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Am. J. Trop. Med. Hyg., 77(4), 2007, pp. 691-698
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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The Immune Response to Toxocariasis Does Not Modify Susceptibility to Mycobacterium tuberculosis Infection in BALB/c Mice

Fabiani Gai Frantz, Rogério Silva Rosada, Walter Miguel Turato, Camila Matias Peres, Arlete Aparecida Martins Coelho-Castelo, Simone Gusmão Ramos, David Michael Aronoff, Célio Lopes Silva, AND Lúcia Helena Faccioli*
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo—Ribeirão Preto, São Paulo, Brazil; Núcleo de Pesquisas em Tuberculose—Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil; Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo—Ribeirão Preto, São Paulo, Brazil; Division of Infectious Disease, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium tuberculosis and helminth infections coincide geographically and are classically described as TH1 and TH2 pathologies. There is much interest in exploring how concurrent worm infections might alter immune responses to mycobacterial infection. To explore this issue, mice were infected with Toxocara canis and co-infected with M. tuberculosis. Mice infected with M. tuberculosis had high numbers of neutrophils and mononuclear cells within the alveolar spaces, with increased parenchymal interferon (IFN)-{gamma} levels. However, in Toxocara-infected mice we detected increased eosinophil numbers in bronchoalveolar lavage fluid (BALF) and increased parenchymal levels of interleukin (IL)-5. In co-infected mice the BALF demonstrated enhanced eosinophil influx with decreased neutrophil and mononuclear cell accumulation. However, co-infected mice had similar mycobacterial proliferation in their lungs accompanied by similar histopathological changes and similar cytokine/nitric oxide production compared with Mycobacterium-only–infected mice. Our results suggest that T. canis infection does not necessarily lead to increased susceptibility to pulmonary tuberculosis.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Helminth parasites cause a significant burden of illness worldwide, with estimates of two billion people infected.1 The largest proportions of human populations infected live in tropical and subtropical regions.2 Helminth infections are potent inducers of TH2-type responses in both humans and experimental models, which are characterized by eosinophilia, high titers of circulating IgE, enhanced TH2 cytokine profile (e.g., increased secretion of interleukin [IL]-4 and IL-5), and reduced TH1 type cytokines (e.g., interferon [IFN]- {gamma}).3 Studies conduced in animal models of TH1-inducing pathogens and/or human preclinical tests of certain vaccines revealed that the potency of TH2 polarization induced by helminthic parasites impaired subsequent TH1 immune responses.4–8

To improve therapies and vaccination protocols against tuberculosis, it is important to investigate the influence of co-infection with different worms on the immune response against mycobacterial pathogens.9 Little is known about the interaction between toxocariasis, caused by the helminths Toxocara canis or Toxocara cati, and tuberculosis along the TH1/TH2 paradigm described above. However, toxocariasis is an important parasitic infection in tropical and subtropical regions10 that induces robust TH2 responses in humans.11 We hypothesized that prior T. canis infection would modify the immune response pattern (TH1 versus TH2) and susceptibility to Mycobacterium tuberculosis (Mtb) infection. We found that infection of BALB/c mice with T. canis elicited a TH2 response, but it did not significantly alter the TH1 immune response (or susceptibility) to subsequent infection with M. tuberculosis.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Specific pathogen-free female 6-week-old BALB/c mice were obtained from the animal facilities of Faculdade de Ciências Farmacêuticas—Universidade de São Paulo and bred in a SPF facility. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of the University. Infected animals were kept in biohazard animal room (Laboratory Bio-safety Level 3) and housed in cages within a laminar-flow safety enclosure.

Experimental infection. T. canis helminth eggs were obtained by the method of Olson and Schulz,12 as modified by Faccioli and others.13 In brief, pregnant female worms were recovered from dogs. The eggs were then rescued from the worm uterus, washed, and sustained in 0.5% formalin at 37°C in shallow dishes, where they were allowed to develop into an infective stage. Before being used, the eggs were thoroughly washed with saline. Infective doses of 500 embryonated eggs in 0.5 mL of saline were prepared. Mice were infected by gastric intubation via a metal cannula. Control group animals received 0.5 mL of saline only.

