New Insights on End-Stage Renal Disease and Healthy Individual Gut Bacterial Translocation: Different Carbon Composition of Lipopolysaccharides and Different Impact on Monocyte Inflammatory Response

Abstract

Chronic kidney disease induces disruption of the intestinal epithelial barrier, leading to gut bacterial translocation. Here, we appreciated bacterial translocation by analyzing circulating lipopolysaccharides (LPS) using two methods, one measuring only active free LPS, and the other quantifying total LPS as well as LPS lipid A carbon chain length. This was done in end-stage renal disease (ESRD) patients and healthy volunteers (HV). We observed both higher LPS concentration in healthy volunteers and significant differences in composition of translocated LPS based on lipid A carbon chain length. Lower LPS activity to mass ratio and higher concentration of high-density lipoproteins were found in HV, suggesting a better plasma capacity to neutralize LPS activity. Higher serum concentrations of soluble CD14 and pro-inflammatory cytokines in ESRD patients confirmed this hypothesis. To further explore whether chronic inflammation in ESRD patients could be more related to LPS composition rather than its quantity, we tested the effect of HV and patient sera on cytokine secretion in monocyte cultures. Sera with predominance of 14-carbon chain lipid A-LPS induced higher secretion of pro-inflammatory cytokines than those with predominance of 18-carbon chain lipid A-LPS. TLR4 or LPS antagonists decreased LPS-induced cytokine production by monocytes, demonstrating an LPS-specific effect. Thereby, septic inflammation observed in ESRD patients may be not related to higher bacterial translocation, but to reduced LPS neutralization capacity and differences in translocated LPS subtypes.

Keywords: HDL - cholesterol; chronic inflammation; end-stage renal disease; gut bacterial translocation; lipid A (LPS).

Figure 1

Gut bacterial translocation in end-stage renal disease (ESRD) patients and healthy volunteers (HV). Comparison of total circulating LPS (A), concentrations (B) and proportions (C) of circulating LPS subtypes between ESRD patients (n = 68) and healthy volunteers (n = 20). LPS quantities were measured in serum by HPLC/MS/MS method. LPS subtypes differed according to the length of lipid A carbon chains (3OHC12:0, 3OHC14:0, 3OHC16:0, 3OHC18:0) and their sum was considered as the total circulating LPS concentration used to determine LPS proportions. Bars and errors bars are expressed as median with interquartile range. Mann-Whitney U test of HV versus ESRD patient’s values, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. HPLC/MS/MS, high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS/MS); LPS, lipopolysaccharide.

Figure 2

Relationship between quantity and activity of circulating LPS in end-stage renal disease (ESRD) patients and healthy volunteers (HV). Comparison of LPS activity (A) and LPS activity to mass ratio (B) between ESRD patients (n = 68) and healthy volunteers (n = 20). LPS quantity and LPS activity were measured in serum by HPLC/MS/MS method and LAL test respectively. Bars and errors bars are expressed as median with interquartile range. Mann-Whitney U test of HV versus ESRD patient’s values, ****p < 0.0001, ns, not significant. HPLC/MS/MS, high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS/MS); LAL, Limulus amebocyte lysate, LPS, lipopolysaccharide.

Figure 3

LPS-induced inflammation in end-stage renal disease (ESRD) patients and healthy volunteers (HV). Comparison of sCD14 (A) and inflammatory cytokines (B) serum levels between ESRD patients (n = 68) and healthy volunteers (n = 20). Soluble CD14 and cytokines (TNF-α, IL-1β, IL-6, IL-10) were determined in serum by enzyme-linked immunosorbent assay (ELISA) and Luminex methods respectively. Bars and errors bars are expressed as median with interquartile range. Mann-Whitney U test of HV versus ESRD patient’s values, **p < 0.01, ****p < 0.0001, ns, not significant. IL, interleukin; LPS, lipopolysaccharide; sCD14, soluble cluster of differentiation 14; TLR4, Toll-like receptor-4; TNF, tumor necrosis factor.

Figure 4

Correlation between LPS subtype’s proportion with sCD14 in end-stage renal disease (ESRD) patients. Correlation between sCD14 serum levels and 3OHC12:0 (A), 3OHC14:0 (B), 3OHC16:0 (C) or 3OHC18:0 (D) LPS proportions in ESRD patients (n = 68). Each cross represents a patient. Linear regression was displayed on graphs. Spearman correlation between sCD14 and LPS proportions, *p < 0.05, **p < 0.01. LPS, lipopolysaccharide; sCD14, soluble cluster of differentiation 14.

Figure 5

Cytokine concentrations in supernatants of monocytes cultured with sera of end-stage renal disease (ESRD) patients (n = 12) according to the highest proportion of each LPS subtype and healthy volunteers (HV) (n = 5). Human monocytes were stimulated with ESRD patient’s serum according to the highest proportion of each LPS subtypes (P/C12, P/C14, P/C16, P/C18, n = 3 for each), with serum of HV (n = 5), with decomplemented autologous serum (AS, n = 3), with AS and LPS from E. coli (LPS-EK, 10ng/mL, n = 3) for the positive control, or monocytes cultured without serum (WS, n = 3) for the negative control. After 12 hours of stimulation, supernatants were collected and the secretion of cytokines TNF-α (A), IL-1β (B), IL-6 (C) and IL-10 (D) were measured by enzyme-linked immunosorbent assay (ELISA). Bars and errors bars are expressed as median with interquartile range. Mann-Whitney U test of pairwise comparison values, **p < 0.01, ***p < 0.001. IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor.

Figure 6

TLR4-dependent inflammation pathway in end-stage renal disease (ESRD) patients and healthy volunteers (HV). Human monocytes were stimulated with ESRD patient’s serum according to the highest proportion of each LPS subtypes (P/C12, P/C14, P/C16, P/C18, n = 3 for each), with HV serum (n = 5), or with decomplemented autologous serum and LPS from E. coli (LPS-EK, 10ng/mL, n = 3) for the positive control. Moreover, cells and sera were cultured with an antagonist of TLR4 (LPS from R. sphaeroides, LPS-RS, 100 ng/mL) or an antibiotic blocking LPS activity (Polymyxin B, PMB, 100 µg/mL). After 12 hours of stimulation, supernatants were collected and the secretion of cytokines TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Bars and errors bars are expressed as median with interquartile range. Mann-Whitney U test of single stimulation (LPS-EK or sera) and co-stimulation with PMB or LPS-RS values, ****p < 0.0001, ns, not significant; LPS, lipopolysaccharide; TLR4, Toll-like receptor-4; TNF, tumor necrosis factor.

Figure 7

New insights on ESRD and HV gut bacterial translocation. Representation of the hypotheses emerging from the GABII cohort study. CKD, Chronic Kidney Disease; ESRD, End-Stage Renal Disease; HV, Healthy Volunteers.