| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1,2Departamento de Microbiologia1 and Departamento de Bioquímica-Imunologia2, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 3Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil
Correspondence Jacques R. Nicoli, jnicoli{at}icb.ufmg.br
Received March 2, 2004
Accepted April 27, 2004
To study the influence of normal associated microbiota on systemic immunological responses during experimental Chagas’ disease, germ-free and conventional NIH Swiss mice were infected with Y strain of Trypanosoma cruzi. Although no statistical differences in mortality and parasitaemia were found, conventional mice showed IFN-
, TNF-
and NO production (P < 0.05) by spleen cell cultures and higher blood levels of immunoglobulins of the IgG2a isotype (P < 0.05) when compared to their germ-free counterparts. Moreover, higher levels of IgG1 were also found in conventional animals. On the other hand, no differences in IL10 production were found between germ-free and conventional mice after infection (P < 0.05). Interestingly, spleen cell cultures from non-infected germ-free mice spontaneously produced higher levels of IL10 than cultures from conventional mice. Moreover, cultures from non-infected germ-free mice responded to T. cruzi antigens with IFN- production, contrary to cultures from conventional animals. In conclusion, the presence of the normal microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in mice. However, the increase in production of cytokines that is linked to resistance to this parasite did not alter the outcome of infection significantly, probably due to high virulence of the Y strain.
 |
INTRODUCTION |
|---|
 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
T. cruzi is the causative agent of Chagas’ disease in man and the protozoa determines a systemic infection that is controlled, although not completely eliminated, by T-cell-dependent immune responses. Control of parasitism in the acute phase of infection is critically dependent on intracellular killing by cytokine-activated macrophages. In this way, different studies indicated the crucial role of IFN-, IL12 (Michailowsky et al., 2001), TNF- (Silva et al., 1995) and NO (Vespa et al., 1994) in host resistance to infection with T. cruzi.
As cited above, infection with the intracellular parasite T. cruzi is more severe in germ-free animals, as shown by a higher mortality when compared with conventional controls (Silva et al., 1987). Germ-free mice also displayed a more precocious and higher parasitism than conventional controls. Moreover, tissues from germ-free mice were more intensively parasitized and presented a more aggressive inflammatory response. Germ-free mice infected with T. cruzi presented a stronger local reaction to subcutaneous injection of formalin-killed parasites, as determined by footpad swelling, than conventional animals (Furarah et al., 1991). In addition, germ-free mice infected with T. cruzi did not survive the subcutaneous injection of antigen but died within 24 h, apparently of shock. Taken together, these data suggest that T. cruzi triggered a stronger cellular response in germ-free mice than in their conventional counterparts. However, cytokine data are not available to date.
In the present study, germ-free and conventional mice were experimentally infected with T. cruzi and used for determination of parasitaemia and survival, of cytokine and NO production by spleen cell cultures and of IgG1 and IgG2a concentrations in blood.
|
METHODS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Parasite.
T. cruzi, Y strain (Laboratório de Parasitologia e Histopatologia, DECBI-ICEB, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil), was used and maintained by weekly successive transfers in NIH conventional mice.
Experimental infection.
To obtain the inocula, infected animals in the acute phase were used. Blood was collected from the axillary plexus of mice with syringes containing 3.8 % sodium citrate in PBS, pH 7.2–7.4. Evaluation of the number of trypomastigotes was performed according to Brener (1962). Adjustment of the number of parasites to the desired inocula was done with PBS. All the manipulations were performed in a laminar flow hood. A sample was seeded in thioglycolate medium and brain heart broth for control of asepsis. Each mouse was inoculated i.p. with 5 x 103 parasites.
Experimental design.
Various germ-free and conventional groups (five to ten mice for each group according to the experiments) were used separately for determinations of parasitaemia, survival, cytokine levels and NO and immunoglobulin concentrations. For survival, the experiments were carried out over 40 days and accumulated mortality was noted. For immunological determinations, groups of mice were sacrificed just before and 7 days after experimental infection. Parasitaemia was determined at day 7 after infection. Three repetitions were performed for each determination.
Obtaining T. cruzi antigens.
Antigens from T. cruzi were obtained from trypomastigotes cultured on VERO cells. Parasites were harvested from the supernatant, centrifuged at 3000 g for 15 min at 4 °C and washed three times in PBS. The number of trypomastigotes was adjusted to 108 ml–1 and submitted to five cycles of freezing. Then, antigens were homogenized, aliquotted and maintained at –20 °C until use.