M. tuberculosis. The H37Rv strain of M. tuberculosis (American Type Culture Collection, Rockville, MD) was grown in 7H9 Middlebrook broth (Difco Laboratories, Detroit, MI) for 7 days. The culture was harvested by centrifugation, and the cell pellet was resuspended in sterile phosphate-buffered saline (PBS) and vigorously agitated. The homogeneous suspension was filtered through 2-µm filters (Millipore, Bedford, MA). Viability of the M. tuberculosis suspension was pretested with fluorescein diacetate (Sigma, St. Louis, MO) and ethidium bromide. An anterior midline incision was made to expose the trachea. A 30-gauge needle attached to a tuberculin syringe was inserted into the trachea, and intratracheal dispersion was used to introduce 105 viable colony-forming units (CFU) of M. tuberculosis H37Rv in 100µL of PBS into the lungs.14 At 30 and 70 days after the M. tuberculosis challenge, mice from all groups were killed. Control mice received intratracheal PBS only. In the co-infected group, infection with intratracheal M. tuberculosis was performed 18 days after the T. canis infection, the time at which the helminth induces a strong TH2 response.15

Bronchoalveolar lavage fluid (BALF). On days 30 and 70 following M. tuberculosis infection, animals were euthanized with sodium pentobarbital. The anterior chest cavity of each animal was carefully opened, and the trachea was exposed and catheterized. The catheter was tied in place, and sterile PBS was infused in three 1-mL aliquots. Lavage fluid was recovered and placed on ice. Total cell counts were immediately performed in a Neubauer Chamber (Boeco, Hamburg, Germany). Differential counts were obtained using Rosenfeld-stained cytospin preparations.16 Lavages were performed prior to removal of lungs for microbiological and histologic analyses (below), and spleens were removed aseptically from the same mice.

Determination of M. tuberculosis CFU in lungs. Recovery of M. tuberculosis was performed as described previously.17 Briefly, the number of live bacteria recovered from the lungs was determined as CFU by plating 10-fold serial dilutions of homogenized tissue on Middlebrook 7H11 agar (Difco) and counting colonies after 28 days at 37°C (expressed as log10 of CFU/g of lung tissue).

Measurement of cytokines and nitrite in lung tissues. For cytokine measurements, lungs were removed on days 30 and 70 post-M. tuberculosis infection. Tissue was homogenized in 2 mL of RPMI 1640 and centrifuged at 450g, and the supernatant was stored at –70°C until assayed. Commercially available enzyme-linked immunosorbent assay (ELISA) antibodies were used to measure IL-5, IL-10, IL-12, and IFN-{gamma} (OptEIA, BD-Pharmingen, San Diego, CA). The plates were coated with 100 µL/well of the capture antibody (1–4 µg/mL) diluted in coating buffer (0.1 M sodium carbonate, pH 9.5) and incubated overnight at 4°C. Plates were then washed 5 times with 300 µL/well of wash buffer (PBS with 0.05% Tween-20), and nonspecific binding was blocked by addition of 200 µL/well assay diluent (PBS with 10% fetal bovine serum, pH 7.0) and incubated at room temperature (RT) for 1 hr. After the plate was washed as described above, 100 µL of each standard, sample, and control was added to appropriate wells followed by a 2-hr incubation at RT. The plates were again washed as above, and 100 µL of working detector solution [biotinylated detection antibody (0.5–2.0 µg/mL) + streptavidin-conjugated horseradish peroxidase reagent] was added to each well with a 1-hr incubation at RT. After 7 total washes, we added 100 µL of substrate solution (TMB substrate reagent set) to each well and incubated the plates for 30 min at RT in the dark. The reaction was stopped by addition of 50 µL of stop solution (2 N H2SO4), and the optical density was measured at 450 nm within 30 min.

