AJTMH Transactions of the Royal Society of Tropical Medicine and Hygiene
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Am. J. Trop. Med. Hyg., 75(4), 2006, pp. 749-752
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT


2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) REDUCES LEISHMANIA MAJOR BURDENS IN C57BL/6 MICE

OWEN J. BOWERS, KIRSA B. SOMMERSTED, RYAN T. SOWELL, GRETCHEN E. BOLING, WILLIAM H. HANNEMAN, RICHARD G. TITUS, AND GREGORY K. DEKREY*
Department of Medicine, University of Colorado at Denver and Health Sciences Center, Denver, Colorado; Department of Biological Sciences, College of Natural and Health Sciences, University of Northern Colorado, Greeley, Colorado; Department of Immunology, University of Texas, Graduate School of Biomedical Sciences at Houston, Houston, Texas; Department of Environmental and Radiological Health Sciences and Department of Microbiology, Immunology and Pathology, College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, Colorado

 

ABSTRACT

Acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can suppress adaptive immunity. In this study, pre-exposure of Leishmania major–infected mice to TCDD caused a dose-dependent and unexpected decrease in parasite burdens on day 20 after infection. In contrast, TCDD-mediated lymphoid atrophy, suppressed antibody levels, and enhanced interleukin-2 production were observed as expected. These results suggest that TCDD may enhance resistance to L. major in the face of immune suppression.

Leishmania major is an obligate intracellular parasite of mammals that resides within parasitophorous vacuoles of phagocytic cells, primarily macrophages. Most strains of domestic mice successfully resist primary subcutaneous infection with L. major by the production of parasite-killing NO within infected macrophages. This resistance is dependent on the development of L. major–specific CD4+ Th1 cells1–3 but does not require B cells or CD8+ T cells.4,5 In contrast, BALB/c mice that are infected in the same way will fail to control L. major growth because of Th2 responses and insufficient interferon (IFN)-{gamma}–induced macrophage activation.1–3

The aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a long-lived, non-metabolized environmental toxicant that is lipophilic and resistant to metabolic degradation, properties that facilitate bioaccumulation in animals.6,7 TCDD exposure can cause multiple toxic responses in humans and animals to varying degrees. These toxic responses include chloracne, teratogenesis, altered metabolism, carcinogenicity, and immunotoxicity. TCDD immunotoxic effects include lymphoid atrophy, especially of the thymus, and suppression of adaptive immunity (cellular and humoral), a response that has been observed in every experimental animal tested.8,9 TCDD toxicity is generally dependent on ligation of TCDD to the aryl hydrocarbon (Ah) receptor.9,10 Immunotoxic mechanisms downstream of the Ah receptor are still unclear but may include altered cytokine or hormone production, altered costimulation, altered phosphorylation activity, altered intracellular calcium homeostasis, and induction of apoptosis.8,9,11–14

The initial goal of this study was to use L. major–infected mice to explore the impact of TCDD on the development of a primary CD4+ T-cell response. To our knowledge, this is the first report of the effects of TCDD in L. major–infected mice. We hypothesized that TCDD exposure would suppress the development of resistant Th1 responses in L. major–infected C57Bl/6 mice, resulting in greater parasite burdens.

C57Bl/6 mice were originally obtained from Jackson Laboratories (Bar Harbor, ME) and used to establish a breeding colony to provide animals for these studies. The maintenance and care of all experimental animals complied with National Institutes of Health guidelines for the humane use of laboratory animals. TCDD was prepared in peanut oil for oral administration to mice as previously described.14 Female mice (6–8 weeks of age) were given peanut oil or TCDD 1 day before infection with 106 L. major promastigotes (LV39, RHO/SU/59/P, Neal, or P strain) by subcutaneous injection into one rear foot pad.15

