ABSTRACT
Intravenous administration to volunteers of an emulsion of medium-chain lipids, but not of an emulsion of pure long-chain lipids or a placebo, increased the growth of Candida albicans in serum and modulated Candida-induced cytokine production by mononuclear cells in a way suggesting that medium-chain, but not long-chain, triglycerides increase the risk for infections by Candida.
The provision of total parenteral nutrition (TPN) is an indispensable strategy to improve the nutritional status of critically ill patients. However, altered immune responses by the TPN lipid component may contribute to the increased rate of infectious complications for these patients (43). It remains unclear whether structurally different lipid emulsions containing either pure long-chain triglycerides (LCT) or mixed long- and medium-chain triglycerides (LCT-MCT) exert distinct immune-modulating effects (1, 3, 4, 7, 8, 10, 12-15, 18, 25, 27, 28, 32, 33, 35-37, 40, 42, 43, 44). Recently, we found that LCT-MCT, unlike LCT, increase in vitro oxygen radical production and adhesion of neutrophils but decrease cellular motility and killing of Candida albicans (20, 45-48). This observation is important because clinical studies indicate that 5% of all patients receiving TPN develop candidemia, with significant mortality and morbidity (38).
In the present study, we exposed LCT and LCT-MCT to the metabolisms and immune systems of healthy volunteers. We investigated the effects of lipids on two pathogenetic aspects of Candida infections: yeast growth and the balance of proinflammatory (gamma interferon [γ-IFN], tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], and IL-6) and anti-inflammatory (IL-10) cytokines (2, 19, 29, 30, 34, 41).
Emulsions containing LCT, LCT-MCT, or saline were administered during 4 h to eight volunteers in a study with a crossover design and a 1-week washout period. Blood samples were taken before and after 4 h of lipid or placebo administration and analyzed as described below. In order to stabilize plasma triglyceride concentrations at a clinically relevant concentration of 3 to 5 mmol/liter, emulsions (overall, ca. 220 ml) were infused according to a triglyceride-clamp schedule (16, 17). For emulsion characteristics, see Table 1.
Leukocytes were isolated from 20 ml of blood anticoagulated with lithium-heparin (6, 21, 45). Peripheral blood mononuclear cells (PBMC) were removed and suspended in medium (RPMI 1640 DM; Flow Laboratories, Irvine, United Kingdom). Heat-killed C. albicans (strain UC820; final concentration, 107 CFU/ml) was used for ex vivo PBMC stimulation in the cytokine assays. After PBMC isolation, cell numbers were adjusted (5 × 106/ml) and cell suspension samples were incubated with Candida (24 h, 37°C). After incubation, the supernatants were frozen (−20°C) until assayed. IL-1β and TNF-α (both in nanograms per milliliter) in supernatants were measured by radioimmunoassays as described previously (23). Detection limits of the assay were 20 pg/ml for TNF-α and 40 pg/ml for IL-1β. Interassay variation was less than 15%, and intra-assay variation was less than 10%. IL-6, IL-8, IL-10, and IFN-γ concentrations were determined in duplicate with commercially available enzyme-linked immunosorbent assay kits (Pelikine Compact human enzyme-linked immunosorbent assay; CLB, Amsterdam, The Netherlands).
After inoculation and overnight culturing, C. albicans was suspended at 106 CFU/ml. The Candida suspension was incubated (24 h, 37°C) with serum samples and Sabouraud medium. After incubation, samples were plated onto Sabouraud agar plates and incubated (29°C) for 8 and 24 h. The colonies were counted (numbers of CFU per milliliter), and growth rates were expressed as ratios of numbers of CFU in samples after lipid administration to those before lipid administration.
Results are expressed as medians (with 25th and 75th percentiles). The statistical significance of treatment effects was determined by analysis of variance with Bonferroni correction for multiple comparisons and by Tukey's posttest.