Nitric oxide (NO) production was assessed by measuring the amount of nitrite in lung homogenates by the Greiss reagent method.18. Data are presented as micromoles of NO2–

Spleen cell cultures and cytokine determination. Spleens from mice killed 30 or 70 days post-M. tuberculosis infection were aseptically removed and minced, and the released cells were washed three times in RPMI 1640 (Gibco BRL, Grand Island, NY). Cells were suspended at 5 x 106 cells per mL in RPMI supplemented with 10% fetal bovine serum (Gibco BRL), penicillin (100 U/mL, Gibco BRL), and streptomycin (100 µg/mL, Gibco BRL) and dispensed into 96-well flat-bottom microtiter plates in a volume of 0.1 mL. Concanavalin A (2 µg/mL, Sigma) and 10 µg/mL of total proteins isolated from T. canis or heat-killed M. tuberculosis 105 bacilli/mL were added to wells (0.1 mL) in triplicate and maintained for 48 hr at 37°C. Heat-killed preparations of M. tuberculosis strain H37Rv (HK-Mtb) were obtained by killing the bacilli at 80°C for 2 hr. Commercially available ELISA antibodies were used to measure IFN-{gamma} and IL-5 (OptEIA, BD-Pharmingen) on supernatants of cultured cells as above.

Preparation of T. canis total antigen. To prepare a total protein lysate from T. canis eggs, a suspension of larvae eggs obtained as described above was washed five times with sterile PBS.19 The suspension was sonicated by a sonic dismem-brator VC 50T (Sonics & Materials, Inc., Danbury, CT) for 15 min with 1-min rests between 1-min bursts. The sonicate was centrifuged at 10,000g for 30 min at 4°C to remove cell debris. The supernatant was filtered through a 0.22-mm-pore-size membrane filter, and the protein content was determined by the Bradford method. The soluble antigen preparations contained 100–150 µg protein/mL and were stored in aliquots at – 70°C.

Histology. At 30 and 70 days post-M. tuberculosis infection, the left upper lobe of each mouse lung was removed and fixed in 10% formalin, embedded in paraffin blocks, prepared routinely, then sectioned for light microscopy. Sections (5 µm each) were stained either with hematoxylin and eosin (H&E) or by the Ziehl–Neelsen (ZN) method for detection of acid-fast bacilli. Slides were evaluated using a Leitz Aristoplan microscope (Leica, Wetzlar, Germany) connected to a color camera (Model DFC280, Leica, Heerbrugg, Germany). Microscope images of the stained tissue sections were captured by a camera linked to a PC computer.

Statistical analysis. Data are represented as mean ± SEM, N = 5 (PBS group) or N = 6 (other groups), and were analyzed with using GraphPad Prism version 4.02 for Windows (GraphPad Software, San Diego, CA). All figures represent data from one representative experiment performed on at least 3 separate occasions. Comparisons were performed with unpaired t-test on CFU analyses or one-way ANOVA with Bonferroni’s post-test. Differences were considered significant if P < 0.05.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Co-infection does not alter M. tuberculosis replication and lung pathology. The results obtained in bacterial CFU from co-infected mice were compared with mice infected with M. tuberculosis alone. The results showed that mice with concurrent T. canis infection presented similar bacterial loads in the lung, compared with M. tuberculosis-infected mice at two time points tested. In the lungs of both groups, bacterial loads showed a slight increase at 70 days (Figure 1Go).


Figure 1
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  		    FIGURE 1. Number of colony forming units (CFU) of M. tuberculosis in the lungs. Co-infection with T. canis was performed 18 days before intratracheal inoculation with M. tuberculosis and the numbers of bacilli in the lungs were determined at 30 ({circ}) and 70 days ({blacksquare}) post-bacterial challenge. Bars represent the arithmetic means.

 
Histologic analysis of the lungs also revealed that cellular accumulation and organization, and the resulting reduction in air exchange areas were similar in co-infected and M. tuberculosis infected mice at both 30 and 70 days post-infection. Figure 2Go represents lung histology at 30 days post-M. tuberculosis infection. Histologic sections of H&E-stained lungs from co-infected and M. tuberculosis-infected mice were characterized predominantly by the presence of macrophages and lymphocytes accumulated mainly in perivascular and peri-bronchial areas (Figure 2AGo). By ZN staining for acid-fast bacilli, there were no obvious differences in the bacillary loads between lung sections of co-infected and M. tuberculosis-infected mice (Figure 2BGo).


