In vivo macrophage function in experimental infection with Trypanosoma cruzi subpopulations

Acta Tropica, 55(1993)171-180 © 1993 Elsevier Science Publishers B.V. All rights reserved 0001-706X/93/$06.00

171

ACTROP 00329

In vivo macrophage function in experimental infection with Trypanosoma cruzi subpopulations Ana M. Celentano* and Stella M. Gonzfilez Cappa Departamento de Microbiologia, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 (piso 13), 1121 Buenos Aires, Argentina (Received 9 November 1992; revised version accepted 5 July 1993)

The macrophage function was investigated in mice infected with Trypanosoma cruzi. Two subpopulations of the parasite were utilized, RA and K98. Strain RA is efficiently internalized by macrophages and is lethal for mice, and clone K98 is poorly phagocytosed by macrophages and is not lethal. Treatment with silica enhanced parasitemia and mortality in mice infected with both parasite subpopulations. Parasitemia kinetics, however, were affected only in mice infected with RA, which suggests that macrophage effector mechanisms may play a more relevant role in this experimental group than in mice infected with K98. Resistance to Salmonella typhimurium infection and bactericidal activity of macrophages depended upon the T. cruzi subpopulation utilized and the infection period. Infection with K98 induced only a trend towards enhanced resistance to bacterial challenge during both the acute and chronic phases, whereas a significantly enhanced bactericidal activity of spleen and liver phagocytes was observed. Mice acutely infected with RA showed significantly enhanced susceptibility to S. typhimurium infection and lower bactericidal activity. Mice surviving infection with this aggressive strain, however, showed significantly enhanced resistance and bactericidal activities. Mice acutely infected with the RA strain displayed a dissociation between macrophage capacities to control S. typhimurium and T. cruzi. A similar phenomenon was also observed in other parasitoses (schistosomiasis, African trypanosomiasis). This fact may be due to differences in the lethal mechanisms through which macrophages control these parasites and S. typhimurium. Key words: Trypanosoma cruzi; Chagas' disease; Macrophage depression; Salmonella typhimurium

Introduction

Chagas' disease affects 16-18 million people in Latin America. Infected people may be asymptomatic or develop diverse pathological manifestations (cardiac or digestive forms). Differences in these manifestations might partially be attributed to the characteristics of parasite subpopulations, since Trypanosoma cruzi exhibits a wide intraspecific variation. Studies in experimental models revealed differences among T. cruzi strains in morphology of bloodstream forms (Brener, 1965), lethality (Andrade et al., 1985), tissue tropism (Melo and Brener, 1978; Gonzfilez Cappa et al., 1981b) and susceptibility to immune mechanisms (Alcantara and Brener, 1978; Krettli et al., 1979). The influence of intraspecific variation in the induction of immune response was *Corresponding author. Fax: + 54 1 9625404.

172 analysed in mice infected with two T. cruzi subpopulations of distinct biological characteristics, RA and K98 (Mfiller et al., 1986; Mfiller and Gonzfilez Cappa, 1987). RA strain, isolated from a child with acute Chagas'disease, is extremely virulent for mice, replicates actively in the reticuloendothelial system (RES) and is an efficient inducer of opsonizing antibodies (Gonzfilez Cappa et al., 1981a,b; Celentano and Gonz~lez Cappa, 1992). The K98 clone derives from and possesses similar features to the CA-I strain (obtained from a patient with chronic cardiomyopathy). It is nonlethal for mice, hardly infects and multiplies in phagocytes and is a poor inducer of opsonizing antibodies (Gonzfilez Cappa et al., 1980, 1981b; Celentano and GonzS.lez Cappa, 1992). Macrophage activation during T. cruzi infection has been reported (Nathan et al., 1979; Russo et al., 1989; Cardoni et al., 1990; Celentano and Gonzfilez Cappa, 1992). Activated but not resident macrophages have been shown to kill the parasite in vitro (Nogueira and Cohn, 1978; Nathan et al., 1979; Celentano and Gonzfilez Cappa, 1992). Chemiluminiscence levels were similarly enhanced in mice infected with both T. cruzi subpopulations during the acute infection. In mice surviving the infection with RA, however, macrophages reached maximum levels of respiratory burst (Celentano and Gonzfilez Cappa, 1992). To support the hypothesis that mice survived infection with RA because of this macrophage activation, studies in vivo were required. Although activated macrophages exert trypanocidal activity, their relevance in resistance remains uncertain. A role for activated macrophages in resistance was suggested by results obtained by blocking the reticuloendothelial system (RES) in T. cruzi infected mice (Goble and Boyd, 1962; Kierszenbaum et al., 1974; Trischmann et al., 1978) and by activation of macrophages with immunomodulators during the infection (Kierszenbaum and Ferraressi, 1979; James et al., 1982a). However, the highest macrophage activation, measured by hydrogen peroxide levels and TNF secretion, was found in the more susceptible mouse strain during experimental T. cruzi infection (Russo et al., 1989). In the present study, in vivo macrophage function in mice infected with RA or K98 T. cruzi subpopulations was evaluated by: (a) macrophage depression by silica administration, and (b) Salmonella typhimurium injection (Blanden et al., 1966; O'Brien et al., 1979).