After 20 days of infection, typical evidence of TCDD-mediated immune suppression was observed. As shown in Table 1Go (line 1), TCDD exposure at 40 µg/kg caused a significant decrease in thymus weights (normalized to body weights) by 22% relative to controls (P = 0.022). TCDD treatment (40 µg/kg) also significantly reduced the numbers of viable cells (measured by trypan blue exclusion) per lesion-draining popliteal lymph node by 47% relative to controls (Table 1Go, line 6; P = 0.046). The levels of serum antibodies specific to soluble L. major antigens were measured using an ELISA16 with a phosphatase-conjugated secondary goat anti-mouse Ig antibody (KPL, Gaithersburg, MD). At a serum dilution of 100:1, L. major–specific antibody levels from TCDD-treated mice (40 µg/kg) were ~37% of control levels (Table 1Go, line 13). These results confirmed previously reported effects of TCDD.8–14


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TABLE 1
TCDD treatment decreased lymphoid cell numbers and increased IL-2 production at 20 days after L. major infection
 
In contrast to our prediction, TCDD exposure did not cause an increase in parasite burdens. Foot parasite burdens were determined by limiting dilution analysis as previously described.17 As shown in Figure 1AGo, on day 20 after infection, a dose-dependent decrease in the number of viable L. major was observed per foot in TCDD-treated mice. Exposure to TCDD at the highest dose (40 µg/kg) resulted in a significant decrease in viable L. major numbers by ~10-fold when compared with control mice (Figure 1BGo). In addition, TCDD exposure at 40 µg/kg significantly delayed the resolution of foot lesions (Figure 1CGo). Lesion size was monitored over time with vernier calipers (lesion size = infected foot – contralateral uninfected foot). The lesions of all infected animals eventually resolved, regardless of treatment, and the numbers of viable L. major in infected feet or lesion-draining lymph nodes on day 139 after infection were < 130 (data not shown). These results indicate that TCDD did not prevent a resistant outcome of L. major infection in C57Bl/6 mice.


Figure 1
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  		    FIGURE 1. TCDD treatment decreased L. major burdens in foot lesions while delaying the resolution of those lesions. Peanut oil or TCDD were given orally 1 day before infection with 106 L. major promastigotes in the left rear foot pad.10,11 Parasite burdens12 were determined on day 20 after infection for pools (two to three mice per pool) of homogenized feet. A, Results from one dose–response experiment with one to two pools of feet per treatment group are shown. B, Results from three independent experiments are shown combined. Data were analyzed by two-way ANOVA and are presented as least square means ± SEM for the effect of TCDD. C, Lesion thickness over time is shown as mean ± SEM for three to five mice per treatment group. Data were analyzed by repeated-measure two-way ANOVA followed by Tukey all-pairwise comparison. *Statistically significant difference from control (P < 0.05).

 
Coordinated cytokine production is essential for control of L. major, and TCDD has been previously shown to alter antigen-driven cytokine production in mice.14,18–20 In particular, interleukin (IL)-2 expression can be directly upregulated by TCDD through activation of the Ah receptor.11 Ex vivo cytokine production was examined in this study on day 20 after L. major infection. Cytokines in supernatants from cultures of L. major–restimulated popliteal lymph node cells were measured by ELISA.21 As shown in Table 1Go (line 8), for cells taken from TCDD-treated mice, the levels of IL-2 in culture were significantly higher (by 91%) than those of controls (P < 0.001). In contrast, no significant TCDD-mediated change in IL-4, IL-5, IL-10, or IFN-{gamma} levels were found (Table 1Go, lines 9–12). In addition, no significant changes in lymphocyte differentials were observed in the popliteal lymph nodes of TCDD-treated mice (CD4+, CD8+, or B220+ cells, measured by flow cytometry22; Table 1Go, lines 3–5).