Figure 2
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  		    FIGURE 2. Leukocyte recruitment and bacillary burden in the lung parenchyma 30 days post-M. tuberculosis infection. Representative lung sections from uninfected mice (PBS), mice infected with T. canis (Tc), M. tuberculosis (Mtb), or both (Tc/Mtb). (A) H&E-stained histopathology of lung section and (B) ZN staining for acid-fast bacilli. This figure appears in color at www.ajtmh.org.

 
Previous T. canis infection modifies leukocyte recruitment induced by M. tuberculosis. Intratracheal inoculation of M. tuberculosis induced significant neutrophil and mononuclear leukocyte accumulation in the lungs at 30 and 70 days post-infection, as judged by BALF, while T. canis infection induced predominantly eosinophils. No such cellular accumulation occurred in the control-uninfected group (Figure 3Go). Also, we compared the cellular accumulation between co-infected mice and animals only infected with M. tuberculosis. Co-infection elicited significantly fewer neutrophils and mononuclear cells at both 30 and 70 days after infection compared with singular M. tuberculosis inoculation. However, at these time points, eosinophil numbers in the bronchoalveolar space of co-infected animals were significantly greater than those of M. tuberculosis infected animals and were similar to the eosinophil counts observed in T. canis-infected mice (Figure 3Go).


Figure 3
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  		    FIGURE 3. Alveolar leukocyte numbers during early ({circ}) and late ({blacksquare}) phases of infection by M. tuberculosis. BALF cells were obtained from mice after intratracheal injection of PBS; oral infection with T. canis (Tc), and intratracheal infection with M. tuberculosis (Mtb) or both (Tc/Mtb). (A) Mononuclear cells; (B) neutrophils; (C) eosinophils. Cells were enumerated and identified after Rosenfeld staining. *PBS vs. other groups; #Tc vs. other groups; §Mtb vs. other groups. P < 0.05.

 
T. canis infection does not alter cytokine or nitric oxide production induced in lung cells by M. tuberculosis. Because proinflammatory and TH1 cytokines play important roles in the immune response against M. tuberculosis, we sought to investigate whether co-infection could alter cytokine production in the lung parenchyma. At 30 and 70 days post-M. tuberculosis infection, lung cells were obtained from animals that were either co-infected or singly infected (M. tuberculosis or T. canis only). In M. tuberculosis-infected animals, IL-12 and IFN-{gamma} levels in the lungs increased at the two analyzed periods, with highest levels being observed at day 30. In the co-infected animals, the levels of IL-12 and IFN-{gamma} were similar to M. tuberculosis-infected animals (Figure 4A and BGo) at both time points. No such increases in IL-12 or IFN-{gamma} levels were observed in T. canis-infected mice above the control group. Levels of IL-10 were similar in T. canis and co-infected animals and greater than PBS-inoculated animals, whereas IL-5 increased only at day 30 in the T. canis-infected group (Figure 4C and DGo).


Figure 4
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  		    FIGURE 4. Cytokine levels and nitrite production in lung tissue 30 ({circ}) and 70 days ({blacksquare}) after infection by M. tuberculosis. Mice were subjected to either intratracheal PBS injection; oral infection with T. canis (Tc); intratracheal infection with M. tuberculosis (Mtb) or both (Tc/Mtb). IL-12 (A), IFN-{gamma} (B), IL-10 (C), and IL-5 (D) levels were determined by ELISA. Nitrite production was quantified by Griess reaction in the supernatants of cellular lysate from lung parenchyma (E). *PBS vs. other groups; #Tc vs. other groups. P < 0.05.

 
As nitric oxide (NO2– ) is an important microbicidal factor, we analyzed its production within the lung parenchyma. M. tuberculosis-infected mice produced increased levels of NO2– compared with cells from control animals. Cells obtained from co-infected animals showed similar NO2– production compared with M. tuberculosis-infected animals, and the lung cells from T. canis-infected mice did not produce NO2– above the levels found in control mice inoculated with PBS (Figure 4EGo).