Infusion of LCT and LCT-MCT equally increased triglyceride concentrations (Table 2). However, ex vivo cytokine production by PBMC was distinctly influenced by lipid treatment (Table 3). Candida-induced production of TNF-α, IL-1β, and IL-10 increased after LCT-MCT exposure. Compared with the placebo, LCT showed no effect, although this might be due to the sample size. Candida-induced IFN-γ production tended to decrease, but values did not reach statistical significance. Infusion of LCT or placebo, with this limited sample size, did not influence the production of any cytokine. With LCT-MCT, we observed a significantly increased rate of growth of Candida after 8 and 24 h compared with rates with the placebo and LCT (Fig. 1).
Infections by C. albicans pose a threat to the use of NADP (9, 38). Outcomes probably depend on yeast growth rates as well as counteractive responses of the innate and adaptive immune systems (30, 31). It appears that the balance of pro- and anti-inflammatory cytokines is altered by LCT-MCT in a way that is known to deactivate innate immunity (30, 31). Also, our results indicate that LCT-MCT, but not LCT, favors the development of Candida infections by enhancing yeast growth rates. Microscopic evaluation (data not shown) revealed the formation of pseudohyphae after lipid administration, indicating that growth rates are underestimated even in our experimental setting. Importantly, inhibitory effects of serum on yeast growth due to iron deprivation were ruled out, as addition of FeCl3 (10 μmol/liter) to serum did not affect test results (data not shown).
Our ex vivo findings support in vitro work where LCT-MCT, but not LCT, impaired neutrophil killing of Candida (48). Previous in vitro studies with LCT showed that Candida grows better in a lipid-rich environment (5, 11, 24, 36). We did not find a growth-enhancing effect on Candida for parenteral LCT, suggesting that with its metabolic breakdown, the effects of LCT on candidal growth disappear, in contrast with what occurs with MCT.
The relative importance of the findings of increased levels of TNF-α, IL-1β, and (to a lesser extent) IL-8 production with MCT, which could be considered protective, remains unclear and probably can be evaluated only in a clinical study.
The altered balance of Candida-induced cytokine production by PBMC, with increased production of IL-10 (by Th2 lymphocytes) and unchanged or decreased production of IFN-γ (by Th1 lymphocytes), results in a decreased IFN-γ/IL-10 ratio. Such an imbalance in Th1 and Th2 responses is considered a major risk factor for the development of fungal infections (30, 34, 41). On the other hand, it is also possible that the IL-10 measured in our experiments was produced by monocytes.
IFN-γ activates phagocytic cells to kill Candida, whereas IL-10 has been shown to inhibit proinflammatory cytokine production and to aggravate the course of disseminated candidiasis (22, 26, 39). The influence of LCT-MCT on the production of IFN-γ (decreased production) and monokines (increased production), in combination with the lack of effects of LCT, suggests that MCT have differential effects on T cells and monocytes. LCT exerted no effects on cytokine production compared with the placebo, making it improbable that components other than the lipids, e.g., an emulsifier and antioxidants, are responsible for the emulsion effects. Finally, it has to be kept in mind that definite proof for the effects of lipids on the susceptibility to fungal infections of humans can be obtained only in large-scale clinical studies.
In conclusion, the results of our study suggest that parenteral MCT, contrary to pure LCT, increase susceptibility to infections with C. albicans by increasing candidal growth rates and by having a detrimental effect on antifungal immune responses.
FIG. 1. Effects of lipid emulsions or the placebo after 8- and 24-h incubations on the growth of samples of C. albicans in cell-free serum. Results are ratios of growth obtained after lipid infusion to growth obtained before lipid infusion. ∗, significance (P ≤ 0.01) of treatment effects relative to results with the placebo and LCT; ∗∗, significance (P ≤ 0.02) of treatment effects relative to results with the placebo and LCT.
TABLE 1. Characteristics of lipid emulsions according to manufacturers
TABLE 2. Effects of lipid administration on triglyceride concentrationslegend
TABLE 3. Effects of lipid administration on cytokine production by PBMC
FOOTNOTES
- Received 26 October 2001.
- Returned for modification 26 December 2001.
- Accepted 5 August 2002.
- Copyright © 2002 American Society for Microbiology
REFERENCES
- 1.↵
Arias Diaz, J., J. M. Rodriguez, E. Vara, C. Garcia, J. Torres Melero, and C. Garcia Carreras.
