swingle J, Wern J. Development and validity of drinking pattern classification: binge, episodic, sporadic, and steady drinkers in treatment for alcohol problems. Addict Behav 2004;29:1745-1761.

[4]Collins RL, Kashdan TB, Koutsky JR, Morsheimer ET, Vetter CJ. A self-administered Timeline Followback to measure variations in underage drinkers' alcohol intake and binge drinking. Addict Behav 2008;33:196-200.

[5]Sobell LC, Agrawal S, Sobell MB, et al. Comparison of a quick drinking screen with the timeline followback for individuals with alcohol problems. J Stud Alcohol 2003;64:858-861.