Immune responses to M. tuberculosis in the spleen are not switched by co-infection. We were interested in characterizing the TH1 versus TH2 immune responses to either M. tuberculosis or T. canis by isolated splenocytes, and testing the hypothesis that prior T. canis infection per se can modulate the TH profile in response to M. tuberculosis antigens. Therefore, spleen cells were collected from either Mtb-infected or co-infected mice and then stimulated in vitro with either heat-killed M. tuberculosis (HK-Mtb), or total proteins from infective eggs of T. canis containing L3 larvae (TP-Tc), or the nonspecific stimulus concanavalin-A (Con-A). We observed similar immune response profiles at days 30 and 70 post-infection, and Figure 5Go represents the data from day 30. Spleen cells from M. tuberculosis-infected mice stimulated in vitro with HK-Mtb or Con-A produced high levels of IFN-{gamma}, indicative of a TH1 response. These cells produced lower amounts of IFN-{gamma} when stimulated in vitro with TP-Tc, when compared with other stimuli. Cells from T. canis-infected mice did not produce IFN-{gamma} after in vitro stimulation. Cells from co-infected mice produced significantly more IFN-{gamma} compared with cells from uninfected animals when stimulated with HK-Mtb or Con-A, but this increase was not as robust as we observed in splenocytes from Mycobacterium-only infected mice. We also observed that spleen cells from T. canis-infected and co-infected mice both produced high levels of IL-5 when stimulated with TP-Tc or Con-A, suggesting a TH2 immune profile (Figure 5Go).


Figure 5
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  		    FIGURE 5. Cytokine levels in the supernatant of spleen cells harvested 30 days after infection by M. tuberculosis. Mice were subjected to either intratracheal PBS injection; oral infection with T. canis (Tc); intratracheal infection with M. tuberculosis (Mtb) or both (Tc/Mtb). The cells were stimulated in culture with total Tc antigens ({triangledown}) or heat-killed TB ({circ}), and IFN-{gamma} (A) and IL-5 (B) levels of culture supernatant were determined by ELISA. Bar graphs represent Con-A stimulation. *PBS vs. other groups; #Tc vs. other groups. P < 0.05.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Toxocariasis is an important helminth pathogen in developing countries20,21 including Brazil, where the prevalence rates reach ~40%.22,23 Tuberculosis remains one of the leading causes of morbidity and mortality in many settings, particularly in the world’s poorest countries. It is estimated that nearly 1.7 million people die each year from tuberculosis, and in 2004 approximately 8.9 million people developed the disease.24 Because mycobacterial and helminth pathogens are coendemic and tend to induce opposing immune responses, we investigated the pattern of inflammatory and immune response in a model of T. canis and M. tuberculosis co-infection. In this report, we studied the effect of previous T. canis infection on the susceptibility of BALB/c mice to subsequent M. tuberculosis infection, by determining the bacterial load, the degree of lung pathology, and the in vivo and in vitro TH1/TH2-type responses of splenic lymphocytes (IFN-{gamma} and IL-5 synthesis, respectively) to mycobacteria and T. canis egg antigens.

Our study was limited by the use of a single, BALB/c, murine strain. We have chosen this strain because it is relatively resistant to lethality from M. tuberculosis,25,26 facilitating long-term studies of systemic and tissue immune responses. Because BALB/c mice are prone to developing TH2-type immune responses to infection,27 it is possible that the use of other mouse strains might yield different results than the present report. Furthermore, for our in vivo infections we used a single inoculum size for both T. canis and M. tuberculosis. We selected these infecting doses based on previously published protocols14,28 with the intention of causing sublethal infections. Whether different inoculum sizes would yield contrary results remains uncertain.

We believe that the present study for the first time demonstrates that mice infected with T. canis are not more susceptible to subsequent M. tuberculosis infection. Co-infection did not increase bacterial propagation in vivo and was associated with the development of IFN-{gamma} producing TH1 cells, similar to mice infected only with M. tuberculosis. Our data agree with literature showing that the rodent hookworm Nippostrongylus brasiliensis did not lead to enhanced susceptibility to mycobacterial infection.29 However, Elias and colleagues30 demonstrated that Schistosoma mansoni infection altered the immune response pattern to M. tuberculosis, leading to an increased bacterial burden during infection. These experimental differences highlight the complex interactions among various parasitic organisms, mycobacteria, and infected hosts.