Materials and Methods Mice

4-week-old male C3H/HeN mice were bred in our laboratory and maintained under standard conditions. Parasites

RA strain and K98 clone T. cruzi subpopulations, which possess contrasting characteristics, were utilized in this study (GonzS.lez Cappa et al., 1980, 1981a,b; Celentano and Gonzfilez Cappa, 1992). Both subpopulations were maintained by serial passages in mice.

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Experimental T. cruzi infection Trypomastigotes were obtained from infected mouse blood at the peak of parasitemia and counted (Pizzi, 1957). The desired density of parasite suspensions for mouse inoculation was adjusted by diluting the blood with PBS supplemented with 5% inactivated normal mouse serum. Mice were inoculated by the intraperitoneal route (ip) with the dose that induced maximal humoral responses in mice for each T. cruzi subpopulation: 10-30 RA trypomastigotes and 104 K98 trypomastigotes (Mfiller and Gonz~.lez Cappa, 1987). Groups of uninfected mice of the same age, body weight and sex were used as controls. Studies were performed during acute (days 7 and 30 for RA and K98 infected mice, respectively) and chronic phases of experimental infection (day 120). Few mice infected with RA exceptionally survived the acute infection. Therefore, to obtain animals with chronic infection several groups of 80-100 mice were required.

Macrophage depression Silica (Merck; minimum particle size 10 la) was used to impair the macrophage function in vivo (Coulombi6 et al., 1986). Mice were injected ip with silica and the dose was adjusted in preliminary experiments. Effectiveness of RES depression was evaluated by injection of the mice with S. typhimurium (O'Brien et al., 1979). The dose selected (20 mg/mouse) exhibited no apparent toxicity for normal animals. Mice were inoculated with silica 24 h before T. cruzi infection, and reinoculated weekly thereafter. A group of sham-inoculated T. cruzi infected mice were used as controls. Parasitemia was followed up throughout the acute period and mortality was recorded daily up to 45 days post-infection.

Bacteria S. typhimurium strain 4066 was obtained from the collection of the Instituto Nacional de Microbiologia Dr.Carlos Malbrfin (Buenos Aires, Argentina). S. typhimurium was propagated by passage on McConkey agar and was periodically tested by Gram stain and biochemical tests. S. typhimurium LD50 was less than 100 colony-forming units (CFU)/mouse. Bacteria were grown overnight in McConkey agar, suspended and diluted in PBS (pH 7.4) to the desired density for mouse inoculation. The number of bacteria was estimated spectrophotometrically before inoculation. The number of CFU was determined 24 h later by plating on McConkey agar. 20-1al blood samples were obtained from the retroorbital sinus under ether anesthesia and diluted in 2 ml PBS. The number of bacteria was determined in blood and spleen-liver samples. The limit of sensitivity was 100 CFU/ml blood and 250 CFU/spleen-liver. Resistance to S. typhimurium Control, RA and K98 T. cruzi infected mice were inoculated i.p. with 103-104 CFU/mouse. Mice infected with T. cruzi but not with S. typhimurium were included in some experiments. Parasitemia was evaluated before bacterial injection in both

