Lack of effect of glutamine administration to boost the innate immune system response in trauma patients in the intensive care unit

Abstract

Introduction: The use of glutamine as a dietary supplement is associated with a reduced risk of infection. We hypothesized that the underlying mechanism could be an increase in the expression and/or functionality of Toll-like receptors (TLR), key receptors sensing infections. The objective of this study was to evaluate whether glutamine supplementation alters the expression and functionality of TLR2 and TLR4 in circulating monocytes of trauma patients admitted to the intensive care unit (ICU).

Methods: We designed a prospective, randomized and single-blind study. Twenty-three patients received parenteral nutrition (TPN) with a daily glutamine supplement of 0.35 g/kg. The control group (20 patients) received an isocaloric-isonitrogenated TPN. Blood samples were extracted before treatment, at 6 and 14 days. Expression of TLR2 and TLR4 was determined by flow cytometry. Monocytes were stimulated with TLR specific agonists and cytokines were measured in cell culture supernatants. Phagocytic ability of monocytes was also determined.

Results: Basal characteristics were similar in both groups. Monocytes from patients treated with glutamine expressed the same TLR2 levels as controls before treatment (4.9 ± 3.5 rmfi vs. 4.3 ± 1.9 rmfi, respectively; P = 0.9), at Day 6 (3.8 ± 2.3 rmfi vs. 4.0 ± 1.7 rmfi, respectively; P = 0.7) and at Day 14 (4.1 ± 2.1 rfim vs. 4.6 ± 1.9 rmfi, respectively; P = 0.08). TLR4 levels were not significantly different between the groups before treatment: (1.1 ± 1 rmfi vs 0.9 ± 0.1 rmfi respectively; P = 0.9), at Day 6 (1.1 ± 1 rmfi vs. 0.7 ± 0.4 rmfi respectively; P = 0.1) and at Day 14 (1.4 ± 1.9 rmfi vs. 1.0 ± 0.6 rmfi respectively; P = 0.8). No differences in cell responses to TLR agonists were found between groups. TLR functionality studied by phagocytosis did not vary between groups.

Conclusions: In trauma patients in the intensive care unit, TPN supplemented with glutamine does not improve the expression or the functionality of TLRs in peripheral blood monocytes.

Trial registration: ClinicalTrials.gov Identifier: NCT01250080.

Figures

Figure 1

Expression of TLR2 in trauma patients treated with and without glutamine. The expression of TLR2 was analyzed in CD14 positive peripheral blood mononuclear cells. rmfi are shown for 23 trauma patients treated with glutamine (black bars) and 20 trauma patients without glutamine and used as controls (white bars). Samples were obtained at the beginning of the treatment (Day 0); at the end of the treatment (Day 6) and at Day 14. Data are given as mean ± SEM.

Figure 2

Expression of TLR4 in trauma patients treated with and without glutamine. The expression of TLR4 was analyzed in CD14 positive peripheral blood mononuclear cells. rmfi are shown for 23 trauma patients treated with glutamine (black bars) and 20 trauma patients without glutamine and used as controls (white bars). Samples were obtained at the beginning of the treatment (Day 0); at the end of the treatment (Day 6) and at Day 14. Data are given as mean ± SEM.

Figure 3

Concentration of TNFα in cell culture supernatants in trauma patients treated with and without glutamine. TLR functionality. Levels of TNFα analyzed by a bead array ELISA (CBA Kit, BD Biosciences), in response to lipopolysaccharide (LPS-100 ng/mL), Pam3CSK4 (PAM-10 pg/mL) and zymosan (ZYM-10 pg/mL) at the beginning of the treatment (Figure 3A); at Day 6 (Figure 3B) and at Day 14 (Figure 3C). Monocytes from trauma patients treated with glutamine subjects (black bars, n = 23) and trauma patients without glutamine (white bars, n = 20). Control bars are samples production of cytokines by unstimulated monocytes. Data are given as mean ± SEM.

Figure 4

Concentration of IL1β in cell culture supernatants in trauma patients treated with and without glutamine. TLR functionality. Levels of IL1β analyzed by a bead array ELISA (CBA Kit, BD Biosciences), in response to lipopolysaccharide (LPS-100 ng/ml), Pam3CSK4 (PAM-10 pg/mL) and zymosan (ZYM-10 pg/mL) at the beginning of the treatment (Figure 4A); at Day 6 (Figure 4B) and at Day 14 (Figure 4C). Monocytes from trauma patients treated with glutamine subjects (black bars, n = 23) and trauma patients without glutamine (white bars, n = 20). Control bars are samples production of cytokines by unstimulated monocytes. Data are given as mean ± SEM.

Figure 5

Concentration of IL6 in cell culture supernatants in trauma patients treated with and without glutamine. TLR functionality. Levels of Cytokines IL 6 analyzed by a bead array ELISA (CBA Kit, BD Biosciences), in response to lipopolysaccharide (LPS-100 ng/ml), Pam3CSK4 (PAM-10 pg/mL) and zymosan (ZYM-10 pg/mL)) at the beginning of the treatment (Figure 5A); at Day 6 (Figure 5B) and at Day 14 (Figure 5C). Monocytes from trauma patients treated with glutamine subjects (black bars, n = 23) and trauma patients without glutamine (white bars, n = 20). Control bars are samples production of cytokines by unstimulated monocytes. Data are given as mean ± SEM.

Figure 6

Concentration of IL10 in cell culture supernatants in trauma patients treated with and without glutamine. TLR functionality. Levels of Cytokines IL 10 analyzed by a bead array ELISA (CBA Kit, BD Biosciences), in response to lipopolysaccharide (LPS-100 ng/ml), Pam3CSK4 (PAM-10 pg/mL) and zymosan (ZYM-10 pg/mL)) at the beginning of the treatment (Figure 6A); at Day 6 (Figure 6B) and at Day 14 (Figure 6C). Monocytes from trauma patients treated with glutamine subjects (black bars, n = 23) and trauma patients without glutamine (white bars, n = 20). Control bars are samples production of cytokines by unstimulated monocytes. Data are given as mean ± SEM.