Although T. canis infection did not modify host susceptibility to tuberculosis infection, we postulated that immune responses characteristic of tuberculosis infection might be altered by the TH2 response elicited by this nematode. Initially, we analyzed the immunopathological features of the infected lungs, including cellular infiltration into infected alveoli (by BALF analyses), and pulmonary parenchymal morphology. M. tuberculosis infection lead to increased neutrophil and mononuclear cell migration into alveoli and an intense inflammatory reaction in the parenchyma, as observed in lung histology sections. These data are in accordance with previous data from our research group.28,31 Moreover, T. canis infection induced enhanced eosinophil accumulation in BALF, as previously described.15 Our results demonstrated that co-infection indeed altered the types of cells migrating into the alveolar space when compared with M. tuberculosis-infected mice. We observed reductions of neutrophils and mononuclear cell numbers and increased numbers of eosinophils. It follows, then, that these alterations in inflammatory cell numbers in the alveoli might not impact bacterial clearance, as CFU numbers recovered from M. tuberculosis-infected and co-infected mice were not significantly different (Figure 1Go). Despite differences in alveolar cell recruitment, we observed similar inflammatory cell recruitment to the lung parenchyma in both M. tuberculosis and co-infected animals (Figure 2Go). The similar profile of parenchymal leukocytes in both singly and doubly infected mice might explain why we failed to detect alterations in IL-12, IFN-{gamma}, and NO2– levels in co-infected mice, in comparison with mice with only M. tuberculosis (Figure 4Go). The importance of NO2–, IL-12, and IFN-{gamma} has been well documented in murine tuberculosis.32–34 IFN-{gamma} is a central factor in the activation of antimycobacterial activities of macrophages and is considered crucial for protection against tuberculosis.35 Nitric oxide is an effective host-defense mechanism against many microbial pathogens and plays an essential role in the killing of M. tuberculosis by mononuclear phagocytes.36 As there is evidence that leukocytes present in the lung parenchyma might be the source of such mediators,37 this lung compartment was chosen for assessing immune responses during infection, to complement our studies of cellular BALF components. Additionally, Franke-Ullmann and others38 have shown that interstitial macrophages are more efficient in releasing immunoregulatory cytokines such as IL-1 and IL-6, expressing MHC class II molecules, and are more effective in functioning as accessory cells for mitogen-stimulated lymphocyte proliferation compared with alveolar macrophages. Therefore, we suggest that cells from either Mycobacterium-infected mice or co-infected mice are equipped to cooperate with interstitial lymphocytes in inducing a specific TH1-immune reaction, independent of the presence of T. canis.

To perform in vitro analyses of immune responses, we stimulated spleen cells with whole T. canis eggs as well as mycobacterial lysates. This approach, in contrast to utilizing recombinant or highly purified proteins/antigens, mimics in vivo infection and allows for a broad array of microbe-associated structures to stimulate cells of the innate and adaptive immune system, including superantigens, Toll-like receptor ligands, and nonprotein signature molecules.3,39 The cell-wall component lipoarabinomannan from M. tuberculosis, for example, is involved in the inhibition of phagosome maturation, apoptosis, and IFN-{gamma} signaling in macrophages and IL-12 secretion of dendritic cells.40 Moreover, complex carbohydrates present in worm antigens are potent inducers of TH2 responses by binding to cell-surface receptors, such as DC-SIGN, L-SIGN, the mannose receptor, macrophage galactose binding lectin, and other lectins, which might contribute to the induction of TH2-associated adaptive responses.41