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chronic and acute T. cruzi stages. In the latter group parasitemia was followed up after bacterial inoculation. Bacteremia was evaluated on days 1, 2, 4 and 7 after S. typhimurium administration. Mortality was recorded daily up to 15 days postbacterial injection. Blood clearance and organ recovery of S. typhimurium Control and T. cruzi infected mice were inoculated by the intravenous (i.v.) route with (1-3) x 106 CFU/mouse. Blood samples were taken at 2, 4, 8 and 12 min after S. typhimurium challenge. Blood clearance values were expressed as the percentage of bacteria recovered. Mice were killed 24 h after S. typhimurium inoculation. Spleen and liver were removed and weighed under sterile conditions. The organs were homogenized together in 15 ml of sterile PBS and plated. The percentage of bacteria recovered from both organs was calculated. Body/organ weight ratios were determined to evaluate hepatomegaly and splenomegaly. Statistical analysis Differences in parasitemia and bacteremia were analyzed by the Mann-Whitney U test for non-parametrics. The Kolmogorov-Smirnov test was used to evaluate differences in cumulative mortality. Clearance, organ bacterial recovery and bodyweight/organ-weight ratios were determined by the Student t-test. The number of animals in each group is given in tables and figures. P levels lower than 0.05 were considered significant.

Results

Parasitemia and mortality in mice treated with silica Macrophage depression enhanced parasitemia levels of mice infected with both T. cruzi subpopulations but the effect of silica on parasitemia was related to the parasite subpopulation. While parasitemia of mice treated with silica and infected with K98 showed a kinetics similar to that of untreated controls, parasitemia increased geometrically in mice treated and infected with RA, and remained almost constant in the control group (Fig. 1). A shorter survival period was seen in mice infected with RA and treated with silica, when compared with the untreated group. In experiments of K98 infection, 4/9 silica-treated mice died while no deaths were recorded in the control group. The difference, however, was not statistically significant (Fig. 1). Effect of T. cruzi infection on resistance to S. typhimurium Studies to determine LDso showed that mortality rates exhibited the best reproducibility when mice were challenged with 103-104 CFU. T. cruzi parasitemia levels were similar in mice receiving S. typhimurium and those without bacterial challenge (data not shown). Only a trend towards lower bacteremia and mortality was observed in

175

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Fig. 1. Parasitemia and mortality of mice infected with different T. cruzi subpopulations and treated with silica. Parasitemia of mice infected with RA strain: x, controls and [], treated with silica; parasitemia of K98-infected mice: +, controls, ~ , treated with silica. Significant differences (P < 0.05) between silicatreated and control groups in: a)RA infection: all time points; b)K98 infection: days 17, 19, 24, 26 and 28 post infection. Mortality rates of mice infected with T. cruzi: RA strain i , controls, [], RA + silica, [], K98 clone+ silica. No deaths occurred in the control group infected with K98. Mortality rates between silica and control groups were significantly different in RA-infected mice only (p<0.01). Number of mice/group: RA strain-infected mice controls: 10; RA + silica: 16; K98 clone-infected mice controls: 8; K98 + silica: 9.

mice infected with K98 when compared with controls, either during acute or chronic periods (Fig. 2). Mice chronically infected with the RA strain survived longer than control mice, whereas mice acutely infected showed a shorter survival period and higher bacteremia (Fig. 2). Effect of T. cruzi infection on clearance and bactericidal capacity of S. typhimurium Bacterial clearance was enhanced in T. cruzi infected mice no matter the parasite subpopulation tested (Table 1). Data in Table 2, however, show differences in the number of CFU recovered from spleen-liver 24 h after S. typhimurium injection, depending on the parasite subpopulation and the T. cruzi infection period. Bacterial proliferation was observed during acute infection with RA while enhanced bactericidal activity was seen during the chronic phase. A better bactericidal activity was observed in mice infected with K98 during both acute and chronic phases as compared with control mice. As expected, splenomegaly "and hepatomegaly were evident in most T. cruzi infected mice no matter the parasite subpopulation employed (Table 2).

Discussion

Results reported here suggest that macrophages play a role in resistance to T. cruzi infection as shown by enhanced parasitemia levels and mortality rates in mice infected with either RA or K98 parasite subpopulations and treated with silica.

