Trypanosoma cruzi: H2 complex and genetic background influence on the humoral immune response against epimastigotes

International Journal for Parasitology 30 (2000) 981±984

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Research note

Trypanosoma cruzi: H2 complex and genetic background in¯uence on the humoral immune response against epimastigotes Juan Carlos AguilloÂn a, Tamara Hermosilla a, MarõÂa Carmen Molina a, Antonio Morello b, È rn c, Arturo Ferreira a,* Yolanda Repetto b, Anders O b

a Programa de InmunologõÂa, Instituto de Ciencias BiomeÂdicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile Programa de FarmacologõÂa ClõÂnica y Molecular, Instituto de Ciencias BiomeÂdicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile c Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden

Received 23 March 2000; received in revised form 13 June 2000; accepted 13 June 2000

Abstract Using A.SW, A.CA, B10.S and B10.M congenic mouse strains, we measured the IgG speci®c humoral immune responses against sonicated and live Trypanosoma cruzi epimastigotes. Genes located in the A background (A.SW and A.CA strains) mediate higher IgG responses against the parasite antigenic complexes than those located in the B background (strains B10.S and B10.M), regardless of the H2 haplotypes. Thus, non H2 genetic elements seem to be more important in determining differences in the total IgG immune response against T. cruzi. Whether a detectable H2 effect, in favor of the H2 s haplotype, occurred in the A or B background, was contingent on the immunisation protocol used. Thus, the H2 s haplotype mediates a higher IgG response in the A background, if immunised with live epimastigotes, and in the B background against sonicated epimastigotes. Most likely this represents a complex sequence of events, controlled by non-MHC genes, involving antigen handling and processing and depending on the physical form of antigen delivery. q 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Major histocompatibility; Immune response

Seldom is the immune response against a complex parasite commanded by a single major gene [1±4]. Rather, multiple genes, interspersed in the genome may in¯uence the resistance / susceptibility to diverse pathogens [5±8]. The role of genetic factors in resistance / susceptibility to infections has been studied by genetically selecting speci®c animal lines, pre-established inbred strains, or animal lines pre-selected for high or low immune response. A consensus has been reached indicating that the genetic basis of the immune response against a complex organism such as an intracellular protozoan parasite, is particularly intricate [9± 11]. In the murine model of Chagas' disease, roles for acquired and innate immunity have been established [12,13]. Even age [14] and sex [15] are important factors. More recently, a few genes affecting susceptibility to infectious pathogens have been identi®ed [16,17]. Large variations in resistance / susceptibility to Trypanosoma cruzi are controlled by genes located near or within the center of the murine major histocompibility complex (MHC), H2, and in the non-H2 genetic background [18±20]. For instance, the * Corresponding author. Fax: 156-2-735-3346. E-mail address: [email protected] (A. Ferreira).

A.SW / A.CA H2 congenic pair, differs drastically in their susceptibility to acute infection with the TulahueÂn strain of T. cruzi. Transfer of puri®ed IgG, from immune A.SW animals, protects A.CA mice against an otherwise lethal parasite challenge [21]. Interestingly, only A.SW animals generate IgG antibodies against calreticulin (formerly known as Tc45), a 45 kDa dimorphic T. cruzi antigen, present in both cultured epimastigotes and trypomastigotes of many T. cruzi strains. [22,23]. The nature of the relationship between this recognition and the resistance mentioned above remains to be determined. Although many studies in the murine model using de®ned synthetic peptides as immunogens have identi®ed H2 as the site where the genes coding for antigen presenting molecules are located, the role of non-MHC genetic factors in response to complex organisms is still poorly understood. In this paper we present a simple quantitative immunogenetic approach for dissecting the in¯uence of the H2 complex and of the genetic background on the total IgG plasma levels against live cultured epimastigotes or their sonicated soluble extracts. Trypanosoma cruzi epimastigotes from the TulahueÂn strain were grown in liver infusion tryptose (LIT) [25],

0020-7519/00/$20.00 q 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00078-3