TCDD is not known to cause generalized activation of macrophages, but some changes in macrophage function have been reported. These include increased superoxide production,23,24 increased tumor necrosis factor (TNF)-{alpha} production,25,26 reduced endocytosis and adherence,27 and reduced B7 expression.13 In contrast, measurable changes in apoptosis,27 phagocytosis, or tumor cell killing28,29 have not been observed. One explanation for the reduced parasite numbers observed in this study (Figure 1Go) may be the altered cytokine environment. Nacy and others30 showed that IL-2 can act as a co-factor, along with IFN-{gamma}, to enhance the NO-mediated killing of L. major by macrophages. Importantly, Nacy and others30 also showed that enhanced TNF production by IL-2 + IFN-{gamma}-–treated macrophages was an essential intermediate step leading to enhanced parasite killing. Thus, TCDD-enhanced IL-2 production and normal IFN-{gamma} production (Table 1Go), coupled with enhanced TNF production,31,32 may enhance NO production, leading to reduced parasite numbers. The role of TNF in reducing parasite burdens in this experimental model will be explored in future studies.

TCDD has been shown to suppress the immune responses of mice to a variety of infectious agents including Listeria monocytogenes, Plasmodium yoelii, murine influenza virus, and others.18,33,34 TCDD-suppressed resistance to experimental infection can result in increased disease severity and mortality.9,18,32 We believe that the data presented here are unique in suggesting enhanced resistance to L. major in TCDD-treated mice, which simultaneously displayed some of the classic signs of TCDD-induced adaptive immune suppression. TCDD is unlikely to cause reduced L. major burdens through direct toxicity to the parasite because, although ligand-activated Ah receptors are known in metazoans, they are not known in protists.35 Thus, any direct toxicity of TCDD in Leishmania would likely be caused through a different mechanism. The authors are unaware of any published studies of the effects of TCDD in protozoans. Preliminary studies in this laboratory have found that L. major proliferation and infectivity were unchanged when the parasites were cultured in the presence of TCDD at concentrations up to 5 x 10–7 mol/L (data not shown), a concentration that is higher than would be expected in the skin of a mouse given TCDD at 40 µg/kg.36 Therefore, the impact of TCDD on L. major burdens in mice is probably caused by the effects of TCDD on mice alone.

Received October 4, 2005. Accepted for publication May 24, 2006.

Financial support: This work was supported by the University of Northern Colorado, Colorado State University, and Public Health Service Grant 29955.

* Address correspondence to Gregory K. DeKrey, School of Biological Sciences, College of Natural and Health Sciences, University of Northern Colorado, 501 20th Street, Greeley, CO 80639. E-mail: gregory.dekrey{at}unco.edu Back

Authors’ addresses: Owen J. Bowers, Division of Infectious Diseases, School of Medicine, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80126, Telephone: 303-315-3558, Fax: 303-315-8054, E-mail: owen.bowers{at}UCHSC.edu. Kirsa B. Sommersted, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, Telephone 970-491-2357, E-mail: kirsa{at}prodigy.net. Ryan T. Sowell, Department of Immunology, University of Texas, Graduate School of Biomedical Sciences at Houston, 6767 Bertner Ave., Houston, TX 77225-0334, Telephone: 303-847-7398, E-mail: rsa7469{at}gmail.com. Gretchen E. Boling, Department of Biological Sciences, College of Arts and Sciences, University of Northern Colorado, 501 20th Street, Greeley, CO 80639, Telephone: 970-351-2921, Fax: 970-351-2335, E-mail: laxcapt21{at}hotmail.com. William H. Hanneman, Department of Environmental and Radiological Health Sciences, College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, CO, 80523, Telephone: 970-491-8635, Fax: 970-491-7569, E-mail: hanneman{at}colostate.edu. Richard G. Titus, Department of Microbiology, Immunology and Pathology, College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, CO, 80523, Telephone: 970-491-4964, Fax: 970-491-0603, E-mail: richard.titus{at}colostate.edu. Gregory K. DeKrey, School of Biological Sciences, College of Natural and Health Sciences, University of Northern Colorado, 501 20th Street, Greeley, CO 80639, Telephone: 970-351-2493, Fax: 970-351-2335, E-mail: gregory.dekrey{at}unco.edu.

 

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