176

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Fig. 2. Bacteremia and mortality of mice inoculated ip with 103-104 S. typhimurium. Bacteremia of x, controls and II, T. cruzi infected mice; each point represents the geometric mean. Significant differences were seen for K98 chronically infected mice on day 1 (P<0.01); RA acutely infected mice on days 1 (P<0.05) and 2 (p<0.01). Mortality rates of El, controls and [], T. cruzi infected mice. Only RA infected mice showed statistically significant differencesin mortality when compared with controls during acute (P<0.01) and chronic (P<0.05) periods. 8-12 mice per group were utilized. However, macrophage function appeared to differ according to the parasite subpopulation assayed. Mice infected with R A and treated with silica displayed a geometrical enhancement of parasitemia levels while the levels remained stable in control mice. Parasitemia kinetics of K98 infected mice were similar in treated and control groups. These findings suggested a more relevant efficacy in macrophage action, as a direct effector mechanism, in those mice infected with the reticulotropic RA strain when compared with those inoculated with K98, which is poorly internalized into macrophages. As silica might only affect peritoneal phagocytes, and parasites were injected intraperitoneally, these results would likely reflect alterations on the primary parasitemacrophage interaction. Our results agree with recent studies showing that gammaI F N , which enhances macrophage trypanocidal activity, played a role in controlling parasitemia and mortality in mice infected with R A but not in those infected with CA-I (from which K98 has been cloned) (Petray et al., 1993). When S. t y p h i m u r i u m was used as a marker of macrophage function, mice infected

177 TABLE 1 Blood clearance of S. typhimurium in mice infected with T. cruzi Mouse group

% S. typhimurium recovered from blood a 2 min b

4 min

8 min

12 min

Control K98 (acute)

43.5+4.7 31.0_+4.2

19.2_+3.1 8.7+0.4 c

9.7_+ 1.1 5.4_+0.7 c

9.0_+2.0 1.I _+0.6'

Control K98 (chronic)

62.4 + 6.9 25.0_+ 4.3 c

26.4 _+3.7 6.0_+ 1.5"

15.6 -+ 1.9 1.5 -+0.2 c

6.9 -+ 1.3 0.9 _+0. I c

Control RA (acute)

35.4_+ 1.6 19.2 _+3.0'

10.0_+0.9 6.8 _+ 1.4

5.7-+0.9 4.6 _+0.8

4.7_+0.8 2.9 _+0.8

Control RA (chronic)

63.4 _+6.4 27.4+ 8.Y

36.2 _+5.3 12.4_+4.9 c

12.3 _+3.2 8.6_+ 3.2

7.4 _+2.0 7.5 _+3.2

aData are expressed as means ___S.E. from 5-9 animals. bMinutes after S. typhimurium injection. cp< 0.05 compared with the control group (Student's t-test). TABLE 2 S. typhimurium recovery from spleen-liver 24 h after injection and body/organ weight ratios in mice infected with T. cruzi Mouse group

% S. typhimurium recovered from organs ~

Weight ratios" body/liver

body/spleen

Control K98 (acute)

31.2+6.4 14.4__+2.5 b

18.6+0.5 15.9 + 0.5 b

389+63 133 + 15b

Control K98 (chronic)

16.4+4.5 5.4+ 1Ab

20.0+ 1.2 15.6+0.8 b

413+41 105+ 12b

19.6-+_3.2 422.0 + 78.0 b

18.9 +0.7 15.3 _+ I. 1b

222 +42 67 ___7b

16.6__ 3.7 2.5__+0.3b

16.9+0.3 16.0_+0.7

251 __+19 124_+ I I b

Control RA (acute) Control RA (chronic)