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Table 1 Comparison of congenic mouse strains in their capacity to generate antitotal Trypanosoma cruzi antigen IgG (sonicated epimastigotes). Effect of genetic background and H2 complex a B10.M (12,840) A.SW (22,892) A.CA (21,427) B10.S (15,622)

b

,0.0001 ,0.0001 b 0.0133 b

B10.S (15,622) b

,0.0001 ,0.0001 b

A.CA (21,427) 0.3420

a Values in parentheses correspond to means (cpm) calculated from 25 observations per strain (®ve mice, ®ve bleedings). The mean for each animal was obtained from three measurements. b Signi®cantly different values.

harvested at the exponential growth phase, washed, resuspended in PBS, subjected to one cycle of freezing and thawing at 2708C, and sonicated (250 W for 30 s). After measuring total protein concentration and adding 1 mM Na -p-tosyl-l-lysine chloromethyl ketone, the samples were aliquoted and frozen at 2708C [24]. Freshly collected live epimastigotes were also used for immunisation of experimental animals. Three month old A.SW and B10.S (both H2 s), and A.CA and B10.M (both H2 f) female mice, obtained from Jackson Laboratories, and then bred in our animal facilities, were used in all experiments described. Two experimental groups per strain, ®ve animals each, were immunised following different protocols (A and B). Preimmune bleedings were obtained from all animals. After import, the animals were kept in quarantine in our animal facility. Filtered air, and sterile food and bedding was provided. The health status of the animals was permanently monitored by quali®ed veterinary personnel. Protocol A involved four immunisations per animal, once a week, with 50 mg of total protein from sonicated epimastigotes per injection. The ®rst one was administered in the rear foot pads; the following two s.c., and the last one i.p. Two weeks later, the animals were bled ®ve times, once a week, through the ventral tail vein and artery. Protocol B involved one i.p. immunisation with 6 £ 10 5 cultured epimastigotes per mouse. After immunisation, serum samples were obtained on ®ve opportunities, at 7 day intervals. No parasitaemia or symptoms of disease were detected in the animals immunised according to this protocol. Sera were stored at 2208C. For immunoradiometric assay (IRMA), 25 ml of sonicated epimastigotes (20 mg total protein per ml) in 0.1 M carbonate buffer (pH 9.6), were delivered into polyvinylchloride wells (PVC plates). The plates were incubated overnight at 48C, washed and saturated for 1 h with PBS with 1% w/v BSA. The plates were then incubated at room temperature, in triplicate, for 1 h with immune and preimmune sera from individual mice. In agreement with pilot titrations of several representative samples, sera from mice immunised with live epimastigotes were used diluted 1/100, while those from animals immunised with parasite extracts were diluted 1/15,000. The plates were washed, and

incubated for 60 min with 10 5 cpm of af®nity puri®ed, 125I radiolabelled, anti murineg -chain, goat IgG. After washing, the radioactivity associated with the individual wells was measured. Negative controls and wells without the ®rst antibody and / or antigen were included [26]. In order to compare the IgG humoral immune responses determined by IRMA, among A.SW, A.CA, B10.S and B10.M strains, we used a split-plot statistical model [27], by means of SAS statistical software. We quanti®ed the average values of IgG, directed against live and sonicated epimastigotes, in 25 samples from each strain (®ve bleedings, ®ve mice). The mean value per animal was obtained from triplicate determinations. The analysis was focussed on the effects of genetic background and H2. Using A.SW, A.CA, B10.S, and B10.M congenic mouse strains we measured the mean values of the intensity of the IgG speci®c humoral immune responses, against sonicated (Table 1) and live epimastigotes (Table 2). We evaluated statistically the magnitude of the differences observed. In order to assess relevant comparisons, we calculated the mean and p values. When comparisons of A.SW versus B10.S or A.CA versus B10.M were made, clearly higher levels (P , 0:0001) of anti-parasite IgG (sonicated parasite form), were associated with the A background in both cases (Table 1). Only the comparison of B10.S with B10.M showed signi®cant (P ˆ 0:0133) differences, in favor of the H2 s haplotype (B10.S strain) (Table 1). Statistical comparison of the responses generated in A.SW strains versus B10.S or A.CA versus B10.M, show signi®cant differences (P , 0:0001 and P ˆ 0:0024, respectively) in favor of background A. The response of the A.SW strain was higher than the A.CA's (P ˆ 0:0059). However, the H2 s haplotype in the B background (B10.S strain) does not mediate a higher response than that of the H2 f haplotype in the same background (B10.M strain) (P ˆ 0:0754). The overall comparison of the results shown in Tables 1 and 2 indicates that genes located in the A background (A.SW and A.CA strains) mediate a higher IgG response against the parasite antigenic complexes than those located in the B background (strains B10.S and B10.M), irrespectively of the H2 haplotypes. In other words, non H2 genetic elements seem to be more important in determining differTable 2 Comparison of congenic mouse strains in their capacity to generate antitotal Trypanosoma cruzi antigen IgG (live epimastigotes). Effect of genetic background and H2 complex a