1996. NO2/NO3 and cytokine plasma profiles under different postoperative parenteral nutrition regimens. Nutrition12:89-92.
- 2.↵
Baggiolini, M., P. Loetscher, and B. Moser.
1995. Interleukin-8 and the chemokine family. Int. J. Immunopharmacol.17:103-108.
- 3.↵
Bellinati Pires, R., D. L. Waitzberg, M. M. Salgado, and M. M. Carneiro Sampaio.
1993. Functional alterations of human neutrophils by medium-chain triglyceride emulsions: evaluation of phagocytosis, bacterial killing, and oxidative activity. J. Leukoc. Biol.53:404-410.
- 4.↵
Braxton, C. C., S. M. Coyle, W. J. Montegut, T. van-der-Poll, M. Roth, and S. E. Calvano.
1995. Parenteral nutrition alters monocyte TNF receptor activity. J. Surg. Res.59:23-28.
- 5.↵
D'Angio, R., R. A., Quercia, N. K. Treiber, J. C. Mclaughlin, and J. Klimek.
1987. The growth of microorganisms in total parenteral admixtures. J. Parenter. Enteral Nutr.11:394-397.
- 6.↵
Drenth, J. P., S. H. Van-Uum, M. Van-Deuren, G. J. Pesman, and J. W. Van-der-Meer.
1995. Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF-alpha and IL-1 beta production. J. Appl. Physiol.79:1497-1503.
- 7.↵
English, D., J. S. Roloff, J. N. Lukens, P. Parker, H. L. Greene, and F. K. Ghishan.
1981. Intravenous lipid emulsions and human neutrophil function. J. Pediatr.99:913-916.
- 8.↵
Fischer, G. W., K. W. Hunter, S. R. Wilson, and A. D. Mease.
1980. Diminished bacterial defences with intralipid. Lancet ii:819-820.
- 9.↵
Forchielli, M. L., K. Gura, E. Anessi-Pessina, D. Richardson, W. Cai, and C. Lo.
2000. Success rates and cost-effectiveness of antibiotic combinations for initial treatment of central-venous-line infections during total parenteral nutrition. J. Parenter. Enteral Nutr.24:119-125.
- 10.↵
Freeman, J., D. A. Goldmann, N. E. Smith, D. G. Sidebottom, M. F. Epstein, and R. Platt.
1990. Association of intravenous lipid emulsion and coagulase-negative staphylococcal bacteremia in neonatal intensive care units. N. Engl. J. Med.323:301-308.
- 11.↵
Gilbert, M., S. C. Gallagher, M. Eads, and M. F. Elmore.
1986. Microbial growth patterns in a parenteral formulation containing lipid emulsion. J. Parenter. Enteral Nutr.10:494-497.
- 12.↵
Gogos, C. A., N. Zoumbos, M. Makri, and F. Kalfarentzos.
1994. Medium- and long-chain triglycerides have different effects on the synthesis of tumor necrosis factor by human mononuclear cells in patients under total parenteral nutrition. J. Am. Coll. Nutr.13:40-44.
- 13.
Gogos, C. A., N. C. Zoumbos, M. Makri, and F. Kalfarentzos.
1992. Tumor necrosis factor production by human mononuclear cells during total parenteral nutrition containing long-chain triglycerides. Nutrition8:26-29.
- 14.
Goldmann, D. A.
1990. Coagulase-negative staphylococci: interplay of epidemiology and bench research. Am. J. Infect. Control18:211-221.
- 15.↵
Huang, Y. C., C. C. Li, T. Y. Lin, R. I. Lien, Y. H. Chou, and J. Wu.
1998. Association of fungal colonization and invasive disease in very low birth weight infants. Pediatr. Infect. Dis. J.17:819-822.
- 16.↵
Iriyama, K., T. Tsuchibashi, C. Miki, I. Kalembeyi, H. Li, and H. Urata.
1996. Elimination rate of fat emulsion particles from plasma in Japanese subjects as determined by a triglyceride clamp technique. Nutrition12:79-82.
- 17.↵
Iriyama, K., C. Miki, T. Inoue, N. Kawarabayashi, H. Urata, and C. Shigemori.