Parasitaemia.
On day 7 after infection, mice were bled from the tail just before sacrifice for the evaluation of the number of circulating parasites, according to the protocol of Brener (1962).
Cytokine quantification in spleen cultures.
Mice from gnotobiotic and conventional groups were sacrificed and the spleen was removed, macerated aseptically in RPMI 1640 (Sigma) and centrifuged at 1000 g for 10 min. Erythrocytes were lysed and spleen cells washed and centrifuged twice, cells were then suspended in RPMI 1640 supplemented with 10 % fetal bovine serum, 0.1 % of 0.05 mM ß-mercaptoethanol (Sigma), 10 mg gentamicin sulfate ml–1 and 3.2 mM L-glutamine (Sigma). Cell number and viability were assessed by trypan blue dye exclusion on a Neubauer hematocytometer and the final cell suspension adjusted to 5 x 106 cells ml–1. Cells were cultured in 24-well tissue culture plates in the absence or presence of the T. cruzi antigen (50 µl antigen preparation ml–1 culture, see above). Triplicate cultures of all experiments, using three animals for each one, were incubated for 72 h at 37 °C in 5 % CO2. After incubation, supernatants were harvested and stored at –86 °C for cytokine assay. Duoset kits for mouse IFN-, TNF- and IL10 (R&D Systems) were used to determine cytokine levels in culture supernatants according to the manufacturer's instructions. Absorbance was read at 450 nm on a microplate reader (Bio-Rad). The sensitivities of the assays were 72, 62 and 36 pg ml–1 for IFN-, TNF- and IL10, respectively.
Detection of NO production in spleen cultures.
NO production was determined according to Green et al. (1982). The nitrite content in the supernatants was measured by adding 50 µl Griess reagent to 50 µl sample in 96-well plates, reading the absorbance at 550 nm 15 min later and comparing with the absorbance curves of serial dilutions of sodium nitrite in complete medium.
Immunoglobulin analysis in serum.
The contents of total and parasite-specific IgG1 and IgG2a in serum were evaluated by capture ELISA. To detect IgG1 and IgG2a isotypes, biotinylated rat anti-mouse IgG1 and IgG2a antibodies (Southern Biotechnology Associates, Birmingham, AL, USA) were used. Absorbance at 492 nm was determined using an ELISA plate reader (Bio-Rad). The concentrations of each immunoglobulin were determined using the respective purified mouse standard (Southern Biotechnology Associates).
Statistical analysis.
The results shown are from one representative of at least three independently performed experiments. Statistical significance of the results was evaluated by Student's t-test and analysis of variance (ANOVA) for all data, except survival, for which the Log-Rank test was used. The level of significance was set at P < 0.05.
|
RESULTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
Fig. 2(a) shows similar basal levels of IFN- in the supernatants of spleen cultures from germ-free and conventional NIH mice before infection. When spleen cells were cultured with T. cruzi antigens for 72 h, a significant increase (P < 0.05) in IFN- production was observed for germ-free animals but not for their conventional counterparts. Seven days after intraperitoneal infection with T. cruzi (Fig. 2b), significantly higher IFN- levels (P < 0.05) were found in spleen culture supernatants of conventional mice, both non-stimulated or stimulated with parasite antigens. In germ-free animals, this increase in IFN- production after experimental infection was not observed, even in supernatants of spleen cells cultured in the presence of T. cruzi antigens.
|
Before experimental infection, basal TNF- production was higher (P < 0.05) in conventional than in germ-free animals. However, similar and higher levels of this cytokine were observed in germ-free and conventional mice when spleen cells were stimulated with parasite antigens (Fig. 2c). After infection (Fig. 2d), higher TNF- levels were found in supernatants of spleen cell cultures from conventional mice, both stimulated and non-stimulated with T. cruzi antigens, when compared with their germ-free counterparts (P < 0.05).