A.SW (8350) A.CA (7392) B10.S (5516) a

B10.M (6171)

B10.S (5516)

A.CA (7392)

, 0.0001 b 0.0024 b 0.0754

, 0.0001 b , 0.0001 b

0.0059 b

Values in parentheses correspond to means (cpm) calculated from 25 observations per strain (®ve mice, ®ve bleedings). The mean for each animal was obtained from three measurements. b Signi®cantly different values.

J.C. AguilloÂn et al. / International Journal for Parasitology 30 (2000) 981±984

ences in the total IgG immune response against T. cruzi. An important part of the non H2 control may be determined by the repertoire of antigenic receptors (T and / or B) present in each strain [28]. Also of importance may be genes that control different stages in antigen processing and macrophage function (antigen processing and capacity to become activated in the presence of cytokines induced by the infection) [29,30]. Future experiments using the recombinant inbred strain panels, AXB and BXA (derived from A/J and C57BL/6) could allow mapping of crucial gene(s). When there was a detectable H2 effect this was in favor of the H2 s haplotype. However, whether this happened in the A or B haplotype depended on the immunisation protocol used. Thus, the H2 s haplotype responds better in the A background, if immunised with live epimastigotes, and in the B background against sonicated epimastigotes. It should be pointed out, however, that these protocols also differ in the route and number of immunisations, as well as in the total amount of antigen administered. Most likely, the role of H-2 is mediated by speci®c class II alleles in their capacity to productively present certain parasitic peptides. This capacity may be increasingly manifest when the response to speci®c epitopes is de®ned in particular parasite antigens. The lack of correlation between these results and those previously reported for Tc45 [23,24] is not surprising, given the fact that here we are measuring the immune response against a multitude of parasite antigens, Tc45 being just one of them. Acknowledgements This work was supported by Research Grants: 1970878, from FONDECYT-Chile; D96T1023, from FONDEFChile; Swedish Agency for Research Co-operation with Developing Countries (SAREC/SIDA) and UNESCO/ RELACIN. We are indebted to Mrs Marisol Briones for excellent technical assistance and to Dr Lorena Ferreira, for editorial suggestions. References [1] Mogensen SC. Genetics of macrophage-controlled resistance to hepatitis induced by herpes simplex virus type 2 in mice. Inf Immun 1977;17:268±73. [2] Bradley DJ. Regulation of Leishmania populations within the host. II. Genetic control of acute susceptibility of mice to Leishmania donovani infection. Clin Exp Immunol 1977;30:130±40. [3] Groves M, Osterman JV. Host defences in experimental scrub typhus: genetics of natural resistance to infection. Inf Immun 1978;19:583±8. [4] Rhodes JC, Wicker LS, Urba WJ. Genetic control of susceptibility to Cryptococcus neoformans in mice. Inf Immun 1980;29:494±9. [5] Williams DM, Grumet FC, Remington JS. Genetic control of murine resistance to Toxoplasma gondii. Inf Immun 1978;19:416±20. [6] Hormaeche CE. Natural resistance to Salmonella typhimurium in different inbred mouse strains. Immunology 1979;37:311±8. [7] Morrison WI, Murray M. Trypanosoma congolense: inheritance of

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