1998. Constant infusion rates of lipid emulsions to stabilize plasma triglyceride concentrations: medium-chain triglyceride/long-chain triglyceride emulsions (MCT/LCT) versus LCT. Surg. Today28:289-292.
- 18.↵
Jarstrand, C., L. Berghem, and G. Lahnborg.
1978. Human granulocyte and reticulo-endothelial system function during intralipid infusion. J. Parenter. Enteral Nutr.2:663-670.
- 19.↵
Kaufmann, S. H.
1995. Immunity to intracellular microbial pathogens. Immunol. Today16:338-342.
- 20.↵
Kruimel, J. W., A. H. Naber, J. H. Curfs, M. A. Wenker, and J. B. Jansen.
2000. With medium-chain triglycerides, higher and faster oxygen radical production by stimulated polymorphonuclear leukocytes occurs. J. Parenter. Enteral Nutr.24:107-112.
- 21.↵
Kuijpers, T. W., A. T. Tool, C. E. van der Schoot, L. A. Ginsel, J. J. Onderwater, and D. Roos.
1991. Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Blood78:1105-1111.
- 22.↵
Kullberg, B. J., J. W. van-'t-Wout, C. Hoogstraten, and R. van-Furth.
1993. Recombinant interferon-gamma enhances resistance to acute disseminated Candida albicans infection in mice. J. Infect. Dis.168:436-443.
- 23.↵
Kullberg, B. J., and E. J. Anaissie.
1998. Cytokines as therapy for opportunistic fungal infections. Res. Immunol.149:478-488.
- 24.↵
Lawrence, J., M. Turner, and P. Gilbert.
1988. Microbial contamination in total parenteral nutrition solutions. J. Clin. Pharm. Ther.13:151-157.
- 25.↵
Lenssen, P., B. A. Bruemmer, R. A. Bowden, T. Gooley, S. N. Aker, and D. Mattson.
1998. Intravenous lipid dose and incidence of bacteremia and fungemia in patients undergoing bone marrow transplantation. Am. J. Clin. Nutr.67:927-933.
- 26.↵
Levitz, S. M., A. Tabuni, S. H. Nong, and D. T. Golenbock.
1996. Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide. Infect. Immun.64:945-951.
- 27.↵
Lin, M. T., H. Saito, R. Fukushima, T. Inaba, K. Fukatsu, and T. Inoue.
1997. Preoperative total parenteral nutrition influences postoperative systemic cytokine responses after colorectal surgery. Nutrition13:8-12.
- 28.↵
Muscaritoli, M., L. Conversano, G. F. Torelli, W. Arcese, S. Capria, and C. Cangiano.
1998. Clinical and metabolic effects of different parenteral nutrition regimens in patients undergoing allogeneic bone marrow transplantation. Transplantation66:610-616.
- 29.↵
Netea, M. G., J. H. Curfs, P. N. Demacker, J. F. Meis, J. W. Van-der-Meer, and B. J. Kullberg.
1999. Infusion of lipoproteins into volunteers enhances the growth of Candida albicans. Clin. Infect. Dis.28:1148-1151.
- 30.↵
Netea, M. G., L. J. van-Tits, J. H. Curfs, F. Amiot, J. F. Meis, and J. W. Van-der-Meer.
1999. Increased susceptibility of TNF-alpha lymphotoxin-alpha double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans. J. Immunol.63:1498-1505.
- 31.↵
Netea, M. G., P. N. Demacker, N. de Bont, O. C. Boerman, A. F. Stalenhoef, and J. W. Van der Meer.
1997. Hyperlipoproteinemia enhances susceptibility to acute disseminated Candida albicans infection in low-density-lipoprotein-receptor-deficient mice. Infect. Immun.65:2663-2667.
- 32.↵
Nijveldt, R. J., A. M. Tan, H. A. Prins, D. de Jong, G. L. van Rij, and R. I. Wesdorp.
1998. Use of a mixture of medium-chain triglycerides and long-chain triglycerides versus long-chain triglycerides in critically ill surgical patients: a randomized prospective double-blind study. Clin. Nutr.17:23-29.