Our in vitro data, obtained by using spleen cells collected 30 (and 70) days after M. tuberculosis infection and stimulated with or without whole immunogens, support our in vivo results obtained in lung tissue at 30 and 70 days post-infection (Figure 5Go). The M. tuberculosis infection stimulated an immune response in the lungs and spleen toward a TH1 profile, as indicated by our finding that cells from Mtb-only infected mice produced higher levels of IFN-{gamma} and lower levels of IL-5 in response to HK-Mtb, when compared with cells stimulated with TP-Tc. We noted that IFN-{gamma} production in response to HK-Mtb (or Con-A) observed in splenocytes obtained from co-infected mice was significantly greater than splenocytes from un-infected mice. Although this was qualitatively similar to our observation in cells from Mtb-only infected animals, the absolute increase in IFN-{gamma} was lower in co-infected than Mtb-only infected splenocytes (Figure 5AGo). These data suggest that prior T. canis infection can partially suppress TH1 responses during subsequent Mtb infection, but this influence was insufficient to alter the pathologic course of the second infection. Furthermore, cells from T. canis or co-infected mice responded in vitro to specific T. canis antigens, as judged by similarly high levels of IL-5 produced. These results indicate that subsequent M. tuberculosis infection (after 18 days) did not interfere the pre-established TH2 response in the spleen. Moreover, TH2 cells present in the lung of co-infected animals were unable to deviate the TH1 immune response against Mtb infection. Our data, in agreement with that of Erb and others,29 show that prior sensitization of the immune system by helminths does not necessarily influence subsequent responses to unrelated pathogens, as was demonstrated by other authors.42–47 It has recently become apparent that regulatory T cells (Treg) might be important in directing immunologic responses to pathogens,48,49 and Oldenhove and others50 clearly demonstrated that enhanced Treg function associated with helminth infection may suppress TH1 responses directed against unrelated antigens and/or pathogens.

It seems that uncontrolled TH2 immune responses induced by certain helminthic infections provide a propitious microenvironment for enhanced mycobacterial growth, which was not observed in our model. We suggest that the immune response induced by toxocariasis, at least in the period analyzed by us, modifies the homeostasis of the immune system but not to such an extent that it impairs the host’s ability to mount an effective TH1 response against M. tuberculosis. Taken together, we propose that the impact of helminthiasis on the host response to intracellular pathogens is not yet clear and is dependent on the nature of the parasite. It is essential to be evaluating the reasons that some helminth infections modify the immune response to tuberculosis while others do not, which might clarify the regulatory mechanisms involved during concurrent infections.

Received January 19, 2007. Accepted for publication May 22, 2007.

Acknowledgments: We are grateful to Carlos Artério Sorgi, Érica Vitaliano Garcia da Silva, Izaíra Tincani Brandão, Ana Paula Massom, and Elaine Medeiros Floriano for their technical assistance.

Financial support: This study was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 00/09663-2 and 03/12887-8) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

* Address correspondence to Lúcia H. Faccioli, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n Ribeirão Preto, São Paulo, Brasil 14.040-903. E-mail: faccioli{at}fcfrp.usp.br Back

Partial results were presented at the 54th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Washington, DC, December 11–15, 2005 (oral presentation during the scientific session Bacteriology III—Respiratory/Other).

Authors’ addresses: Fabiani G. Frantz, Walter M. Turato, Camila M. Peres, and Lúcia H. Faccioli, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo—Av. do Café, s/n. Ribeirão Preto, São Paulo, Brasil, 14.040-903, Telephone: +55-16-3602 4303, Fax: +55-16-3602 4725, E-mails: frantz{at}fcfrp.usp.br (F.G.F.), turato{at}rpm.fmrp.usp.br (W.M.T.), pcamila{at}usp.br (C.M.P.), and faccioli{at}fcfrp.usp.br (L.H.F.). Rogério S. Rosada, Arlete A.M. Coelho-Castelo, and Célio Lopes Silva, Núcleo de Pesquisas em Tuberculose—Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo—Av. dos Bandeirantes, 3900 Ribeirão Preto, São Paulo, Brasil, 14.049-900, Telephone/Fax: +55-16-3602 3228, E-mails: rosada{at}cpt.fmrp.usp.br (R.S.R.), accastel{at}rpm.fmrp.usp.br (A.A.M.C-C.), and clsilva{at}cpt.fmrp.usp.br (C.L.S.). Simone G. Ramos, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo—Av. dos Bandeirantes, 3900 Ribeirão Preto, São Paulo, Brasil, 14.049-900, Telephone: +55-16-3602 3341, Fax: +55-16-3633 1068, E-mail: sgramos{at}fmrp.usp.br. David Michael Aronoff, Division of Infectious Diseases Department of Internal Medicine, 5220-D MSRB III, 1500 W. Medical Center Drive, Ann Arbor, MI 48109-0640, Telephone: +1 (734) 647-1786, Fax: +1 (734) 764-4556, E-mail: daronoff{at}umich.edu.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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