Fig. 3(a) shows similar NO levels in supernatants of spleen cultures of germ-free and conventional animals, both in the presence or absence of parasite antigens, before infection. Seven days after experimental infection (Fig. 3b), an increase in NO production was observed in spleen culture from conventional animals but not in the germ-free ones (P < 0.05).
|
Before infection (Fig. 4a), background production of IL10 was higher in supernatants of spleen cell cultures of germ-free mice than in conventional ones. Furthermore, the presence of T. cruzi antigen in culture induced increased IL10 production. After infection (Fig. 4b), a statistically significant increase over spontaneous production in culture was observed only when cells from conventional mice were stimulated with T. cruzi antigens.
|
Since the ratio of IgG2a and IgG1 is an indication of the prevalence of type 1 or type 2 responses in vivo, we assayed total and T. cruzi-specific IgG subclasses in sera of mice. Higher concentrations of total IgG1 were found in the serum of germ-free mice than in conventional animals, before infection (P < 0.05), but after the experimental challenge, the levels of this immunoglobulin decreased to similar values in the two groups (Fig. 5a). Inversely, higher concentrations of total IgG2a were observed in the serum of conventional mice than in their germ-free counterparts, before infection (P < 0.05), but after the experimental challenge, the levels of this immunoglobulin increased to similar values (Fig. 5b).
|
Finally, Figs 5(c) and 5(d) show lower anti-T. cruzi IgG1 and IgG2a levels in germ-free mice than in the conventional ones, before as well as after the experimental infection.
|
DISCUSSION |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
, which activates macrophages to produce NO and kill the obligate intracellular amastigote form of the parasite (Vespa et al., 1994). In addition, TNF- provides a second signal stimulating NO production and anti-T. cruzi activity in IFN--activated macrophages (Silva et al., 1995). On the other hand, the down-regulatory cytokines IL10 and TGF-ß are associated with susceptibility to infection by inhibiting IFN--mediated macrophage activation (Cardillo et al., 1996).
In the present study, germ-free and conventional mice were used to determine the influence of the normal microbiota on the survival and production of cytokines and immunoglobulins during the course of experimental Chagas’ disease. A less severe T. cruzi experimental infection in conventional mice than in germ-free animals has been described repeatedly in different reports published by our group over the last 15 years (Cintra et al., 1998; Furarah et al., 1991; Pedrosa et al., 1993; Santos et al., 1992; Silva et al., 1987). In this study, a difference in survival was not observed between conventional and germ-free mice, probably due the difference in the strain of T. cruzi used (CL in all the previous studies versus Y herein). However, a clear skew towards Th1 response was found in the conventional group after infection. Thus, higher IFN-, TNF- and NO productions by spleen cell cultures and higher blood levels of specific immunoglobulins of isotype IgG2a were observed 7 days after infection in conventional animals when compared to their germ-free counterparts. Basal production of IFN- by spleen cells was similar between germ-free and conventional mice before the infection and increased (P < 0.05) only in conventional animals after the infectious challenge. Spleen cells from germ-free mice were unable to produce IFN- even after antigen stimulation in vitro, as opposed to spleen cells from conventional animals. Similar results were obtained for TNF- production after infection. In accordance with the higher IFN- and TNF- levels in spleen cell cultures from conventional mice infected with T. cruzi, an increase in NO production was also noted in these animals under the same conditions. Furthermore, no differences in IL10 production were observed after infection between conventional and germ-free mice. No IL4 was detected in cell cultures from any of the groups. Hence, a stronger skew towards a type 1 response was found in cultures from infected conventional mice. Accordingly, infected conventional mice showed higher levels of T. cruzi-specific IgG2a in sera. These results are in accordance with the seminal work by MacDonald & Carter (1979) who showed that germ-free mice had a deficiency in mounting a cell-mediated immune response, compared with conventional controls. The reason for the increase of T. cruzi-specific antibodies of the IgG1 subclass in both germ-free and conventional mice in the absence of detectable levels of IL4 is not clear.
IFN- production by spleen cells from uninfected germ-free mice in response to T. cruzi antigen was higher than in conventional mice. This unpredicted observation might be explained by lower stimulation by lipopolysaccharide (LPS) in germ-free mice compared to conventional animals. Glycophosphatidylinositol-anchored mucin-like glycoconjugates from T. cruzi (tGPI mucins) induce pro-inflammatory cytokines (Camargo et al., 1997). According to Ropert et al. (2002), the recognition system triggered by tGPI mucins appears to share much in common with the recognition system for LPS. In fact, LPS can induce tolerance to tGPI mucins, and vice versa (Ropert et al., 2002). Hence, it is possible that conventional mice are exposed to low levels of LPS from the normal microbiota and were, therefore, less reactive to T. cruzi antigens (which contain tGPI mucins). Germ-free mice are exposed to very low levels of LPS and may be more sensitive to it and to tGPI mucins. Hence, they produce high levels of pro-inflammatory cytokines in response to T. cruzi before infection, even to the low levels of tGPI mucins present in the antigen preparation added to cultures. This increased production of pro-inflammatory cytokines, however, was not sufficient to prevent parasite dissemination and disease progression. The reason for the apparent anergy that infection with T. cruzi caused in germ-free mice is unknown and cannot be explained by the production of IL10.