- 33.↵
Rasmussen, A., I. Hessov, and E. Segel.
1988. The effect of intralipid on polymorphonuclear leukocytes. Clin. Nutr.7:37-41.
- 34.↵
Romani, L.
2000. Innate and adaptive immunity in Candida albicans infections and saprophytism. J. Leukoc. Biol.68:175-179.
- 35.↵
Sedman, P. C., S. S. Somers, C. W. Ramsden, T. G. Brennan, and P. J. Guillou.
1991. Effects of different lipid emulsions on lymphocyte function during total parenteral nutrition. Br. J. Surg.78:1396-1399.
- 36.↵
Sedman, P. C., C. W. Ramsden, T. G. Brennan, and P. J. Guillou.
1990. Pharmacological concentrations of lipid emulsions inhibit interleukin-2-dependent lymphocyte responses in vitro. J. Parenter. Enteral Nutr.14:12-17.
- 37.↵
Snydman, D. R., S. A. Murray, S. J. Kornfeld, J. A. Majka, and C. A. Ellis.
1982. Total parenteral nutrition-related infections. Prospective epidemiologic study using semiquantitative methods. Am. J. Med.73:695-699.
- 38.↵
Stratov, I., T. Gottlieb, R. Bradbury, and G. M. O'Kane.
1998. Candidaemia in an Australian teaching hospital: relationship to central line and TPN use. J. Infect.36:203-207.
- 39.↵
Tonnetti, L., R. Spaccapelo, E. Cenci, A. Mencacci, P. Puccetti, and R. L. Coffman.
1995. Interleukin-4 and -10 exacerbate candidiasis in mice. Eur. J. Immunol.25:1559-1565.
- 40.↵
Tufano, M. A., F. Rossi, F. Rossano, P. Catalanotti, L. Stella, and G. Servillo.
1995. Survival to lipopolysaccharide, cytokine release and phagocyte functions in mice treated with different total parenteral nutrition regimens. Immunopharmacol. Immunotoxicol.17:493-509.
- 41.↵
van Enckevort, F. H., M. G. Netea, A. R. Hermus, C. G. Sweep, J. F. Meis, and J. W. van der Meer.
1999. Increased susceptibility to systemic candidiasis in interleukin-6 deficient mice. Med. Mycol.37:419-426.
- 42.↵
Vazquez, W. D., G. Arya, and V. F. Garcia.
1994. Long-chain predominant lipid emulsions inhibit in vitro macrophage tumor necrosis factor production. J. Parenter. Enteral Nutr.18:35-39.
- 43.↵
The Veterans Affairs Total Parenteral Nutrition Cooperative Study Group.
1991. Perioperative total parenteral nutrition in surgical patients. N. Engl. J. Med.325:525-532.
- 44.↵
Waitzberg, D. L., R. Bellinati Pires, M. M. Salgado, I. P. Hypolito, G. M. Colleto, and O. Yagi.
1997. Effect of total parenteral nutrition with different lipid emulsions of human monocyte and neutrophil functions. Nutrition13:128-132.
- 45.↵
Wanten, G. J., A. H. Naber, J. W. Kruimel, A. T. Tool, D. Roos, and J. B. Jansen.
1999. Influence of structurally different lipid emulsions on human neutrophil oxygen radical production. Eur. J. Clin. Investig.29:357-363.
- 46.
Wanten, G. J., T. B. Geijtenbeek, R. A. Raymakers, Y. van Kooyk, D. Roos, and J. B. Jansen.
2000. Medium-chain triglyceride emulsions increase neutrophil adhesion and degranulation. J. Parenter. Enteral Nutr.24:228-233.
- 47.
Wanten, G. J., D. Roos, and A. H. Naber.
2000. Effects of structurally different lipid emulsions on human neutrophil migration. Clin. Nutr.19:327-331.
- 48.↵
Wanten, G. J., J. H. Curfs, J. F. Meis, and A. H. Naber.
2001. Phagocytosis and killing of Candida albicans by human neutrophils after exposure to structurally different lipid emulsions. J. Parenter. Enteral Nutr.25:9-13.