"Values are expressed as means+ S.E. from 5-9 animals. bP<0.05 compared with the control group (Student's t-test). with K98 displayed enhanced microbicidal activity of spleen-liver phagocytes without s i g n i f i c a n t differences in r e s i s t a n c e in b o t h a c u t e a n d c h r o n i c T.cruzi i n f e c t i o n . O n the other hand, macrophages from mice inoculated with the aggressive RA strain s h o w e d a d u a l b e h a v i o u r . F i r s t , a f a i l u r e in t h e i r f u n c t i o n w a s seen d u r i n g the a c u t e i n f e c t i o n j u s t b e f o r e the p e a k o f l e t h a l i t y w a s r e a c h e d , f o l l o w e d by a n e n h a n c e m e n t o f r e s i s t a n c e a n d b a c t e r i c i d a l a c t i v i t y a m o n g t h e s u r v i v o r s . T h e significant e n h a n c e d r e s i s t a n c e d i s p l a y e d o n l y by m i c e c h r o n i c a l l y i n f e c t e d w i t h R A , t o g e t h e r w i t h t h e l o w e s t v a l u e s o f b a c t e r i a l r e c o v e r y f r o m spleen-liver, is c o i n c i d e n t w i t h the best m a c r o p h a g e a c t i v a t i o n p r e v i o u s l y r e p o r t e d f o r the few s u r v i v i n g m i c e i n f e c t e d w i t h this s t r a i n ( C e l e n t a n o a n d G o n z ~ l e z C a p p a , 1992). T h e f a i l u r e in m a c r o p h a g e f u n c t i o n , r e s p o n s i b l e o f the e n h a n c e d s u s c e p t i b i l i t y to

178

S. typhimurium during the acute infection with RA, seemed to be restricted to the bactericidal effector mechanisms because total phagocytic activity measured by clearance studies remained unchanged. Macrophages may be exerting different lethal mechanisms to control T. cruzi and S. typhimurium. Oxygen-derived products have been linked to the control of T. cruzi by macrophages (Nathan et al. 1979; Reed et al. 1987), whereas there is no correlation between resistance to S. typhimurium challenge and oxygen metabolite levels (Blumenstock and Jann, 198 l; Kagaya et al., 1989). Recently, it has been reported a role for reactive nitrogen intermediates related to trypanocidal activities in vitro (Gazzinelli et al., 1991). The relevance of these products in vivo and the possible differences in their induction by diverse parasite subpopulations remain to be elucidated. Macrophage activation, evidenced by enhanced respiratory burst (Celentano and Gonzfilez Cappa, 1992) and effective trypanocidal capacity, contrast with the macrophage failure in exerting bactericidal activity showed by mice acutely infected with RA. This discrepancy, however, agrees with results communicated for other parasites. In this regard, it has been reported that activated macrophages are capable of killing efficiently Toxoplasma gondii trophozoites with enhanced hydrogen peroxide levels, but are unable to control S. typhimurium (Van Dissel et al., 1987; Langermans et al., 1990). A similar decreased resistance and bactericidal capacity of spleen and liver phagocytes was demonstrated in Schistosoma mansoni infection (Rocha et al., 1971; Bomfim de Lima et al., 1982), despite their enhanced schistosomula killing ability (James et al., 1982b). Decreased phagocyte function was also seen in African trypanosome-infected mice (Glick and Jones, 1984) in spite of their enhanced hydrogen peroxide levels (Grossinsky et al. 1983). S. typhimurium inhibits phagolysosome fusion, an inhibition that might play a crucial role in survival of these bacteria within macrophages (Ishibashi and Arai, 1990; Buchmeier and Heffron, 1991). In this regard, in vitro macrophage infection by metacyclic trypomastigotes has been shown to trigger an inhibitory effect on phagolysosomal fusion (Osuna et al., 1986). The RA strain but not K98 might be exerting such inhibitory effect because only RA is internalized efficiently within macrophages (Gonzfilez Cappa et al. 1981b). If this hypothesis is confirmed, differences in immunogenicity reported for different T. cruzi subpopulations (Miiller et al., 1986; Mfiller and Gonz53ez Cappa, 1987; Celentano and GonzS.lez Cappa, 1992) may be explained by differences in the antigenic processing pathways, later reflected in the outcome of the T. cruzi infections.

Acknowledgements This research was supported by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) and Universidad de Buenos Aires. S.M.G.C. is a Member of the Research Career and A.M.C. is a fellow of the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET), Buenos Aires, Argentina. Silica was kindly supplied by Dr.F61ix Coulombi& The bacterial strain was generously provided by Dr. Teresa Eiger. We thank Dr. Gerardo Mirkin and Dr. Oscar Campetella for critical comments and Dr. Daniel Sordelli for critical reading of the manuscript.

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