In spite of the higher production of protective cytokines by conventional animals in response to infection, no protection as measured by parasitaemia and mortality was found in our experiments. This is surprising, since the presence of these pro-inflammatory cytokines have been extensively associated with resistance to T. cruzi (Hoft et al., 2000; Michailowsky et al., 2001). Moreover, our own data with a different strain of the parasite suggested that germ-free mice are more susceptible to infection with T. cruzi (Cintra et al., 1998; Furarah et al., 1991; Pedrosa et al., 1993; Santos et al., 1992; Silva et al., 1987). The reasons for the similar susceptibility found here are, in our view, the high dose of parasites and the high virulence of the Y strain. We chose this strain and a high number of parasites in the hope of exacerbating the effects we had seen of the normal microbiota on the outcome of infection with T. cruzi. Although our data does suggest that a more prompt type 1 response is mounted by conventional mice upon infection with T. cruzi, the high virulence of the parasite used herein did not allow us to correlate this response to protection. Thus, more studies are necessary to clarify these issues.
It is possible that priming of T cells by the indigenous microbiota influences the memory cell repertoire in conventional mice, hence influencing their immune response. Indeed, an exacerbated IL4 production in response to infection with Leishmania major has been associated with a CD4+ T cell population expressing the memory phenotype, in BALB/c mice. According to the authors, this T cell population would be primed by a peptide that is present in E. coli and in the LACK protein of L. major (Julia et al., 2000). However, more recent data has associated the early IL4 production in response to the parasite with a naïve phenotype (Stetson et al., 2002), probably ruling out the role of the normal microbiota in skewing the immune response during infection with L. major. The numbers of T cells expressing the memory phenotype in germ-free mice are also controversial: some authors find that there are more cells expressing memory markers when mice are exposed to the normal microbiota (Inagaki et al., 1996; Lee et al., 1990; Price & Cerny, 1999), while others do not (Bonorino et al., 1998; Dobber et al., 1992; Park et al., 2000). Hence, the extent to which the normal microbiota can change the host T cell repertoire and influence its response to pathogens is still not clear.
Another possible influence that the microbiota may have is in the regulatory cell repertoire. Several studies have provided evidence for the ‘hygiene hypothesis', in which low exposure to micro-organisms would induce an exacerbated immune response, either of the Th1 or the Th2 type (Wills-Karp et al., 2001; Yazdanbakhsh et al., 2002). However, our data show that the lack of exposure to antigenic stimuli from the normal microbiota does not induce an exacerbated immune response against a single invasive micro-organism, suggesting that there might be a threshold of antigenic stimulation below which an inflammatory response is not fully mounted. Recent data has pointed to IL6 as a putative regulator of the inflammatory response, before it is triggered (Hori et al., 2003; Pasare & Medzhitov, 2003; Powrie & Maloy, 2003). Hence, it seems that the microbiota would provide the necessary stimulus to surpass a non-inflammatory state enabling the organism to achieve optimum levels of inflammatory cytokine production and protection against an infecting agent.
In conclusion, this study provides evidence that normal associated microbiota plays an important role in the development of an immune system that is competent to react against an acute infection. Experiments with gnotoxenic animals monoassociated with bacterial strains representative of the predominant gut microbiota of humans are being carried out currently in our laboratory to determine their individual influence on the immune response during the course of experimental Chagas’ disease and to identify which strains are most effective to redirect the Th1–Th2 balance.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
|
REFERENCES |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
T cell involvement. J Immunol 161, 5210–5216.-primed macrophages. J Immunol 159, 6131–6139.[Abstract] axis in controlling tissue parasitism and inflammation in the heart and central nervous system during Trypanosoma cruzi infection. Am J Pathol 159, 1723–1733.14+ NKT cells in germ-free mice. Eur J Immunol 30, 620–625.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
) and conventional (•) mice after i.p. challenge with 5 x 103 T. cruzi trypomastigotes. Data from three identical experiments performed independently (ten mice per experiment) are shown.
